Human Transplantation Research Articles

Combination of reduced post-transplant cyclophosphamide and early tacrolimus initiation increases the incidence of chronic graft-versus-host disease in human leukocyte antigen-haploidentical peripheral blood stem-cell transplantation.

We evaluated the clinical impacts of the concurrent modification of post-transplant cyclophosphamide (PTCy) dose and tacrolimus (Tac)-initiation timing in 61 patients with human leukocyte antigen-haploidentical transplantation. Reduced-dose PTCy (80mg/kg) was associated with a higher incidence of moderate-to-severe chronic graft-versus-host disease (GVHD) than standard-dose PTCy (100mg/kg) (35.0%vs. 26.6%, p=0.053). Notably, early-initiation Tac (day -1) increased moderate-to-severe chronic GVHD than standard-initiation Tac (day 5) in the reduced-dose PTCy group (p=0.032), whereas Tac-initiation timing did not impact chronic GVHD in the standard-dose PTCy group. These data indicate that the combination of reduced-dose PTCy and early-initiation Tac can amplify chronic GVHD.

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.

1504-P: AdoShell, a Nonfibrotic Encapsulation System for Human Islets Transplantation, Shows Promising Results for Clinical Application as a Cure for People with Type 1 Diabetes

1504-P: AdoShell, a Nonfibrotic Encapsulation System for Human Islets Transplantation, Shows Promising Results for Clinical Application as a Cure for People with Type 1 Diabetes

A comprehensive analysis of m6A/m7G/m5C/m1A-related gene expression and immune infiltration in liver ischemia–reperfusion injury by integrating bioinformatics and machine learning algorithms

BackgroundLiver ischemia–reperfusion injury (LIRI) is closely associated with immune infiltration, which commonly occurs after liver surgery, especially liver transplantation. Therefore, it is crucial to identify the genes responsible for LIRI and develop effective therapeutic strategies that target immune response. Methylation modifications in mRNA play various crucial roles in different diseases. This study aimed to identify potential methylation-related markers in patients with LIRI and evaluate the corresponding immune infiltration.MethodsTwo Gene Expression Omnibus datasets containing human liver transplantation data (GSE12720 and GSE151648) were downloaded for integrated analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to investigate the functional enrichment of differentially expressed genes (DEGs). Differentially expressed methylation-related genes (DEMRGs) were identified by overlapping DEG sets and 65 genes related to N6-methyladenosine (m6A), 7-methylguanine (m7G), 5-methylcytosine (m5C), and N1-methyladenosine (m1A). To evaluate the relationship between DEMRGs, a protein–protein interaction (PPI) network was utilized. The core DEMRGs were screened using three machine learning algorithms: least absolute shrinkage and selection operator, random forest, and support vector machine-recursive feature elimination. After verifying the diagnostic efficacy using the receiver operating characteristic curve, we validated the expression of the core DEMRGs in clinical samples and performed relative cell biology experiments. Additionally, the immune status of LIRI was comprehensively assessed using the single sample gene set enrichment analysis algorithm. The upstream microRNA and transcription factors of the core DEMRGs were also predicted.ResultsIn total, 2165 upregulated and 3191 downregulated DEGs were identified, mainly enriched in LIRI-related pathways. The intersection of DEGs and methylation-related genes yielded 28 DEMRGs, showing high interaction in the PPI network. Additionally, the core DEMRGs YTHDC1, METTL3, WTAP, and NUDT3 demonstrated satisfactory diagnostic efficacy and significant differential expression and corresponding function based on cell biology experiments. Furthermore, immune infiltration analyses indicated that several immune cells correlated with all core DEMRGs in the LIRI process to varying extents.ConclusionsWe identified core DEMRGs (YTHDC1, METTL3, WTAP, and NUDT3) associated with immune infiltration in LIRI through bioinformatics and validated them experimentally. This study may provide potential methylation-related gene targets for LIRI immunotherapy.

