Search

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. [ABSTRACT FROM AUTHOR]

This is a brief expository paper on reparameterized intercepts under constrained variants of the nominal response model, including the generalized partial credit and partial credit models. Such parameterizations are commonly found in item response theory software packages such as flexMIRT\textregistered , IRTPRO, and OpenMx / \emph {rpf}, and both these models are highly popular in educational and psychological testing. A heuristic graphical interpretation is provided. We give examples of how intercepts may be easily generated for Monte Carlo simulation studies, including a brief study to increase generalizability and explore limitations of a recently developed information matrix test to detect misspecification when collapsing adjacent response categories.

Early graft rejection is a common cause of early postoperative mortality after heart transplantation. Viral myocarditis persisting in the donor organ is a possible exclusion criterion, but often difficult to exclude completely. A combination of allograft rejection with prior, undiagnosed viral replication in the donor heart is challenging, with competing and seemingly conflicting treatment options. We report the case of a 45-year-old woman with ischemic cardiomyopathy who received a heart from a 27-year-old donor who underwent resuscitation during exercise but suffered irreversible brain damage. The recipient developed early combined cellular and humoral allograft rejection (G1R/pAMR 2) accompanied by high levels of preformed and de novo HLA-specific antibodies. As a result of clinical deterioration, treatment with human immunoglobulin and high-dose cortisone, followed by immunapheresis, was initiated and anti-thymocyte globulin was administered in addition to standard immunosuppressive therapy. While recovering clinically, she developed high replication of human herpesvirus (HHV)-6 with ∼200 × 103 copies/μg tissue. Eight days after maximal rejection therapy, graft rejection was significantly reduced (G1R/pAMR 0), but HHV-6 viral load doubled. Repeated flow cytometric analysis of recipient blood revealed transiently dysregulated lymphocyte composition at the level of T, B, and NK cells, as well as particular lymphocytes of the CD38hi and CD25hi lineages. Because it is still unclear whether this population is indicative of both antiviral activity and rejection-based immune activation, we are currently performing single-cell RNA sequencing in combination with antibody-based detection of surface molecules in cryopreserved leukocytes from blood and endomyocardial tissue to characterize these and other observable populations. Whereas inflammatory T-cell activity after heart transplantation is usually associated with early allograft rejection, we report a case in which both acute rejection and (preformed) HHV-6 myocarditis in the donor heart occurred, challenging all treatment decisions. Further experimental steps need to be taken to gain insight into the underlying (patho)physiological role of the previously unrecognized lymphocyte population in this patient. [ABSTRACT FROM AUTHOR]

Patients with end-stage lung diseases on the MHH waiting list for lung transplantation (LTx) have been vaccinated against the SARS-CoV-2 spike protein with usually three doses of the mRNA vaccine. Hence, they are supposed to develop robust antibody and T cell responses when immunized prior to LTx without the influence of immunosuppression. Therefore, we hypothesized the induction of high spike-specific IgG levels and protection against SARS-CoV-2 infection and severe COVID-19. To proof this, we aimed to analyze the IgG levels specific for the spike S1-, RBD- and S2-domains in patients on a waiting list (WL-LTx) longitudinally before and after their transplantation. Plasma obtained pre (n=70) and post LTx (n=28) of WL-LTx patients was analyzed for spike-specific IgG by Luminex-based multiplex assays. The threshold for positivity was set separately for each spike domain based on the median MFI +2σ in a healthy, unexposed pre-pandemic control group. Patients with previous SARS-CoV-2-infection were excluded. 