Normal Recipients Research Articles

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

Right Ventricular Function on Cardiovascular Magnetic Resonance Imaging and Long-Term Outcomes in Stable Heart Transplant Recipients.

In heart transplant recipients, right ventricular (RV) dysfunction may occur for a variety of reasons. Whether RV dysfunction in the stable phase after heart transplantation is associated with long-term adverse outcomes is unknown. We aimed to determine the long-term prognostic significance of RV dysfunction identified on cardiovascular magnetic resonance imaging (CMR) at least 1 year after heart transplantation. In consecutive heart transplant recipients who underwent CMR for surveillance, we assessed 2 CMR measures of RV function: RV ejection fraction and RV global longitudinal strain (RVGLS). We investigated associations between RV dysfunction and a composite end point of death or major adverse cardiac events, including retransplantation, nonfatal myocardial infarction, coronary revascularization, and heart failure hospitalization. A total of 257 heart transplant recipients (median age, 59 years; 75% men) who had CMR at a median of 4.3 years after heart transplantation were included. Over a median follow-up of 4.4 years after the CMR, 108 recipients experienced death or major adverse cardiac events. In a multivariable Cox regression analysis adjusted for age, time since transplantation, indication for transplantation, cardiac allograft vasculopathy, history of rejection, and CMR covariates, RV ejection fraction was not associated with the composite end point, but RVGLS was independently associated with the composite end point with a hazard ratio of 1.08 per 1% worsening in RVGLS ([95% CI, 1.00-1.17]; P=0.046). RVGLS provided incremental prognostic value over other variables in multivariable analyses. The association was replicated in subgroups of recipients with normal RV ejection fraction and recipients with late gadolinium enhancement imaging. A similar association was seen with a composite end point of cardiovascular death or major adverse cardiac events. CMR feature tracking-derived RVGLS assessed at least 1 year after heart transplantation was independently associated with the long-term risk of death or major adverse cardiac events. Future studies should investigate its role in guiding clinical decision-making in heart transplant recipients.

Conclusive demonstration of iatrogenic Alzheimer’s disease transmission in a model of stem cell transplantation

The risk of iatrogenic disease is often underestimated as a concern in contemporary medical procedures, encompassing tissue and organ transplantation, stem cell therapies, blood transfusions, and the administration of blood-derived products. In this context, despite the prevailing belief that Alzheimer's disease (AD) manifests primarily in familial and sporadic forms, our investigation reveals an unexpected transplantable variant of AD in a preclinical context, potentially indicating iatrogenic transmission in AD patients. Through adoptive transplantation of donor bone marrow stem cells carrying a mutant human amyloid precursor protein (APP) transgene into either APP-deficient knockout or normal recipient animals, we observed rapid development of AD pathological hallmarks. These pathological features were significantly accelerated and emerged within 6-9months post transplantation and included compromised blood-brain barrier integrity, heightened cerebral vascular neoangiogenesis, elevated brain-associated β-amyloid levels, and cognitive impairment. Furthermore, our findings underscore the contribution of β-amyloid burden originating outside of the central nervous system to AD pathogenesis within the brain. We conclude that stem cell transplantation from donors harboring a pathogenic mutant allele can effectively transfer central nervous system diseases to healthy recipients, mirroring the pathogenesis observed in the donor. Consequently, our observations advocate for genomic sequencing of donor specimens prior to tissue, organ, or stem cell transplantation therapies, as well as blood transfusions and blood-derived product administration, to mitigate the risk of iatrogenic diseases.

Exosomal microRNA-92b Is a Diagnostic Biomarker in Breast Cancer and Targets Survival-Related MTSS1L to Promote Tumorigenesis.