Human Amniotic Epithelial Cell Transplantation is Safe and Well Tolerated in Patients with Compensated Cirrhosis: A First-in-Human Trial.

Placenta-derived human amniotic epithelial cells (hAEC) exhibit anti-inflammatory and anti-fibrotic effects in cirrhosis models. We conducted a first-in-human phase I clinical trial to assess the safety and tolerability of hAEC in adults with compensated cirrhosis. We examined increasing and repeated doses of hAEC in 9 patients in 3 cohorts. Cohort 1 patients received 0.5 × 106/kg hAEC in one IV infusion. Cohort 2 patients received 1 × 106/kg hAEC in one IV infusion. The patients in cohort 3 received 1 × 106/kg hAEC on days 0 and 28. Here, we report follow-up to post-infusion day 56 (D56), during which no serious adverse events occurred. Six patients experienced no study-related adverse events, while 3 patients reported mild (grade 1) headaches that were possibly infusion-related. A transient decrease in serum platelet count occurred in all patients, which returned to baseline screening values by day 5. FIB-4 values to assess fibrosis were significantly lower at D56. Although not statistically significant, serum AST levels and liver stiffness measurements at D56 were lower than those at baseline. The hepatic venous pressure gradient, a measure of portal hypertension, declined in 4 patients, did not change in 3 patients, and increased in 2 patients. In conclusion, intravenous infusion of allogeneic hAEC in patients with compensated cirrhosis at the doses used in this study was safe and well tolerated, with no difference observed between 1 and 2 doses. Decreased hepatic inflammation, liver stiffness, and portal hypertension support larger studies aimed at identifying patients who may benefit from this therapy. Clinical Trial registration: The trial was prospectively entered on the Australian Clinical Trials Registry (ANZCTR12616000437460).

Human umbilical cord mesenchymal stem cell transplantation for the treatment of acute-on-chronic liver failure: protocol for a multicentre random double-blind placebo-controlled trial

IntroductionAcute-on-chronic liver failure (ACLF) is a prevalent and life-threatening liver disease with high short-term mortality. Although recent clinical trials on the use of mesenchymal stem cells (MSCs) for ACLF treatment...

A phase 1 study to evaluate the safety and preliminary efficacy of minnelide given alone or in combination with paclitaxel in advanced gastric cancer.

4033 Background: Minnelide is a water-soluble disodium salt variant of triptolide, an HSP70 inhibitor. Studies using pancreatic cancer cell lines and human xenograft transplants demonstrated that Minnelide prevents tumor progression, increases survival, and causes tumor regression. This study aims to evaluate the safety and antitumor activity of Minnelide and its combination with paclitaxel in patients with advanced gastric cancer (AGC). Methods: This open-label, single-center, dose-escalation phase I study recruited adult patients with previously treated advanced gastric cancer. Patients received Minnelide per oral (PO) daily starting with 1.0 mg for 3weeks and 1week rest schedule, escalating to 1.25mg, and 1.5mg. In combination Regimen, patients received Minnelide starting at a dose of 0.25 mg and escalating by 0.25mg to 1.25mg plus paclitaxel 60-80 mg/m2 intravenously on Day 1, 8, and 15 of each cycle, administered in 4-week cycles. The primary objective was to assess the safety and determine the recommended phase II dose (RP2D). Secondary objectives included preliminary efficacy using RECIST v1.1, and survival analysis. Results: Among Regimen A (n = 11), two patients experienced dose-limiting toxicity (DLTs) (Minnelide 1.5 mg: Grade 3 abdominal pain). No DLTs occurred in combination treatment, Regimen B & C (n=25). The MTD was Minnelide 1.25 mg once daily for 21days Q4 weeks as monotherapy. The most common Grade ≥3 AEs were neutropenia (19.4%) and abdominal pain (11.1%). In Regimen C, 71.5% achieved either a partial response or a stable disease with the median PFS of 4.5 months, and the median OS of 10.7 months. Notably, patients without previous exposure to paclitaxel (n = 4) in the last 2 cohorts of Regimen C, had a confirmed ORR of 50.0%, DCR of 75.0%, the median PFS was 5.3 months (95% CI NE-NE) and the median OS has not reached as some of the patients are still receiving treatment. Conclusions: The combination of Minnelide plus paclitaxel as salvage treatment in AGC patients showed meaningful clinical activity with a manageable safety profile. Based on this Phase I experience a Phase II will be initiated in AGC patients. Clinical trial information: NCT05566834 .

