Bruton's tyrosine kinase ablation inhibits B cell responses and antibody production for the prevention of chronic rejection in cardiac transplantation.

Chronic rejection is the primary cause of late allograft failure, however, the current treatments for chronic rejection have not yielded desirable therapeutic effects. B cell activation and donor-specific antibody (DSA) production are the primary factors leading to chronic rejection. Bruton's tyrosine kinase (BTK) plays a key role in the activation and differentiation of B cells and in antibody production. This study investigated the efficacy of blocking BTK signalling in the prevention of chronic rejection. BTK signalling was blocked using the BTK inhibitor ibrutinib and gene knockout. In vitro assays were conducted to examine the consequences and underlying mechanisms of BTK blockade in regards to B cell activation, differentiation, and antibody secretion. Additionally, we established a cardiac transplantation mouse model of chronic rejection to explore the preventive effects and mechanisms of BTK ablation on chronic rejection. Ablating BTK signalling in vitro resulted in the inhibition of B cell activation, differentiation, and antibody production. In vivo experiments provided evidence that ablating BTK signalling alleviated chronic rejection, leading to reduced damage in myocardial tissue, neointimal hyperplasia, interstitial fibrosis, inflammatory cell infiltration, and C4d deposition. Allograft survival was prolonged, and B cell responses and DSA production were inhibited as a result. We confirmed that ablation of BTK signalling inhibited B cell response by blocking downstream PLCγ2 phosphorylation and inhibiting the NF-κB, NFAT, and ERK pathways. Our findings demonstrated that ablation of BTK signalling inhibited B cell activation and differentiation, reduced DSA production, and effectively prevented chronic rejection.

A Novel In-Home Sleep Monitoring System Based on Fully Integrated Multichannel Front-End Chip and Its Multilevel Analyses.

OBJECTIVE: A novel in-home sleep monitoring system with an 8-channel biopotential acquisition front-end chip is presented and validated via multilevel data analyses and comparision with advanced polysomnography. METHODS AND PROCEDURES: The chip includes a cascaded low-noise programmable gain amplifier (PGA) and 24-bit [Formula: see text]-[Formula: see text] analog-to-digital converter (ADC). The PGA is based on three op-amp structure while the ADC adopts cascade of integrator feedforward and feedback (CIFF-B) architecture. An innovative chopper-modulated input-scaling-down technique enhances the dynamic range. The proposed system and commercial polysomnography were used for in-home sleep monitoring of 20 healthy participants. The consistency and significance of the two groups' data were analyzed. RESULTS: Fabricated in 180 nm BCD technology, the input-referred noise, input impedance, common-mode rejection ratio, and dynamic range of the acquisition front-end chip were [Formula: see text]Vpp, 1.25 GN), 113.9 dB, and 119.8 dB. The kappa coefficients between the sleep stage labels of the three scorers were 0.80, 0.76, and 0.79. The consistency of the slowing index, multiscale entropy, and percentile features between the two devices reached 0.958, 0.885, and 0.834. The macro sleep architecture characteristics of the two devices were not significantly different (all p [Formula: see text] 0.05). CONCLUSION: The proposed chip was applied to develop an in-home sleep monitoring system with significantly reduced size, power, and cost. Multilevel analyses demonstrated that this system collects stable and accurate in-home sleep data. CLINICAL IMPACT: The proposed system can be applied for long-term in-home sleep monitoring outside of laboratory environments and sleep disorders screening that with low cost.

Ex vivo anchored PD-L1 functionally prevent in vivo renal allograft rejection.

Organ transplantation is the optimal treatment for patients with end-stage diseases. T cell activation is a major contributing factor toward the trigger of rejection. Induction therapy with T cell depleting agent is a common option but increases the risk of severe systemic infections. The ideal therapy should precisely target the allograft. Here, we developed a membrane-anchored-protein PD-L1 (map-PD-L1), which effectively anchored onto the surface of rat glomerular endothelial cells (rgEC). The expression of PD-L1 increased directly with map-PD-L1 concentration and incubation time. Moreover, map-PD-L1 was even stably anchored to rgEC at low temperature. Map-PD-L1 could bind to PD-1 and significantly promote T cell apoptosis and inhibited T cell activation. Using kidney transplantation models, we found that ex vivo perfusion of donor kidneys with map-PD-L1 significantly protected grafts against acute injury without using any immunosuppressant. We found map-PD-L1 could reduce T cell graft infiltration and increase intragraft Treg infiltration, suggesting a long-term effect in allograft protection. More importantly, modifying donor organs in vitro was not only safe, but also significantly reduced the side effects of systemic application. Our results suggested that ex vivo perfusion of donor organ with map-PD-L1 might provide a viable clinical option for organ-targeted induction therapy in organ transplantation.

