Small extracellular vesicles (sEVs) isolated from plasma of lung transplant recipients (LTRs) with chronic lung allograft dysfunction (CLAD) contain increased levels of lung associated self-antigens, Kα1 tubulin and collagen V, and decreased expression of the tumor suppressor liver kinase B1 (LKB1). In this study, sEVs were isolated from plasma collected from LTRs with or without cystic fibrosis (CF) from multiple centers at the onset of CLAD and 6 and 12 months before clinical diagnosis of CLAD (n = 32) as well as from time-matched stable controls (n = 25). sEVs were analyzed for Kα1 tubulin, collagen V, and LKB1 by western blot. Exoview R200, a functionalized microarray chip was employed to characterize the LKB1 in sEVs. EVs from non-CF LTRs had higher levels of lung self-antigens (p < 0.05) and lower levels of LKB1 (p = 0.024) 12 months before CLAD diagnosis than those from time-matched stable LTRs; however, in CF LTRs, only LKB1 levels were lower (p = 0.0005) 6 months before diagnosis. Further characterization of sEVs 6 months before CLAD in CF LTRs also demonstrated significantly lower numbers of LKB1 and LKB1/CD9 + sEV particles. Reduced LKB1 in circulating sEVs offers a potential biomarker for the risk of CLAD in LTRs with CF.

Extracellular vesicles (EVs) are nanosized vesicles that are secreted by all cells into the extracellular space. EVs are involved in cell-to-cell communication and can be found in different bodily fluids (bronchoalveolar lavage fluid, sputum, and urine), tissues, and in circulation; the composition of EVs reflects the physiological condition of the releasing cell. The ability to use EVs from bodily fluids for minimally invasive detection to monitor diseases makes them an attractive target. EVs carry a snapshot of the releasing cell’s internal state, and they can serve as powerful biomarkers for diagnosing diseases. EVs also play a role in the body’s immune and pathogen detection responses. Pathogens, such as bacteria and viruses, can exploit EVs to enhance their survival and spread and to evade detection by the immune system. Changes in the number or contents of EVs can signal the presence of an infection, offering a potential avenue for developing new diagnostic methods for infectious diseases. Ongoing research in this area aims to address current challenges and the potential of EVs as biomarkers in diagnosing a range of diseases, including infections and infectious diseases. There is limited literature on the development of EVs as diagnostic biomarkers for infectious diseases using existing molecular biology approaches. We aim to address this gap by reviewing recent EV-related investigations in infectious disease studies.

INTRODUCTION:A better understanding of the immune mechanisms involved in allograft rejection after transplantation is urgently needed to improve patient outcomes. As microRNA-155 (miR155) plays a critical role in inflammation, we postulated that a deficiency of miR155 will improve cardiac allograft survival and enhance tolerance induction after heart transplantation. METHODS:We developed an acute rejection mouse model through heterotopic BALB/c cardiac transplantation to C57BL/6 (wild-type) and C57BL/6 miR155 knock-out (miR155KO) mice. Further, we induced tolerance in both groups through a costimulatory blockade with CTLA4-Ig (200 μg; post-transplant day 2) and MRI antibodies (250 μg; post-transplant day 0), targeting CD28/B7 and CD40/CD154 signals, respectively. Finally, we examined the effects of injecting 100 μg of small extracellular vesicles (sEVs) isolated from wild-type mice undergoing rejection into tolerant miR155KO mice. RESULTS:Mean survival time (MST) of the cardiac allografts in wild-type and miR155KO mice was 7 and 15 days, respectively (p < 0.0001). Costimulatory blockade increased MST to 65 days and > 100 days in the wild-type and miR155KO recipients, respectively (p < 0.001). Injection of sEVs isolated from wild-type mice undergoing rejection into tolerant miR155KO mice decreased the allograft survival to 9 days, significantly lower than the tolerant miR155KO mice without injection of sEVs (>100 days; p < 0.0001). CONCLUSION:miR155KO mice have improved cardiac allograft survival and enhanced induction of tolerance after heterotopic cardiac transplantation. Injection of sEVs from wild-type mice undergoing rejection into the miR155KO mice reversed these benefits.

In the realm of biomedical advancement, extracellular vesicles (EVs) are revolutionizing our capacity to diagnose, monitor, and predict disease progression. However, the comprehensive exploration and clinical application of EVs face significant limitations due to the current isolation techniques. The size exclusion chromatography, commercial precipitation reagents, and ultracentrifugation are frequently employed, necessitating skilled operators and entailing challenges related to consistency, reproducibility, quality, and yields. Notably, the formidable challenge of extracellular vesicle isolation persists when dealing with clinical samples of limited availability. This study addresses these challenges by aiming to devise a rapid, user-friendly, and high-recovery EVs isolation technique tailored for blood samples. The NTI-EXO precipitation method demonstrated a 5-fold increase in the recovery of serum EVs compared to current methodologies. Importantly, we illustrate that a mere two drops of blood (∼100 µL) suffice for the recovery of enriched EVs. The integrity and quality of these isolated EVs were rigorously assessed for the size, purity, and contaminants. This method was validated through the successful isolation of EVs from organ transplant recipients to detect disease-specific exosomal markers, including LKB1, SARS-CoV-2 spike protein, and PD-L1. In conclusion, NTI-EXO method can be used for small clinical samples, thereby advancing discoveries in the EV-centric domain and propelling the frontiers of biomedical research and clinical applications.