Evaluating longitudinal cytomegalovirus-specific humoral immune responses and association with DNAemia risk in seropositive lung transplant recipients

BackgroundCytomegalovirus (CMV) is the most common viral infection among lung transplant recipients and is associated with chronic lung allograft dysfunction. There is a need for better therapeutics as well as biomarkers to enable effective stratification of CMV seropositive patient risk for developing CMV DNAemia to inform prophylaxis duration. MethodsCMV-specific IgG binding and functional responses were evaluated in a discovery cohort of longitudinal plasma samples from 51 CMV seropositive human lung transplant recipients, collected as part of the CTOT-20 and CTOT-22 consortium studies. Pre-transplant plasma from an additional 43 CMV seropositive lung transplant recipients were evaluated as a validation cohort. ResultsIn the discovery cohort with longitudinal samples, pre-transplant plasma IgG binding to CMV surface glycoproteins gH/gL, gH/gL/gO, and pentameric complex (PC), as well as neutralization of CMV in epithelial cells are associated with increased risk of CMV DNAemia post-prophylaxis. However, these results were not confirmed by the validation cohort. ConclusionWhile quantification of pre-transplant CMV-specific antibody responses showed association with DNAemia in the discovery cohort, additional clinical variables and/or known risk factors for CMV, such as patient CMV-specific T-cell responses, may need to be considered in combination with humoral immunity to effectively stratify risk of CMV DNAemia.

Human-derived fecal microbiota transplantation alleviates social deficits of the BTBR mouse model of autism through a potential mechanism involving vitamin B6 metabolism.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by social communication deficiencies and stereotypic behaviors influenced by hereditary and/or environmental risk factors. There are currently no approved medications for treating the core symptoms of ASD. Human fecal microbiota transplantation (FMT) has emerged as a potential intervention to improve autistic symptoms, but the underlying mechanisms are not fully understood. In this study, we evaluated the effects of human-derived FMT on behavioral and multi-omics profiles of the BTBR mice, an established model for ASD. FMT effectively alleviated the social deficits in the BTBR mice and normalized their distinct plasma metabolic profile, notably reducing the elevated long-chain acylcarnitines. Integrative analysis linked these phenotypic changes to specific Bacteroides species and vitamin B6 metabolism. Indeed, vitamin B6 supplementation improved the social behaviors in BTBR mice. Collectively, these findings shed new light on the interplay between FMT and vitamin B6 metabolism and revealed a potential mechanism underlying the therapeutic role of FMT in ASD.IMPORTANCEAccumulating evidence supports the beneficial effects of human fecal microbiota transplantation (FMT) on symptoms associated with autism spectrum disorder (ASD). However, the precise mechanism by which FMT induces a shift in the microbiota and leads to symptom improvement remains incompletely understood. This study integrated data from colon-content metagenomics, colon-content metabolomics, and plasma metabolomics to investigate the effects of FMT treatment on the BTBR mouse model for ASD. The analysis linked the amelioration of social deficits following FMT treatment to the restoration of mitochondrial function and the modulation of vitamin B6 metabolism. Bacterial species and compounds with beneficial roles in vitamin B6 metabolism and mitochondrial function may further contribute to improving FMT products and designing novel therapies for ASD treatment.