95.7% of WL-LTx patients had seroconverted for either RBD-, S1- or S2-specific IgG pre LTx and still 92.86% were positive post LTx. Overall, S1-, S2- and RBD-specific IgG MFI values did not significantly differ between pre vs. post LTx. A subanalysis of matched plasma samples (n=25) revealed that 52% of the WL-LTx patients showed a higher IgG response pre than post LTx for all three spike protein domains and 28% showed even elevated antibody levels post LTx. Interestingly, S2-specific IgG MFI values were significantly elevated compared to RBD-specific IgG MFI values, both pre (S2 vs. RBD p<0.0001) and post LTx (S2 vs. RBD p=0.0225). The majority of WL-LTx patients mounted high SARS-CoV-2 spike-specific IgG responses following vaccination pre LTx. Based on the more efficient antibody production against the S2-domain compared to RBD- and S1-domains, S2-specific IgG responses should be included also in the general evaluation of humoral immune responses to SARS-CoV-2. As expected, WL-LTx patients showed a superior antibody response to vaccination compared to LTx-recipients vaccinated only after LTx, which could even be maintained after LTx in some patients. Therefore, both patients on waiting list and LTx recipients may benefit from additional booster vaccinations after LTx. [ABSTRACT FROM AUTHOR]

The development of anti-HLA donor specific antibodies detected early after lung transplantation (eDSA) has been associated with antibody-mediated rejection (AMR) and poor graft survival. At our institution, since 2013, successive infusions of IgA- and IgM-enriched human intravenous immunoglobulins (IgGAM, first infusion: 2gr/kg, then 0.5gr/kg once every 4 weeks for a maximum of 6 months) formed the backbone of eDSA therapy. Actually, patients with only evidence of eDSA (possible subclinical AMR) are treated with IgGAM only. Patients with concomitant graft dysfunction (possible clinical AMR) or preformed DSA (positive virtual crossmatch) receive additionally plasmapheresis (PE) or immunoabsorption before the first IgGAM dose, and a single dose of anti-CD20 antibody (Rituximab) thereafter. Aims of this study were to present the 9-year results of the IgGAM-based therapy. Records of patients transplanted between 02/2013 and 10/2022 were reviewed. Outcomes were compared between patients with eDSA and treated with IgGAM (IgGAM group) and without eDSA (control group). Median (IQR) follow-up was 51 (23-79) months. During the study period, among the 1,078 transplanted patients, 270 (25%) patients formed the IgGAM group and 785 (73%) the control group. The remaining 23 (2%) patients (14 patients with eDSA but not treated, and 9 patients treated only with tPE and Rituximab) were excluded. Sixty-five IgGAM patients (24%) showed pre-formed eDSA and 43 (16%) patients showed possible clinical AMR. Median time to eDSA detection was 14 days. At follow-up end, treatment was completed in 246 (91%) patients (still on treatment, n=9; in-hospital deaths, n=4; treatment interrupted earlier as intended by protocol, n=11). In these patients, IgGAM treatment cleared eDSA in 220 (89%) patients, 38 (17%) patients showing eDSA recurrence a median of 15 months after treatment end. In IgGAM vs. control patients and at 5- and 9-year follow-up, respectively, graft survival (%) was 77 vs. 76 and 60 vs. 64 (p=0.83), and freedom from CLAD was 76 vs. 74 and 65 vs. 66 (p=0.74), respectively. After lung transplantation, an IgGAM-based treatment protocol for eDSA yielded high eDSA clearance. Patients with eDSA and IgGAM-treatment have good 9-year graft survival similar to control patients. [ABSTRACT FROM AUTHOR]

Pretransplant sensitization to human leukocyte antigens (HLA) increases waiting list time and mortality in lung transplantation (LTx). Instead of awaiting crossmatch-negative donors, since 2013, our recipients with preformed anti-HLA donor-specific antibodies (pfDSA) have been managed peri-transplant with a combination of repeated IgA/IgM-enriched intravenous immunoglobulin infusions (IgGAM, first infusion 2g/kg, then 0.5g/kg every 4 weeks (wk) to max. 6 months), preceded by repeated plasmapheresis (PE), and a single dose of anti-CD20 antibody (375mg/m2, Rituximab) after the first IgGAM infusion. We hypothesized that the composition of plasma cytokines/chemokines was altered in pfDSA patients due to this treatment compared to DSA-free patients without this peri-transplant immunomodulation. Plasma cytokines and chemokines of subgroups of LTx patients with pfDSA (n=8 of 65) were compared to patients without preformed or de novo DSA (n=26 of 780) before and after LTx. 50 cytokines/chemokines in recipient plasma were quantified