Purpose: Bile Acid (BA) aspiration is an established marker of aspiration and known risk factor for chronic lung allograft dysfunction (CLAD) and reduced survival of lung transplant recipients.The BA action on human alveolar cell components is poorly explored although its understanding could contribute to define mechanisms for CLAD.BA acting as inflammatory molecules on lung cells may trigger a pro-fibrotic phenotype and also affect surfactant protein homeostasis Methods: H441 human lung epithelial cells were used as in vitro model.According to our previous clinical study, we tested the most relevant unconjugated (CA, CDCA) and conjugated (GCA, TCDCA) bile acids at several concentrations, depending on the assay performed (from 6mM to 1000mM).Cell viability and cell toxicity have been performed with WST1 and LDH assay respectively.TGFb1, ACTA2, CTGF, SPA1-2 gene expression levels were measured with real-time qRT-PCR using TaqMan Gene Expression Assays.mRNA analysis is relative to GAPDH control gene and normalized on non-treated (NT) level (Figure ) Results: We showed that BAs treatment affects cell viability and cell toxicity in human lung epithelial cells in a dose-dependent manner.The unconjugated and conjugated BA treatment increased fibrosis marker gene expression, as TGFB1, CTGF, ACTA2 after 24h compared to control cells in a dose dependent fashion.Moreover, with the increase of BAs doses we observed a lower SP-A genes level expression in H441cells Conclusion: Bile acids detected in bronchial washings and lavage have been described as markers of adverse lung allograft outcomes.Here we show that BAs increase fibrosis in epithelial lung cells and decrease the protective SP-A role consequently altering the surfactant homeostasis.These, in vitro findings may provide insights into the mechanism of CLAD injury occurring in lung transplant recipients

Supplementary Figure 1: Expression of MAM-A and HLA-A2 in breast cancer cell lines. Supplementary Figure 2: MAM-A DNA vaccination increases breast cancer-induced TNF-α production by MAM-A-specific CD8 T cells. Supplementary Figure 3: MAM-A DNA vaccination increases NKG2D expression in MAM-A-specific CD8 T cells. Supplementary Figure 4: MAM-A DNA vaccination increases DAP10 adapter protein expression in MAM-A-specific CD8 T cells. Supplementary Figure 5: MAM-A DNA vaccination increases perforin expression in MAM-A-specific CD8 T cells. Supplementary Figure 6: Therapy received by the patients (screen failed and vaccinated).

There is a lower incidence of antibody-mediated rejection (AMR) after simultaneous liver-kidney transplantation (SLKT) than after kidney-only transplantation. It has been suggested that soluble human leukocyte antigen (sHLA) produced by the liver protects the kidney from AMR. However, this hypothesis has not been tested after SLKT. We present a case of SLKT with 2 donor-specific antibodies (DSAs) (DR53, 12,364 mean fluorescence intensity [MFI]; DQ7, 1253 MFI) that displayed a decrease by day 7 (DR53, 2747 MFI; DQ7, 107 MFI). On day 351, the patient was diagnosed with kidney AMR associated with high levels of DSA (DR53, 18,542 MFI; DQ7, 22,007 MFI) that persisted until day 531. High levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were also detected on day 398. Consequently, the patient underwent treatment with plasmapheresis, intravenous immunoglobulin, prednisone, and rituximab. On day 752, biopsy results were negative for AMR. Moderate levels of DSA (DR53, 9798 MFI; DQ7, 1271 MFI), and baseline levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were observed. Increases in CD4+CD25+FOXP3+ regulatory T cell marker-containing exosomes (CD73, programmed death-ligand 1) were observed on day 752 compared to day 398. These data show a direct correlation between sHLA and HLA-containing exosomes and an inverse correlation between tolerance marker-containing exosomes and kidney AMR after SLKT.

Purpose Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in lung transplant recipients (LTxR) under immunosuppression carries higher risk with 14-39% mortality. Immune responses of LTxR under immunosuppression following SARS-CoV-2 infection or vaccination remains unknown. Our goal is to determine the humoral and cellular immunity to SARS-CoV-2 in LTxR with infection and following vaccination. Methods We performed a single center analysis to determine immune responses of LTxR with infection and following BNT162b2 mRNA vaccination. The results were compared with controls (non-transplant individuals). ELISA was developed to determine the antibody (Ab) concentration (IgG) to SARS-CoV-2 spike (CSP) and nucleocapsid (CNP) antigens. PBMCs from LTxR were isolated by ficoll-hypaque centrifugation to determining the frequency of cells secreting IFNγ and TNFα to CSP and CNP by ELISpot. Results Concentration of Abs developed and T-cell frequencies secreting TNFα and IFNγ against CSP and CNP in LTxR and controls are given in Table 1. Infected LTxR and controls developed Abs to both CSP and CNP. In contrast, vaccinated LTxR induced 10 fold less Abs to CSP in comparison to control. Frequencies of cells secreting TNFα for both CSP and CNP were significantly reduced in LTxR with infection. However, vaccination of both LTxR and control induced similar levels of TNFα secreting cells upon stimulation with both CSP and CNP. It is of interest that frequency of IFNγ producing cells against both CSP and CNP were significantly higher in LTxR in comparison to control. Conclusion Infection with SARS-CoV-2 in LTxR and controls produced comparable levels of Abs both against CSP and CNP. However, vaccinated LTxR didn`t induce significant levels of Abs against CSP. Frequency of T-cells, secreting IFNγ were significantly increased by vaccination in LTxR and in controls suggesting that T cell responses against SARS-CoV-2 has been induced in LTxR by mRNA vaccine.