#1945 Comparative analysis of kidney transplantation modeled using precision-cut kidney slices and kidney transplantation in pigs

Abstract Background and Aims Kidney transplants are at risk for so far unavoidable ischemia-reperfusion injury. Several experimental kidney transplantation models are available to study this injury, but all have their own limitations. Here, we describe precision-cut kidney slices (PCKS) as a novel model of kidney ischemia-reperfusion injury in comparison with pig and human kidney transplantation. Method Following bilateral nephrectomy in pigs, we applied warm ischemia (1 h), cold ischemia (20 h) and a reperfusion period (4 h) to one whole kidney undergoing transplantation to a recipient pig and, in parallel, established PCKS undergoing ischemia and modeled reperfusion. Tissues were analyzed using standard histopathology assessment by two blinded pathologists, and bulk RNAseq with gene set enrichment analysis with comparison to human kidney biopsy gene sets obtained from Gene Expression Omnibus. Results Histopathological assessment revealed the presence of some but not all morphological features of tubular injury in PCKS as seen in pig kidney transplantation. RNAseq demonstrated that the majority of changes occurred after reperfusion only, with a partial overlap between PCKS and kidney transplantation, with some differences in transcriptional response attributable to systemic inflammatory responses and immune cell migration. Comparison of PCKS and pig kidney transplantation with RNAseq data from human kidney biopsies by gene set enrichment analysis revealed that both PCKS and pig kidney transplantation reproduced the post-reperfusion pattern of human kidney transplantation. In contrast, only post-cold ischemia PCKS and pig kidney partially resembled the gene set of human acute kidney injury. Conclusion Overall, the present study established that a PCKS protocol can model kidney transplantation and its reperfusion-related damage on a histological and a transcriptomic level. PCKS may thus expand the toolbox for developing novel therapeutic strategies against ischemia-reperfusion injury.

Alginate Hydrogel Integrated with a Human Fibroblast-Derived Extracellular Matrix Supports Corneal Endothelial Cell Functionality and Suppresses Endothelial-Mesenchymal Transition.

Human corneal transplantation is still the only option to restore the function of corneal endothelial cells (CECs). Therefore, there is an urgent need for hCEC delivery systems to replace the human donor cornea. Here, we propose an alginate hydrogel (AH)-based delivery system, where a human fibroblast-derived, decellularized extracellular matrix (ECM) was physically integrated with AH. This AH securely combined with the ECM (ECM-AH) was approximately 50 μm thick, transparent, and permeable. The surface roughness and surface potential provided ECM-AH with a favorable microenvironment for CEC adhesion and growth in vitro. More importantly, ECM-AH could support the structural (ZO-1) and functional (Na+/K+-ATPase) markers of hCECs, as assessed via western blotting and quantitative polymerase chain reaction, which were comparable with those of a ferritic nitrocarburizing (FNC)-coated substrate (a positive control). The cell density per unit area was also significantly better with ECM-AH than the FNC substrate at day 7. A simulation test of cell engraftment in vitro showed that hCECs were successfully transferred into the decellularized porcine corneal tissue, where they were mostly alive. Furthermore, we found out that the endothelial-mesenchymal transition (EnMT)-inductive factors (Smad2 and vimentin) were largely declined with the hCECs grown on ECM-AH, whereas the EnMT inhibitory factor (Smad7) was significantly elevated. The difference was statistically significant compared to that of the FNC substrate. Moreover, we also observed that TGF-β1-treated hCECs showed faster recovery of cell phenotype on the ECM. Taken together, our study demonstrates that ECM-AH is a very promising material for hCEC culture and delivery, which endows an excellent microenvironment for cell function and phenotype maintenance.

#2906 Kidney immune cells and endothelial cells can be modulated by low-salt and high-salt diets

Abstract Background and Aims Kidney immune cells and endothelial cells are involved in pathogenesis of kidney ischemia-reperfusion injury and potential therapeutic targets to improve kidney transplant outcomes. We hypothesized that dietary salt intake can change kidney immune cells and endothelial cells. Method C57BL/6 mice were treated with normal diet, low-salt diet, or high-salt diet. After 6 weeks of allocated diets, mice were euthanized, and their kidneys were collected. Leukocyte infiltration was assessed with CD45 immunohistochemistry. Lymphocytes were isolated from kidneys and were studied by flow cytometry. Human kidney transplant tissues obtained by a protocol biopsy at 2 weeks after kidney transplantation were stained with CD45, CD3, CD20, and CD31. Expression of each marker was quantified using an automated imaging analysis system and analyzed per donors’ pre-donation 24 h urine sodium levels. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and end-stage kidney disease (ESKD) patients were treated with different sodium concentrations, and their proliferation was assessed. Results Kidneys from mice fed with high-salt diet had higher kidney leukocytes (CD45+) than those fed with normal diet (1.9 ± 0.3% vs 0.9 ± 0.1%, P = .043). Effector-memory CD4+ T cells (normal 44.2 ± 2.6%; low-salt 57.1 ± 3.5%, P = .020; high-salt 62.1 ± 2.5% of CD4+ T cells, P = .002) and mature B cells (70.0 ± 1.5%; 79.1 ± 1.4%, P = .001; 76.7 ± 1.6% of CD19+ cells, P = .015) increased by low-salt and high-salt diets, whereas naïve CD4+ T cells deceased (45.9 ± 2.1%; 32.7 ± 3.2%, P = .016; 27.8 ± 3.1% of CD4+ T cells, P = .001) in kidneys. Human kidney transplants from donors under low-salt diet showed lower total leukocytes (CD45+, 0.3 ± 0.1% vs 0.9 ± 0.3%, P = .019) with lower T and B cells (CD3+, 1.8 ± 0.2% vs 3.5 ± 0.4%, P<.001; CD20+, 0.8 ± 0.1% vs 2.1 ± 0.6%, P = .011) and higher capillary density (CD31+, 29.0 ± 2.2% vs 16.1 ± 1.8%, P = .009) compared to those from donors under high-salt diet. The proliferation of iPSC-ECs from healthy individuals and ESKD patients after hypoxia was reduced under higher sodium concentrations. Conclusion High-salt and low-salt diets affect kidney immune cell compositions and numbers as well as endothelial cells. Reconstitution of kidney microenvironment by modifying dietary salt intake could be a novel approach to improve kidney transplant outcomes.

Crowdsourcing Opinions and Awareness of Upper Extremity Transplantation in the United States.

Introduction As of 2008, the United States had 41,000 people living with upper extremity amputation. This number is projected to reach300,000 by 2050. Human upper extremity transplantation (HUET) may become a more common treatment option with the potential to significantly improve the quality of life for certain amputees. Awareness and opinions regarding HUETamong Americans, particularly in Veterans/Service Members (VSM) affiliates, are largely unknown. Materials and methods We administered a survey on Amazon Mechanical Turk (MTurk) workers. Eligible participants were US citizens aged ≥18 years; MTurk worker selection targeted workers who self-reported being a VSM. We used descriptive statistics to summarize study findings and Fisher's exact and Wilcoxon's rank-sum tests for between-group comparisons. Results The survey was completed by 764 individuals, 604 (79.1%) of whom reported being aware of HUET. Among those familiar versus unfamiliar, a significantly higher proportion were aged ≤35 years (n=385, 64.0% vs. n=86, 53.7%; p=0.017), employed (n=523, 86.6% vs. n=114, 71.3%; p<0.001), and aware of their religion's stance on organ/tissue donation (n=341, 54.5% vs. n=62, 38.8%; p<0.001). Amputees and/or respondents related to an amputee were more likely to be aware of HUET than individuals who were amputation naive (n=211, 90.6% vs. n=393, 74.0%, respectively; p<0.001), as were individuals with a personal or familial military affiliation (n=286, 85.4% with vs. n=318, 74.1% with no affiliation; p<0.001). The most reported HUET information sources were digital media (n=157, 31.2%) and internet (n=137, 27.2%). Conclusions Our survey of MTurk workers found greater awareness of HUET among individuals with a VSM or amputee connection. Our additional findings that the internet and academic sources, such as journals or reputable medical publications, were respondents' preferred sources of HUET information emphasize the importance of vascularized composite allotransplantation (VCA) centers' involvement in creating accurate and accessible content to help educate the public about this treatment.