Exosomal microRNAs (miRNAs) are novel, non-invasive biomarkers for facilitating communication and diagnosing cancer. However, only a few studies have investigated their function and role in the clinical diagnosis of breast cancer. To address this gap, we established a stable cell line, MDA-MB-231-CD63-RFP, and recruited 112 female participants for serum collection. We screened 88 exosomal miRNAs identified through microarray analysis of 231-CD63 and literature screening using real-time PCR; only exosomal miR-92b-5p was significantly increased in patients with breast cancer. It had a significant correlation with stage and discriminated patients from the control with an AUC of 0.787. Exosomal miR-92b-5p impacted the migration, adhesion, and spreading ability of normal human mammary epithelial recipient cells through the downregulation of the actin dynamics regulator MTSS1L. In clinical breast cancer tissue, the expression of MTSS1L was significantly inversely correlated with tissue miR-92b-5p, and high expression of MTSS1L was associated with better 10-year overall survival rates in patients undergoing hormone therapy. In summary, our studies demonstrated that exosomal miR-92b-5p might function as a non-invasive body fluid biomarker for breast cancer detection and provide a novel therapeutic strategy in the axis of miR-92b-5p to MTSS1L for controlling metastasis and improving patient survival.

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway.

Introduction: Exosomes, pivotal in intercellular communication during skin disease pathogenesis, have garnered substantial attention. However, the impact of environmental pollutants, such as benzo[a]pyrene (BaP) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), on exosome release amid inflammatory skin diseases remains unexplored. This study addresses this gap by examining the influence of BaP and TCDD on exosome function, specifically focusing on immune-related pathway alterations in normal recipient keratinocytes and peripheral blood mononuclear cells (PBMCs). Methods: HaCaT cells were treated with exosomes from BaP- or TCDD-treated keratinocytes. Proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5, were assessed. The involvement of the p65NF-κB/p38MAPK/ERK signaling pathway in recipient keratinocytes was investigated. Aryl hydrocarbon receptor (AhR) silencing was employed to elucidate its role in mediating the proinflammatory response induced by exosomes from BaP- or TCDD-treated keratinocytes. Results and discussion: Treatment with exosomes from BaP- or TCDD-treated keratinocytes induced a significant increase in proinflammatory cytokines and chemokines in HaCaT cells. The upregulation implicated the p65NF-κB/p38MAPK/ERK signaling pathway. AhR silencing attenuated this response, suggesting a role for AhR in mediating this response. In PBMCs from healthy controls, exosomes from BaP-stimulated PBMCs of psoriatic patients led to increased expression of proinflammatory cytokines and modulation of Th1/Th17 cell distribution via AhR activation. These findings unveil a novel dimension in the interplay between environmental xenobiotic agents (BaP and TCDD) and exosomal functions. The study establishes their influence on psoriatic inflammatory responses, shedding light on the underlying mechanisms mediated through the AhR signaling pathway in recipient keratinocytes and PBMCs.

Autophagy reduces aortic calcification in diabetic mice by reducing matrix vesicle body-mediated IL-1β release

Vascular calcification (VC) is a common pathological process of cardiovascular disease that occurs in patients with type 2 diabetes mellitus (T2DM). However, the molecular basis of VC progression remains unknown. A GEO dataset (GSE146638) was analyzed to show that microbodies and IL-1β may play important roles in the pathophysiology of VC. The release of matrix vesicle bodies (MVBs) and IL-1β and the colocalization of IL-1β with MVBs or autophagosomes were studied by immunofluorescence in an in vivo diabetes mouse model with aortic calcification and an in vitro high glucose cell calcification model. MVB numbers, IL-1β levels and autophagy were increased in calcified mouse aortas and calcified vascular smooth muscle cells (VSMCs). IL-1β colocalized with MVBs and autophagosomes. The MVBs from calcified VSMCs induced the calcification of normal recipient VSMCs, and this effect was alleviated by silencing IL-1β. The autophagy inducer rapamycin reduced IL-1β expression and calcification in VSMCs, while these processes were induced by the autophagy inhibitor chloroquine. In conclusion, our results suggested that MVBs could carry IL-1β out of cells and induce VC in normal VSMCs, and these processes could be counteracted by autophagy. These results suggested that MVB-mediated IL-1β release may be an effective target for treating vascular calcification.