before, directly after, 24h, 3, 6wk and 12 months after LTx in these pfDSA+/- subgroups using multiplex assays. Clinical follow-up of all LTx patients was 51 (22-80) months. Of the 1072 LTx patients, 65 (6%) patients exhibited pfDSA, primarily against HLA class II (n=42, 65%), followed by HLA class I (n=19, 29%) and both classes (n=4, 6%), while 780 (73%) did not develop pf or early DSA (control group). In pfDSA vs. control patients, prevalence of primary graft dysfunction grade 3 at 72 hours was 10.8% vs. 4.5% (p=0.036). Graft survival and freedom from chronic lung allograft dysfunction did not differ between groups (9 years, 75% vs. 66%, p=0.41; 65% vs. 65%, p=0.59). The pfDSA treatment protocol resulted in significant changes in the plasma cytokine/chemokine composition with significantly reduced concentrations of several pro-inflammatory factors such as TNF-α, IFN-a2, IL-12p70, CXCL8/9 and FGF-β continuously from pre-Tx to at least 6 months later (all p< 0.05). A treatment protocol based on IgG/AM with PE allowed transplantation of patients with preformed DSA with a 9-year graft survival similar to non-DSA-control patients. The pfDSA treatment caused consistent reduction of multiple cytokines, thereby, potentially contributing to this improved graft survival. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level, independent of alternative splicing of TBKBP1 transcripts. Notably, the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients' blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke., (© 2024. The Author(s).)

Steroid-free immunosuppression protocols gained popularity in pediatric liver transplantation (pLT) after the introduction of IL-2-receptor blockade for induction therapy. We analyzed the clinical and immunologic outcome data of the multicenter prospective observational ChilSFree study to compare the impact of steroid-free versus steroid-containing immunosuppressive therapy following pLT in a real-life scenario. Two hundred forty-six children [55.3% male, age at pLT median: 2.4 (range: 0.2-17.9) y] transplanted for biliary atresia (43%), metabolic liver disease (9%), acute liver failure (4%), hepatoblastoma (9%), and other chronic end-stage liver diseases (39%) underwent immune monitoring and clinical data documentation over the first year after pLT. Patient and graft survival at 1 year was 98.0% and 92.7%, respectively. Primary immunosuppression was basiliximab induction followed by tacrolimus (Tac) monotherapy (55%), Tac plus steroid tapering over 3 months (29%), or cyclosporine and steroid tapering (7%). One center used intraoperative steroids instead of basiliximab followed by Tac plus mycophenolate mofetil (7% of patients). N = 124 biopsy-proven T-cell-mediated rejections were documented in n = 82 (33.3%) patients. T-cell-mediated rejection occurred early (median: 41 d, range: 3-366 d) after pLT. Patients initially treated with Tac plus steroids experienced significantly fewer episodes of rejection than patients treated with Tac alone (chi-square p <0.01). The use of steroids was associated with earlier downregulation of proinflammatory cytokines interferon (IFN)-γ, Interleukin (IL)-6, CX motif chemokin ligand (CXCL)8, IL-7, and IL-12p70. Both primary immunosuppression with Tac plus steroids and living donor liver transplantation were independent predictors of rejection-free survival 1 year after pLT on logistic regression analysis. Adjunctive steroid therapy after pLT leads to earlier suppression of the post-pLT proinflammatory response and significantly reduced rejection rates during the first year after pLT (15.9%). Fifty-one percent of patients initially treated without steroids remain steroid-free over the first 12 months without rejection., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Background: Human immune responses to COVID-19 vaccines display a large heterogeneity of induced immunity and the underlying immune mechanisms for this remain largely unknown., Methods: Using a systems biology approach, we longitudinally profiled a unique cohort of female high and low responders to the BNT162b vaccine, who were known from previous COVID-19 vaccinations to develop maximum and minimum immune responses to the vaccine. We utilized high dimensional flow cytometry, bulk and single cell mRNA sequencing and 48-plex serum cytokine analyses., Findings: We revealed early, transient immunological and molecular signatures that distinguished high from low responders and correlated with B and T cell responses measured 14 days later. High responders featured a distinct transcriptional activity of interferon-driven genes and genes connected to enhanced antigen presentation. This was accompanied by a robust cytokine response related to Th1 differentiation. Both transcriptome and serum cytokine signatures were confirmed in two independent confirmatory cohorts., Interpretation: Collectively, our data contribute to a better understanding of the immunogenicity of mRNA-based COVID-19 vaccines, which might lead to the optimization of vaccine designs for individuals with poor vaccine responses., Funding: German Center for Infection Research, German Center for Lung Research, German Research Foundation, Excellence Strategy EXC 2155 "RESIST" and European Regional Development Fund., Competing Interests: Declaration of interests G.H. reports grants from the German Federal Ministry of Education and Research, German Center for Lung Research, and German Research Foundation as well as personal fees from Sanofi GmbH, MedUpdate, and Abbvie. A.D-J served as an advisor or speaker from Novartis and Astra-Zeneca unrelated to this work. Funding parties had no role in study design, data collection, data analyses, interpretation, or writing of report. The other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pretransplant allosensitization to human leukocyte antigens (HLA) increases the recipient's waiting list time and mortality in lung transplantation. Rather than waiting for crossmatch-negative donors, since 2013, recipients with preformed donor-specific antiHLA antibodies (pfDSA) have been managed with repeated IgA- and IgM-enriched intravenous immunoglobulin (IgGAM) infusions, usually in combination with plasmapheresis before IgGAM and a single dose of antiCD20 antibody. This retrospective study presents our 9-year experience with patients transplanted with pfDSA. Records of patients transplanted between February 2013 and May 2022 were reviewed. Outcomes were compared between patients with pfDSA and those without any de novo donor-specific antiHLA antibodies. The median follow-up time was 50 months. Of the 1,043 patients who had undergone lung transplantation, 758 (72.7%) did not develop any early donor-specific antiHLA antibodies, and 62 (5.9%) patients exhibited pfDSA. Among the 52 (84%) patients who completed treatment, pfDSA was cleared in 38 (73%). In pfDSA vs control patients and at 8-year follow-up, respectively, graft survival (%) was 75 vs 65 (P = .493) and freedom from chronic lung allograft dysfunction (%) was 63 vs 65 (P = .525). In lung transplantation, crossing the preformed HLA-antibody barrier is safe using a treatment protocol based on IgGAM. Patients with pfDSA have a good 8-year graft survival rate and freedom from chronic lung allograft dysfunction, similar to control patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fabio Ius reports financial support was provided by Biotest AG. Christine Falk reports financial support was provided by Biotest AG. Christine Falk reports financial support was provided by SFB 738 B3. Fabio Ius reports a relationship with Biotest AG that includes: consulting or advisory, funding grants, speaking and lecture fees, and travel reimbursement. Gregor Warnecke and Wiebke Sommer declare congress fees, study grants and honorary for lectures paid by Biotest AG., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Configurational comparative methods (CCMs) and logic regression methods (LRMs) are two families of exploratory methods that employ very different techniques to analyze data generated by causal structures featuring conjunctural causation and equifinality. Aiming for the same by different means carries a substantive synergy potential, which, however, remains untapped so far because representatives of the two frameworks know little of each other. The purpose of this article is to change that. We first level the field for readers from both backgrounds by providing brief introductions to the basic ideas behind CCMs and LRMs. Then, we carve out the strengths and weaknesses of the two method families by benchmarking their performance when applied to binary data under a variety of different discovery contexts. It turns out that CCMs and LRMs have complementary strengths and weaknesses. This creates various promising avenues for cross-validation.