Transcriptional Profiling Underscores the Role of Preprocurement Allograft Metabolism and Innate Immune Status on Outcomes in Human Liver Transplantation.

The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT. We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups. We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways. We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT.

Single-Cell Transcriptome Landscape and Cell Fate Decoding in Human Brain Organoids after Transplantation.

Human stem cells and derivatives transplantation are widely used to treat nervous system diseases, while the fate determination of transplanted cells is not well elucidated. To explore cell fate changes of human brain organoids before and after transplantation, human brain organoids are transplanted into prefrontal cortex (PFC) and hippocampus (HIP), respectively. Single-cell sequencing is then performed. According to time-series sample comparison, transplanted cells mainly undergo neural development at 2 months post-transplantation (MPT) and then glial development at 4MPT, respectively. A different brain region sample comparison shows that organoids grafted to PFC have obtained cell fate close to those of host cells in PFC, other than HIP, which may be regulated by the abundant expression of dopamine (DA) and acetylcholine (Ach) in PFC. Meanwhile, morphological complexity of human astrocyte grafts is greater in PFC than in HIP. DA and Ach both activate the calcium activity and increase morphological complexity of astrocytes in vitro. This study demonstrates that human brain organoids receive host niche factor regulation after transplantation, resulting in the alignment of grafted cell fate with implanted brain regions, which may contribute to a better understanding of cell transplantation and regenerative medicine.

A novel intralamellar semi‐bioresorbable keratoprosthesis—Part A: Design conception, material perspective, and device manufacturing

AbstractCorneal diseases are a significant cause of visual impairment and blindness. The first‐line treatment for corneal opacity is penetrating keratoplasty (human donor cornea transplantation). At present, keratoprosthesis (KPro), an artificial cornea, is the last resort for correcting end‐stage corneal blindness and is usually supported by donor tissue. This article describes a new intralamellar tissue‐free KPro design concept and its preparation method. Wherein, an injection‐molding route is adopted to create a mechanically and structurally stable near‐KPro geometry using a photo‐polymerized poly(2‐hydroxyethyl methacrylate) (pHEMA) with 4% Bisphenol A dimethacrylate (BisMA) crosslinked (PC4) hydrogel composition. Prior to this, the physico‐mechanical properties of crosslinked hydrogels matching corneal tissue are identified, and the surface morphological characteristics of silk cocoon membranes are ascertained in choosing suitable KPro materials. Cytocompatibility tests on PC4 and silk‐incorporated PC4 hydrogels using rabbit corneal fibroblast cell‐line evidenced enhancement in cell growth on silk‐PC4 surfaces. Furthermore, near KPro geometry is surface‐profiled to create a one‐of‐a‐kind design with clear optics and a silk‐bioactivated composite‐based haptic‐flange hydrogel network containing site‐specific submillimeter‐scale perforations to improve tissue integration. Considering this unique KPro geometry, the optic‐haptic‐flange construct is a tissue‐free semi‐bioresorbable hydrogel device presumed to provide stability under the influence of intraocular pressure (IOP) and eyelid shear. Through this study, it is identified new KPro materials facilitate significant cytocompatibility while complimented with site‐specific novel design would offer tissue ingrowth with gradual resorption of silk, leaving behind a stable intralamellar tissue integrated with hydrogel when implanted in the corneal niche.

Genome-wide DNA methylation and transcriptomic analysis of liver tissues subjected to early ischemia/reperfusion injury upon human liver transplantation

Introduction and ObjectivesEpigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. Patients and Methods12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. ResultsSignificant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. ConclusionsIn the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI.