Association between oocyte donors’ or recipients’ body mass index and clinical outcomes after first single blastocyst transfers—the uterus is the most affected

ObjectiveTo assess if high BMI in either oocyte donors or recipients is associated with poorer outcomes after first single blastocyst transfers. DesignRetrospective study including 1394 first blastocyst single embryo transfers (SETs) conducted by 1394 recipients during oocyte donation cycles with the gametes retrieved from 1394 women (Jan2019-July2021). Four BMI clusters were defined for both donors and recipients (underweight: <18.5; normal weight: 18.5-24.9; over-weight: 25-29.9; obese: ≥30). Subjects1394 recipients, 42.4±4.0 years old and with a BMI 23.2±3.8, and 1394 donors, 26.1±4.2 years old and with a BMI 21.9±2.5. InterventionAll oocytes were vitrified at two egg banks and warmed at eight IVF clinics part of the same network. ICSI, blastocyst culture and either fresh or vitrified-warmed SETs were conducted. Putative confounders were investigated, and the data adjusted through regression analyses. Main outcome measuresThe primary outcome was the live birth rate (LBR) per SET according to donors’ and/or recipients’ BMI. The main secondary outcome was the miscarriage rate (<22nd gestational weeks) per clinical pregnancy. ResultsThe LBR per blastocyst SET showed no significant association with donors’ BMI. Regarding recipients’ BMI, instead, the multivariate-OR was significant in obese versus normal weight recipients (0.58, 95%CI 0.37-0.91, p=0.019). The miscarriage rate per clinical pregnancy was also significantly associated with recipients’ obesity with a multivariate-OR of 2.31 (95%CI 1.18-4.51, p=0.014) versus normal weight patients. A Generalized Additive Model (GAM) method was used to represent the relationship between predicted LBR or miscarriage rates with donors’ or recipients’ BMI; it pictured a scenario where the former outcome moderately but continuously decreases with increasing recipients’ BMI to then sharply decline in the BMI range 25-35. The miscarriage rate, instead, increases almost linearly with respect to both donors’ and recipients’ increasing BMI. ConclusionObesity mostly effects the uterus, especially due to higher miscarriage rates. Yet, poorer outcomes can be appreciated already with a BMI of 25 in both oocyte donors and recipients. Finer markers of nutritional homeostasis are therefore desirable, recipients should be counselled about poorer expected outcomes in case of overweight/obesity, and oocyte banks should avoid assigning oocytes from overweight donors to overweight/obese recipients.

Identification of common sequence motifs shared exclusively among selectively packed exosomal pathogenic microRNAs during rickettsial infections.

We previously reported that microRNA (miR)23a and miR30b are selectively sorted into exosomes derived from rickettsia-infected endothelial cells (R-ECExos). Yet, the mechanism remains unknown. Cases of spotted fever rickettsioses have been increasing, and infections with these bacteria cause life-threatening diseases by targeting brain and lung tissues. Therefore, the goal of the present study is to further dissect the molecular mechanism underlying R-ECExos-induced barrier dysfunction of normal recipient microvascular endothelial cells (MECs), depending on their exosomal RNA cargos. Infected ticks transmit the rickettsiae to human hosts following a bite and injections of the bacteria into the skin. In the present study, we demonstrate that treatment with R-ECExos, which were derived from spotted fever group R parkeri infected human dermal MECs, induced disruptions of the paracellular adherens junctional protein VE-cadherin, and breached the paracellular barrier function in recipient pulmonary MECs (PMECs) in an exosomal RNA-dependent manner. We did not detect different levels of miRs in parent dermal MECs following rickettsial infections. However, we demonstrated that the microvasculopathy-relevant miR23a-27a-24 cluster and miR30b are selectively enriched in R-ECExos. Bioinformatic analysis revealed that common sequence motifs are shared exclusively among the exosomal, selectively-enriched miR23a cluster and miR30b at different levels. Taken together, these data warrant further functional identification and characterization of a monopartition, bipartition, or tripartition among ACA, UCA, and CAG motifs that guide recognition of microvasculopathy-relevant miR23a-27a-24 and miR30b, and subsequently results in their selective enrichments in R-ECExos.

Autoantibodies Drive Heart Damage Caused by Concomitant Radiation and PD-1 Blockade.