Corrigendum: Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

[This corrects the article DOI: 10.3389/fimmu.2021.778359.].

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Plasma Macrophage Migration Inhibitory Factor Predicts Graft Function Following Kidney Transplantation: A Prospective Cohort Study.

Background: Delayed graft function (DGF) is a common complication after kidney transplantation (KT) with a poor clinical outcome. There are no accurate biomarkers for the early prediction of DGF. Macrophage migration inhibitory factor (MIF) release during surgery plays a key role in protecting the kidney, and may be a potential biomarker for predicting post-transplant renal allograft recovery. Methods: Recipients who underwent KT between July 2020 and December 2020 were enrolled in the study. Plasma MIF levels were tested in recipients at different time points, and the correlation between plasma MIF and DGF in recipients was evaluated. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2000035596). Results: Intraoperative MIF levels were different between immediate, slowed, and delayed graft function groups (7.26 vs. 6.49 and 5.59, P < 0.001). Plasma MIF was an independent protective factor of DGF (odds ratio = 0.447, 95% confidence interval [CI] 0.264-0.754, P = 0.003). Combining plasma MIF level and donor terminal serum creatinine provided the best predictive power for DGF (0.872; 95%CI 0.795-0.949). Furthermore, plasma MIF was significantly associated with allograft function at 1-month post-transplant (R 2 = 0.42, P < 0.001). Conclusion: Intraoperative MIF, as an independent protective factor for DGF, has excellent diagnostic performance for predicting DGF and is worthy of further exploration.

Artemisinin Attenuates Transplant Rejection by Inhibiting Multiple Lymphocytes and Prolongs Cardiac Allograft Survival.

Immunological rejection is an important factor resulting in allograft dysfunction, and more valid therapeutic methods need to be explored to improve allograft outcomes. Many researches have indicated that artemisinin and its derivative exhibits immunosuppressive functions, apart from serving as a traditional anti-malarial drug. In this assay, we further explored the therapeutic effects of artemisinin for transplant rejection in a rat cardiac transplantation model. We found that it markedly attenuated allograft rejection and histological injury and significantly prolonged the survival of allograft. Upon further exploring the mechanism, we demonstrated that artemisinin not only attenuated T cell-mediated rejection (TCMR) by reducing effector T cell infiltration and inflammatory cytokine secretion and increasing regulatory T cell infiltration and immunoregulatory cytokine levels, but also attenuated antibody-mediated rejection (ABMR) through inhibition of B cells activation and antibody production. Furthermore, artemisinin also reduced macrophage infiltration in allografts, which was determined to be important for TCMR and ABMR. Moreover, we demonstrated that artemisinin significantly inhibited the function of pure T cells, B cells, and macrophages in vitro. All in all, this study provide evidence that artemisinin significantly attenuates TCMR and ABMR by targeting multiple effectors. Therefore, this agent might have potential for use in clinical settings to protect against transplant rejection.

Donor plasma mitochondrial DNA is associated with antibody-mediated rejection in renal allograft recipients.

We previously showed that donor plasma mitochondrial DNA (dmtDNA) levels were correlated with renal allograft function. The aim of the current study was to determine whether dmtDNA levels are associated with the occurrence of antibody-mediated rejection (ABMR). This is a retrospective open cohort study comprised of 167 donors and 323 recipients enrolled from January 2015 to December 2017. We quantified the mtDNA level present in donor plasma using quantitative real-time polymerase chain reaction. The average plasma dmtDNA level in the acute rejection (AR) group was higher than that of the control group (0.156 versus 0.075, p<0.001). Multivariate logistic regression analysis showed that dmtDNA levels were also significantly associated with AR (OR=1.588, 95% CI 1.337-4.561, p<0.001). When the dmtDNA level was >0.156, the probability of AR was 62.9%. The plasma dmtDNA level in the ABMR group was significantly higher than that of the T cell-mediated rejection group (0.185 versus 0.099, p=0.032). The area under the receiver operating characteristic curve of dmtDNA for prediction of ABMR was as high as 0.910 (95% CI 0.843-0.977). We demonstrated that plasma dmtDNA was an independent risk factor for ABMR, which is valuable in organ evaluation. dmtDNA level is a possible first predictive marker for ABMR.

Characterization of small GTPase Rac1 and its interaction with PAK1 in crayfish Procambarus clarkii.

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii.