Pattern Recognition Receptor-reactivity Screening of Liver Transplant Patients

Objective and backgroundPattern recognition receptors (PRRs) on immune and parenchymal cells can detect danger-associated molecular patterns (DAMPs) released from cells damaged during ischemia-reperfusion injury (IRI), in heart attack or stroke settings, but also as an unavoidable consequence of solid organ transplantation. Despite IRI being a significant clinical problem across all solid organ transplants, there are limited therapeutics and patient-specific diagnostics currently available.MethodsWe screened portal blood samples obtained from 67 human liver transplant recipients both pre- [portal vein (PV) sample] and post-(liver flush; LF) reperfusion for their ability to activate a panel of PRRs, and analyzed this reactivity in relation to biopsy-proven IRI.ResultsPV samples from IRI+ orthotopic liver transplantation (OLT) patients (n = 35) decreased activation of hTLR4- and hTLR9-transfected cells, whereas PV from IRI- patients (n = 32) primarily increased hTLR7 and hNOD2 activation. LF samples from OLT-IRI patients significantly increased activation of hTLR4 and hTLR9 over IRI- LF. In addition, the change from baseline reactivity to hTLR4/9/NOD2 was significantly higher in IRI+ than IRI- OLT patients.ConclusionsThese results demonstrate that TLR4/7/9 and NOD2 are involved in either promoting or attenuating hepatic IRI, and suggest a diagnostic screening of portal blood for reactivity to these PRRs might prove useful for prediction and/or therapeutic intervention in OLT patients before transplantation

Association of pro-inflammatory cytokines and monocyte subtypes in older and younger patients on clinical outcomes after mechanical circulatory support device implantation

Noninvasive immunologic analysis of peripheral blood holds promise for explaining the mechanism of development of adverse clinical outcomes, and may also become a method for patient risk stratification before or after mechanical circulatory support device (MCSD) implantation. Dysregulation of the innate immune system is associated with increased patient age but has yet to be evaluated in the older patient with advanced heart failure undergoing MCSD surgery. Patients pre- and post-MCSD implantation had peripheral blood mononuclear cells (PBMC) and serum isolated. Multiparameter flow cytometry was used to analyze markers of innate cell function, including monocyte subtypes. Multiplex cytokine analysis was performed. MELD-XI and SOFA scores were utilized as surrogate markers of outcomes. Increased levels of pro-inflammatory cytokines including IL-15, TNF-α, and IL-10 were associated with increased MELD-XI and SOFA scores. IL-8, TNF- α, and IL-10 were associated with risk of death after MCSD implantation, even with correction for patient age. Increased frequency of 'classical' monocytes (CD14 + CD16-) were associated with increased MELD-XI and SOFA scores. This suggests that inflammation and innate immune system activation contribute to progression to multiorgan system failure and death after MCSD surgery. Development of noninvasive monitoring of peripheral blood holds promise for biomarker development for candidate selection and patient risk stratification

T cell dysfunction and patient age are associated with poor outcomes after mechanical circulatory support device implantation

Immunologic impairment may contribute to poor outcomes after implantation of mechanical circulatory support device (MCSD), with infection often as a terminal event. The study of immune dysfunction is of special relevance given the growing numbers of older patients with heart disease. The aim of the study was to define which immunologic characteristics are associated with development of adverse clinical outcomes after MCSD implantation. We isolated peripheral blood mononuclear cells (PBMC) from patients pre- and up to 20 days post-MCSD implantation and analyzed them by multiparameter flow cytometry for T cell dysfunction, including terminal differentiation, exhaustion, and senescence. We used MELD-XI and SOFA scores measured at each time point as surrogate markers of clinical outcome. Older patients demonstrated increased frequencies of terminally differentiated T cells as well as NKT cells. Increased frequency of terminally differentiated and immune senescent T cells were associated with worse clinical outcome as measured by MELD-XI and SOFA scores, and with progression to infection and death. In conclusion, our data suggest that T cell dysfunction, independently from age, is associated with poor outcomes after MCSD implantation, providing a potential immunologic mechanism behind patient vulnerability to multiorgan dysfunction and death. This noninvasive approach to PBMC evaluation holds promise for candidate evaluation and patient monitoring

Disulfide High‐Mobility Group Box 1 Drives Ischemia‐Reperfusion Injury in Human Liver Transplantation

Background and aimsSterile inflammation is a major clinical concern during ischemia-reperfusion injury (IRI) triggered by traumatic events, including stroke, myocardial infarction, and solid organ transplantation. Despite high-mobility group box 1 (HMGB1) clearly being involved in sterile inflammation, its role is controversial because of a paucity of patient-focused research.Approach and resultsHere, we examined the role of HMGB1 oxidation states in human IRI following liver transplantation. Portal blood immediately following allograft reperfusion (liver flush; LF) had increased total HMGB1, but only LF from patients with histopathological IRI had increased disulfide-HMGB1 and induced Toll-like receptor 4-dependent tumor necrosis factor alpha production by macrophages. Disulfide HMGB1 levels increased concomitantly with IRI severity. IRI+ prereperfusion biopsies contained macrophages with hyperacetylated, lysosomal disulfide-HMGB1 that increased postreperfusion at sites of injury, paralleling increased histone acetyltransferase general transcription factor IIIC subunit 4 and decreased histone deacetylase 5 expression. Purified disulfide-HMGB1 or IRI+ blood stimulated further production of disulfide-HMGB1 and increased proinflammatory molecule and cytokine expression in macrophages through a positive feedback loop.ConclusionsThese data identify disulfide-HMGB1 as a mechanistic biomarker of, and therapeutic target for, minimizing sterile inflammation during human liver IRI

Association between preoperative peripheral blood mononuclear cell gene expression profiles, early postoperative organ function recovery potential and long-term survival in advanced heart failure patients undergoing mechanical circulatory support

Multiorgan dysfunction syndrome contributes to adverse outcomes in advanced heart failure (AdHF) patients after mechanical circulatory support (MCS) implantation and is associated with aberrant leukocyte activity. We tested the hypothesis that preoperative peripheral blood mononuclear cell (PBMC) gene expression profiles (GEP) can predict early postoperative improvement or non-improvement in patients undergoing MCS implantation. We believe this information may be useful in developing prognostic biomarkers.We conducted a study with 29 patients undergoing MCS-surgery in a tertiary academic medical center from 2012 to 2014. PBMC samples were collected one day before surgery (day -1). Clinical data was collected on day -1 and day 8 postoperatively. Patients were classified by Sequential Organ Failure Assessment score and Model of End-stage Liver Disease Except INR score (measured eight days after surgery): Group I = improving (both scores improved from day -1 to day 8, n = 17) and Group II = not improving (either one or both scores did not improve from day -1 to day 8, n = 12). RNA-sequencing was performed on purified mRNA and analyzed using Next Generation Sequencing Strand. Differentially expressed genes (DEGs) were identified by Mann-Whitney test with Benjamini-Hochberg correction. Preoperative DEGs were used to construct a support vector machine algorithm to predict Group I vs. Group II membership.Out of 28 MCS-surgery patients alive 8 days postoperatively, one-year survival was 88% in Group I and 27% in Group II. We identified 28 preoperative DEGs between Group I and II, with an average 93% prediction accuracy. Out of 105 DEGs identified preoperatively between year 1 survivors and non-survivors, 12 genes overlapped with the 28 predictive genes.In AdHF patients following MCS implantation, preoperative PBMC-GEP predicts early changes in organ function scores and correlates with long-term outcomes. Therefore, gene expression lends itself to outcome prediction and warrants further studies in larger longitudinal cohorts