Concurrent PD-1 blockade and thoracic radiotherapy is being investigated in clinical trials for locally advanced, non-small cell lung cancer and small cell lung cancer, despite a potential overlapping risk of cardiotoxicity. Our prior studies demonstrate that cardiotoxicity from concurrent cardiac irradiation and anti-PD-1 administration in a mouse model is CD8+ T-cell dependent. The objective of this study was to determine whether humoral immunity contributed to the observed cardiac tissue damage, as measured by creatine kinase MB and cardiac troponin 1 release and decline in cardiac function. In the current study, we demonstrate the presence of cardiac autoantibodies, which were essential for the occurrence of cardiotoxicity from the combined therapy. Mice subjected to cardiac irradiation, while being treated with anti-PD-1, developed high levels of antibodies that reacted with cardiac tissues in vivo and cardiac antigens in vitro. Moreover, mice deficient in B cells were protected against cardiotoxicity, whereas the transfer of autoantibody-containing sera from mice that had received combined treatment reproduced the same pathologic phenotype in mice exposed to cardiac irradiation but was not observed in normal recipients. The cardiotoxic effect of the sera, which associated with CD8+ T-cell accumulation in cardiac tissue, was limited by IgG depletion. In conclusion, concurrent cardiac irradiation and PD-1 blockade leads to production of cardiac autoantibodies, likely due to antigen exposure within the irradiated cardiac tissues, which play a key role in the resulting cardiotoxicity.

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

Associations of Minor Histocompatibility Antigens with Clinical Outcomes Following Allogeneic Hematopoietic Cell Transplantation

Introduction Donor T-cell recognition of tumor-presented minor histocompatibility antigens (mHAs) is thought to be a central mediator of the therapeutic graft versus leukemia (GvL) effect following allogeneic hematopoietic cell transplantation (alloHCT). In contrast, graft versus host disease (GvHD) develops when donor T-cells react to mHAs presented by normal recipient cells. While the role of mHAs in outcomes following alloHCT is recognized, it has not been well-characterized. It remains unclear whether GvL and GvHD are mediated by the cumulative effect of total mHAs in a donor-recipient pair (DRP) or individual immunodominant mHAs. We hypothesized that an increased mHA count and individual mHAs would be associated with lower relapse and disease-related mortality (DRM) and/or increased GvHD mortality. To this end, we predicted genome-wide mHA peptides in a large patient population and analyzed the associations between clinical outcomes and 1) the total number of predicted class I and class II mHAs, and (2) individual and groups of mHAs. Methods The study population consisted of 2249 European American DRPs in the DISCOVeRY-BMT dataset treated for Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) with alloHCT. To predict mHAs in a DRP, we translated nonsynonymous SNP alleles present in alloHCT recipients, but absent in their donor, into all possible peptides of varying lengths that would encompass the altered amino acid. To identify mHAs most likely to be immunogenic, peptides were filtered based on (1) source protein gene expression data in GvL and GvHD target tissues, including AML, liver, skin, and colon, from UCSC's Xena Dataset repository and (2) recipient HLA binding affinity from netMHCpan. To test the association of mHA count and individual mHAs with all-cause mortality (OS) and leukemia-free survival (LFS) we used cox proportional hazard models and competing risk models for DRM, treatment-related mortality (TRM), and GvHD mortality. Individual mHA analyses were stratified by HLA type to recognize that T-cell responses depend on HLA-mHA complexes. Models were adjusted for clinical covariates. Bonferroni correction for multiple testing was based on the number of independent SNPs. Results Patients with a class I mHA count greater than the population median had a 39% (HR=1.39, 95%CI 1.005,1.772, P=0.046) increase in hazard of GvHD mortality compared to those with a count less than the median. Patients with a class II mHA count greater than the population median had a decreased overall survival compared to those with a count less than the median (Fig. 1, P=0.029). In recipients with HLA-B*08:01, the class I mHA DLRCKYISL was associated with increased risk of GVHD mortality (HR=2.84, 95%CI 1.52,5.31, P=0.01). Within HLA-B*40:01, WEHGPTSLL was associated with decreased LFS (HR=1.94, 95%CI 1.27,2.95, P=0.044). Within HLA-C*03:04, STSPTTNVL was associated with increased risk of DRM (HR=2.32, 95%CI 1.5,3.6, P=0.008). In recipients with HLA-DRB1*15:01, the class II mHA YQEIAAIPSAGRERQ was associated with increased risk of TRM (HR=3.05, 95%CI 1.75,5.31, P=0.02). Two mHAs, WEHGPTSLL and STSPTTNVL, were found within the HLA haplotype B*40:01-C*03:04. Recipients with both mHAs (versus neither) had a greater hazard of 1-year all-cause mortality (HR=3.58, P=7.9×10-5) and DRM (HR=3.65, P=0.003) (Fig. 2). Patients with either mHA (versus neither) had greater 1-year all-cause mortality (HR=1.69, P=0.008) and increased DRM (HR=2.05, P=0.008) Conclusions The number of class I mHAs in a DRP was associated with increased GvHD mortality, which is consistent with our hypothesis. We also found two mHAs correlated with GvHD mortality and DRM which may represent increased GvH effects driven by these peptides. However, contrary to our hypothesis, an increased number of class II mHAs was correlated with greater risk of all-cause mortality that may be due to decreased LFS. Similarly, we found two mHAs correlated with increased DRM and decreased LFS. These two mHAs are found in an HLA haplotype where they exhibit a dose effect relationship with the risk of DRM and decreased OS. These findings may reflect an unexpectedly attenuated GvL effect that requires further investigation. Our study reports the first large scale investigation of the role of predicted mHA peptides in clinical outcomes following alloHCT. If validated, these results may be used to help guide clinical decision making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Perfusion and oxygenation in allografts with transplant renal artery stenosis: Evaluation with functional magnetic resonance imaging.