Renin-Angiotensin System Signaling in Parietal Epithelial Cells

INTRODUCTION. Bowman's capsule surrounds the kidney glomerular tuft and consists of a monolayer of parietal epithelial cells (PECs) creating a physiologically functioning barrier between the urinary space and the renal interstitium. Pathophysiological PEC activation describes the phenotypic switch from their normal quiescent state to one of proliferation and migration to the glomerular tuft, leading to glomerular injury. The development of several proteinuric kidney diseases is closely associated with PEC activation, however, the mechanisms of functional receptor signaling in these cells are not well described. This study investigates the functional presence of renin-angiotensin system (RAS) components and corresponding intracellular mechanisms in PECs. Methods. The vibrodissociation method was utilized to obtain freshly isolated Bowman’s capsules from discarded adult male human transplant tissues and 12-week old Wistar male and female rats. Confocal fluorescent microscopy of freshly isolated Bowman’s capsule PECs was performed using Fluo-8 or DAF-FM fluorescent dyes to detect changes in intracellular Ca2+ concentrations and nitric oxide (NO) response to the variety of RAS peptides. RESULTS. In separate experiments on freshly isolated rodent and human Bowman’s capsule PECs, we demonstrate the changes in redox response to the stimulation of RAS components. In particular, the acute application of Ang II peptide caused a rapid increase in NO production. Interestingly, we did not indicate significant changes in intracellular Ca2+ influx. To find out which receptor is responsible for the NO release we preincubated PECs with AT1R and AT2R blockers (Losartan and PD123319 respectively). Our data indicate that RAS mediates NO production through the AT2R activation. There is no sex difference was observed in these experiments. Further experiments with the variety of nitric oxide synthase (NOS) pharmacology reveal the functional distribution of NOS in rodent PECs. CONCLUSION. Our study provides an overview of the RAS signaling within PECs. For the first time, we present evidence demonstrating a direct connection between Ang II and redox homeostasis in PECs. Further investigation using the developed approach will be essential for understanding the role of Bowman's capsule cells in the glomerulus and the development of potential treatment against the hyperplasia of activated PECs and corresponding chronic kidney pathologies. Funding Sources: R01 DK129227, R01 DK126720, VA CDA-2 1IK2BX005605-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Leukotriene B4: A potential mediator and biomarker for cardiac allograft vasculopathy

BackgroundCardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to-date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in development of CAV. MethodsWe used an established pre-clinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy non-transplant controls. ResultsWe showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found non-significantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, non-transplant controls. ConclusionsThis study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development, and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.

Spectrum of chronic lung allograft dysfunction pathology in human lung transplantation

BackgroundLong-term survival after lung transplantation (LTx) remains limited by Chronic Lung Allograft Dysfunction (CLAD), which includes two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), with possible overlap. We aimed to detail and quantify pathological features of these CLAD sub-types. MethodsPeripheral and central paraffin-embedded explanted lung samples were obtained from 20 consecutive patients undergoing a second LTx for CLAD, from 3 lobes. Thirteen lung samples, collected from non-transplant lobectomies or donor lungs, were used as controls. Blinded semi-quantitative grading was performed to assess airway fibrotic changes, parenchymal and pleural fibrosis, as well as epithelial and vascular abnormalities. ResultsCLAD lung samples had higher scores for all airway- and lung-related parameters compared to controls. There was a notable overlap in pathological scores between BOS and RAS, with a wide range of scores in both conditions. Parenchymal and vascular fibrosis scores were significantly higher in RAS compared to BOS (p=0.003 for both). We observed a significant positive correlation between the degree of inflammation around each airway, the severity of epithelial changes and airway fibrosis. Immunofluorescence staining demonstrated a trend towards a lower frequency of club cells in CLAD, and a higher frequency of apoptotic club cells in BOS samples (p=0.01). ConclusionsCLAD is a spectrum of airway, parenchymal, and pleural fibrosis, as well as epithelial, vascular, and inflammatory pathological changes, where BOS and RAS overlap significantly. Our semi-quantitative grading score showed a generally high inter-reader reliability and may be useful for future CLAD pathological assessments.