Background: Chronic lung allograft dysfunction (CLAD) is a leading cause of graft loss in lung transplantation. Despite this, convincing treatment data is lacking, and protocols vary widely between centers. CLAD phenotypes exist, but phenotype transitioning has increased the challenge of designing clinically relevant studies. Extracorporeal photopheresis (ECP) has long been a suggested salvage treatment, but efficacy appears unpredictable. This study describes our experiences with photopheresis, using novel temporal phenotyping to illustrate the clinical course., Methods: Retrospective analysis of patients completing ≥3 months of ECP for CLAD between 2007 and 2022 was performed. A latent class analysis employing a mixed-effects model was performed, deriving patient subgroups based on spirometry trajectory over the 12 months prior to photopheresis until graft loss or 4 years post photopheresis initiation. The resulting temporal phenotypes were compared in terms of treatment response and survival outcomes. Linear discriminatory analysis was used to assess phenotype predictability, relying solely on data available at photopheresis initiation., Results: Data from 5,169 outpatient attendances in 373 patients was used to construct the model. Five trajectories were identified, with uniform spirometry changes evident following 6 months of photopheresis. Outcomes were poorest in Fulminant patients (N = 25, 7%) with median survival of 1 year. In the remainder, poorer lung function at initiation led to poorer outcomes. The analysis revealed important confounders, affecting both decision-making and outcome interpretation., Conclusions: Temporal phenotyping provided novel insights into ECP treatment response in CLAD, particularly the importance of timely intervention. Limitations in % Baseline values in guiding treatment decisions warrant further analysis. Photopheresis may have a more uniform effect than previously thought. Predicting survival at ECP initiation appears feasible., Competing Interests: Disclosure statement MG has previously received speaker fees from Therakos. Neither Therakos nor any of their partners were involved or indeed had any prior knowledge of the manuscript at any stage prior to publication. JMM reports institutional grants and speaker fees from Boehringer Ingelheim. FI reports consulting and speaker fees as well as travel support grants from Biotest AG. TW has received research grants from the German Ministry for Research and Education for projects unrelated to this manuscript. None of the other authors reported any relevant financial interests., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: The current gold standard to diagnose T-cell-mediated acute rejection (TCMR) requires liver histology. Using data from the ChilSFree study on immune response after paediatric liver transplantation (pLT), we aimed to assess whether soluble cytokines can serve as an alternative diagnostic tool in children suspected to have TCMR., Methods: A total of n = 53 blood samples obtained on the day of or up to 3 days before liver biopsy performed for suspected TCMR at median 18 days (range 7-427) after pLT in n = 50 children (38% female, age at pLT 1.8 (0.5-17.5) years) were analysed for circulating cytokine levels using Luminex-based Multiplex technology. Diagnostic accuracy of cytokine concentrations was assessed using a multivariable model based on elastic net regression and gradient boosting machine analysis., Results: TCMR was present in 68% of biopsies. There was strong evidence that patients with TCMR had increased levels of soluble CXCL8, CXCL9, CXCL10, IL-16, IL-18, HGF, CCL4, MIF, SCGF-β, and HGF before biopsy. There was some evidence for increased levels of sCD25, ICAM-1, IL-6, IL-3, and CCL11. Diagnostic value of both single cytokine levels and a combination of cytokines and clinical markers was poor, with AUROCs not exceeding 0.7., Conclusion: Patients with TCMR showed raised levels of cytokines and chemokines reflective of T-cell activation and chemotaxis. Despite giving insight into the mechanisms of TCMR, the diagnostic value of soluble cytokines for the confirmation of TCMR in a clinical scenario of suspected TCMR is poor.