Transplant renal artery stenosis (TRAS) has been shown to reduce kidney perfusion leading to post-operative hypertension. We aimed to measure the perfusion and oxygenation changes in TRAS with arterial spin labeling (ASL) and blood oxygen level-dependent (BOLD) imaging, respectively. In this single-center prospective study, a total of seven patients with TRAS and seven age- and sex-matched normal kidney transplant recipients underwent both ASL and BOLD imaging. Moreover, measurements of ASL and BOLD were also performed in five patients after successful angioplasty for TRAS. Allograft cortical perfusion as measured by ASL in the TRAS group was significantly decreased as compared with normal control group (129.9±46.6ml/100g vs. 202.4±47.7ml/100g, P=.01). Interestingly, allograft oxygenation as indicated by R2* derived from BOLD in both the cortex (16.42±1.90Hz vs. 18.25±4.34Hz, P=.33) and the medulla (30.34±2.35Hz vs. 30.43±6.85Hz, P=.97) showed no statistical difference between the TRAS and normal control group. In addition, both cortical and medullary oxygenation remained unchanged despite significantly improved cortical perfusion in those undergone successful angioplasty. Cortical and medullary oxygenation were preserved in the presence of reduced allograft perfusion in clinically significant TRAS. Prospective larger studies are needed to conclusively establish perfusion and oxygenation changes in TRAS.

Presume Why Probiotics May Not Provide Protection in Inflammatory Bowel Disease through an Azoxymethane and Dextran Sodium Sulfate Murine Model.

Recent studies have shown dysbiosis is associated with inflammatory bowel disease (IBD). However, trying to restore microbial diversity via fecal microbiota transplantation (FMT) or probiotic intervention fails to achieve clinical benefit in IBD patients. We performed a probiotic intervention on a simulated IBD murine model to clarify their relationship. IBD was simulated by the protocol of azoxymethane and dextran sodium sulfate (AOM/DSS) to set up a colitis and colitis-associated neoplasm model on BALB/c mice. A single probiotic intervention using Clostridium butyricum Miyairi (CBM) on AOM/DSS mice to clarify the role of probiotic in colitis, colitis-associated neoplasm, gut microbiota, and immune cytokines was performed. We found dysbiosis occurred in AOM/DSS mice. The CBM intervention on AOM/DSS mice failed to improve colitis and colitis-associated neoplasms but changed microbial composition and unexpectedly increased expression of proinflammatory IL-17A in rectal tissue. We hypothesized that the probiotic intervention caused dysbiosis. To clarify the result, we performed inverse FMT using feces from AOM/DSS mice to normal recipients to validate the pathogenic effect of dysbiosis from AOM/DSS mice and found mice on inverse FMT did develop colitis and colon neoplasms. We presumed the probiotic intervention to some extent caused dysbiosis as inverse FMT. The role of probiotics in IBD requires further elucidation.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

Mitigation of Post-Transplant Mortality Risk by Appropriate Donor-to-Recipient Size Matching Using Predicted Heart Mass

<h3>Purpose</h3> Obesity is a known risk factor for post-transplant mortality. Obesity epidemic in U.S. has led to a higher prevalence of obese patients as potential heart transplant recipients. It is unclear if appropriate size matching of donor-to-recipient using predicted heart mass (PHM) can mitigate this risk among obese recipients. <h3>Methods</h3> We selected all recipients with heart transplants performed between 1995 and 2020 (n=34,812) using the United Network for Organ Sharing database to examine the risk of death in 6 separate categories of recipient's BMI (Table) according to PHM matching. PHM-based appropriate match happened when Donor PHM/Recipient PHM x 100% was within 86% to 114% whereas under- and over-match were defined as <86% and >114%, respectively. Multivariable Cox regression models were created examining long-term risk of death in appropriately matched and over-matched categories compared to under-matched category in the overall cohort and separately within each BMI group. <h3>Results</h3> Upon follow-up (mean 5.29 years), 9,810 patients died. Recipients appropriately matched to donor hearts had an 8% lower risk of death compared to recipients matched with underweight donors by PHM (Table). Compared to those who had under-matched hearts, normal or overweight recipients who received appropriately matched donor hearts based on PHM had a 10-13% lower risk of death post-transplant (Table). In addition, morbidly obese recipients who were appropriately matched with donor hearts also had a 17% lower risk of death post-transplant (borderline statistically significant p=0.06; Table). The risk of death among obese or severely obese recipients did not differ with appropriate PHM matching or over matching compared to under-matching. <h3>Conclusion</h3> Although pretransplant obesity continues to be a risk factor for death post-transplant, appropriate size matching for donor hearts by PHM may lower the risk of death compared to under-matching specifically among normal and overweight recipients.

Reducing Morbidity in High Panel Reactive Antibody Heart Transplant Recipients Through Human Leukocyte Antigen Matching

<h3>Purpose</h3> A high PRA prior to heart transplantation places recipients at risk for rejection and mortality. The purpose of this study was to assess the impact donor-recipient HLA matching has on post-transplant survival and morbidity. <h3>Methods</h3> A retrospective review of heart transplant recipients in the UNOS database was performed from 2000-2021. Patients were stratified by normal (<25%) and high (≥25%) PRA reactivity. Recipients HLA matched to their donors at ≥ 3 alleles in the high PRA subgroup were identified. Primary outcome was Kaplan-Meier survival estimates at 1, 3 and 5 years post-transplant. Multivariable regression was also performed on high PRA-HLA matched recipients to determine if matching reduced the odds of rejection or infection. <h3>Results</h3> A high PRA was identified in 4,214 heart transplant recipients of which 678 (16%) were HLA matched (Table 1a). Survival estimates were significantly different between normal and high PRA recipients at all primary time points. High PRA-HLA matched patients did not have a significantly different survival than high PRA-HLA mismatched recipients at any time point (Figure 1). On multivariable analysis (Table 1b), HLA matching significantly reduced the odds of graft rejection (OR 0.62) and infection (OR 0.73) <h3>Conclusion</h3> A high PRA places recipients at an increased risk for post-transplant mortality. HLA matching does not improve survival but does significantly reduce the odds of rejection and infection. HLA matching donors to high PRA recipients should be considered to reduce future morbidity, lost time hospitalized and financial burden for these patients.

Optimizing nerve transfer surgery in tetraplegia: clinical decision making based on innervation patterns in spinal cord injury.

Nerve transfers are increasingly being utilized in the treatment of chronic tetraplegia, with increasing literature describing significant improvements in sensorimotor function up to years after injury. However, despite technical advances, clinical outcomes remain heterogenous. Preoperative electrodiagnostic testing is the most direct measure of nerve health and may provide prognostic information that can optimize preoperative patient selection. The objective of this study in patients with spinal cord injury (SCI) was to determine various zones of injury (ZOIs) via electrodiagnostic assessment (EDX) to predict motor outcomes after nerve transfers in tetraplegia. This retrospective review of prospectively collected data included all patients with tetraplegia from cervical SCI who underwent nerve transfer at the authors' institution between 2013 and 2020. Preoperative demographic data, results of EDX, operative details, and postoperative motor outcomes were extracted. EDX was standardized into grades that describe donor and recipient nerves. Five zones of SCI were defined. Motor outcomes were then compared based on various zones of innervation. Nineteen tetraplegic patients were identified who underwent 52 nerve transfers targeting hand function, and 75% of these nerve transfers were performed more than 1 year postinjury, with a median interval to surgery following SCI of 24 (range 8-142) months. Normal recipient compound muscle action potential and isolated upper motor neuron injury on electromyography (EMG) were associated with greater motor recovery. When nerve transfers were stratified based on donor EMG, greater motor gains were associated with normal than with abnormal donor EMG motor unit recruitment patterns. When nerve transfers were separated based on donor and recipient nerves, normal flexor donors were more crucial than normal extensor donors in powering their respective flexor recipients. This study elucidates the relationship of the preoperative innervation zones in SCI patients to final motor outcomes. EDX studies can be used to tailor surgical therapies for nerve transfers in patients with tetraplegia. The authors propose an algorithm for optimizing nerve transfer strategies in tetraplegia, whereby understanding the ZOI and grade of the donor/recipient nerve is critical to predicting motor outcomes.

Exosomes Derived from Hydroquinone-transformed Human Bronchial Epithelial Cells Inhibited Recipient Cell Apoptosis by transferring miR-221

ObjectiveAlthough benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown. MethodsIn the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry. ResultsThe amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells. ConclusionExosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.

Airway Hyper-Responsiveness Associated With Sickle Cell Disease Is Mediated By Placenta Growth Factor

Airway hyper-responsiveness (AHR) affects about 55-77% of children with sickle cell disease (SCD) and is a risk factor for asthma, increasing the risk of vaso-occlusive events and acute chest syndrome in this patient population. Although in the non-SCD pediatric population AHR is associated with an active allergic response, the molecular mechanisms underlying the high incidence of AHR in a red blood cell disorder remain unclear. Our laboratory has reported that placenta growth factor (PlGF), an erythroid cell secreted-growth factor belonging to the vascular endothelial growth factor family is found at high levels in patients with SCD, mediates increased 5-lipoxygenase (5LO) expression in vitro (Patel et al, 2009). 5LO is the key enzyme in the leukotriene synthetic pathway (LT) and LT levels have been reported to be elevated in SCD (Setty et al, 2002; Jennings et al, 2008). We hypothesized that PlGF contributes to AHR in SCD and investigated the direct contribution of PlGF to AHR in mice with genetically imposed deficiency of PlGF and in the well characterized Berkeley sickle mouse model (HbS mice). We have previously reported that imposing PlGF deficit in HbS mice resulted in extremely small litters and the rare HbS/PlGF-/- mice born were infertile (Patel et al, 2010). Hence the PlGF deficient and HbS mice were studied separately. We utilized a well-established murine model of allergen induced AHR using the highly AHR susceptible mouse strain (BALB/c), that either had normal levels of PlGF (PlGFWT) or had a genetically imposed deficit of PlGF (PlGF-/- mice). Results were validated in HbS mice by using pharmacological blockade of this mechanistic pathway. Following allergen treatment with either IL-13 or house dust mites, mice were subjected to a cholinergic challenge and AHR was assessed using either APTI or airway resistance, using an established AHR protocol. Upon allergen challenge, PlGF-/- BALB/c mice showed significantly blunted AHR compared to PlGFWT BALB/c mice: the airway resistance was 683 ± 93.5 vs 1283 ± 229 cm2xs; p≤0.04. PlGF neutralization with a blocking antibody (kindly provided by Thrombogenics, Inc.) reduced the severity of AHR in PlGFWT mice to levels similar to PlGF-/- mice. Consistent with the importance of eosinophils in driving AHR development, bronchoalveolar lavage fluid (BALF) from PlGF-/- mice revealed reduced eosinophils (9.3 ± 6.4 x104 vs. 27.5 ± 4.8 x 104 cell/ml P≤0.05), and decreased leukocyte infiltration of lungs by histology. Mechanistically, PlGF deficiency was associated with decreased 5LO expression in lungs both at the mRNA and protein level. We also found that in addition to 5LO, genetically imposed deficiency of PlGF or its pharmacological inhibition were associated with a 2.5-5 fold decrease in IL17A expression in CD4+ lymphocytes from BALF. AHR susceptibility across mouse strains varies tremendously, and has been reported to be mediated via IL-13 and IL-17A (Lajoie and Wills Karp, Nature Immunology, 2010). The HbS mice were originally outbred and have been crossed to C57Bl/6 mice for six generations. To study AHR in HbS mice while eliminating confounding variables attributable to genetic background difference, we transplanted sickle hematopoietic stem cells from HbS donors into Bl/6 (BoyJ) mice, and derived HbS/Bl/6 chimeric mice that had donor derived sickle hematopoiesis replaced the normal recipient hematopoiesis. C57Bl/6 mice transplanted into congenic BoyJ (Bl/6) mice served as controls. Four months after transplant when the mice were totally chimeric, allergen challenge revealed significant increased AHR in HbS/Bl/6 mice compared to C57/Bl/6 mice. Interestingly, IL17A levels were similar between HbS/Bl/6 and C57/Bl/6mice; whereas they were significantly lower in HbS mice compared to control C57/Bl6 mice prior transplant. Consistent with the role of PlGF in AHR in SCD, PlGF neutralizing antibody reduced the AHR severity in chimeric HbS/Bl/6 mice; and pharmacological blockade of 5-LO using zileuton, a 5-LO inhibitor significantly decreased the severity of AHR in HbS/Bl/6 mice in comparison to vehicle treated HbS/Bl/6 mice. Overall, our results indicate that PlGF expression exacerbates AHR via regulating the leukotriene and IL-17A pathways. Leukotriene antagonists are licensed for asthma and PlGF antibody is in clinical trials for cancer. These can be used to reduce the pulmonary morbidity in susceptible patients with moderate to severe SCD. Disclosures:No relevant conflicts of interest to declare.

Change of Safety Interval from Hyperviscosity Problem in COVID-19 Vaccine Recipient with Underlying Diabetic Problem.

Sir, The new coronavirus disease 2019 (COVID-19) vaccine is widely used in the present day. Since COVID-19 is novel, it is necessary to have a continuous study on the effectiveness and safety of the vaccine. After the COVID-19 vaccination, an important clinical complication is a clot and thrombosis.[1] Rheologically, there is a change of blood viscosity after the COVID-19 vaccination. According to a recent report, the blood viscosity in a normal COVID-19 vaccine recipient increases by +2.4 cp from the normal level.[2] If there is a very high increase, it might reach 5 cp,[3] which might result in vascular thrombotic complications. For any COVID-19 recipient, there will be a safety interval for which there is no hyperviscosity problem. This value is equal to the difference between hyperviscosity level minus the elevated viscosity level after vaccination. This problem might occur in a person with an underlying disease. A diabetic patient is presently examined about the risk. In this study, the authors estimated the safety interval from high viscosity problem for a diabetic patient after receiving the COVID19 vaccine. The results of a previous publication on background blood viscosity among diabetic patients are used as primary data for modeling calculation.[4] The estimated safety intervals for diabetic patients and healthy persons are shown in Table 1.Table 1: Estimated safety margin for no hyperviscosity problem after COVID-19 vaccinationAccording to this study, there is a difference in safety interval among different groups. Diabetic patients with retinopathy form the group with the narrowest safety interval. Nevertheless, there is still a safety interval for any diabetic patient to receive the COVID-19 vaccine. Based on the present findings, there should be close monitoring of a diabetic patient with poor diabetic control after COVID-19 vaccination. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.