Application progress on contrast-enhanced ultrasound in acute rejection after kidney transplantation / 器官移植

Early diagnosis of acute rejection is of significance for the protection of renal allograft function. Pathological puncture biopsy is the gold standard for the diagnosis of acute rejection of renal allografts. Nevertheless, it may provoke multiple complications, such as bleeding, infection and renal parenchymal injury, which limit its widespread application. In recent years, the sensitivity of contrast-enhanced ultrasound in the diagnosis of acute rejection has been constantly improved. Ultrasound-targeted microbubble technique has further enhanced the diagnostic specificity of contrast-enhanced ultrasound, making it possible to replace pathological puncture biopsy. Besides, in the field of acute rejection treatment, microbubble ultrasonic cavitation may promote local delivery of immunosuppressants by inducing sonoporation and exhibit anti-rejection effect. In this article, the application of contrast-enhanced ultrasound in the diagnosis and treatment of acute rejection after kidney transplantation was reviewed, aiming to provide reference for widespread application of contrast-enhanced ultrasound in kidney transplantation.

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1highCD11b+ cells elimination in hepatocellular carcinoma

Cryoablation (CRA) and microwave ablation (MWA) are two main local treatments for hepatocellular carcinoma (HCC). However, which one is more curative and suitable for combining with immunotherapy is still controversial. Herein, CRA induced higher tumoral PD-L1 expression and more T cells infiltration, but less PD-L1highCD11b+ myeloid cells infiltration than MWA in HCC. Furthermore, CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models. Mechanistically, anti-PD-L1 antibody facilitated infiltration of CD8+ T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy. On the other hand, anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1highCD11b+ myeloid cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect after CRA therapy. Both aspects relieved the immunosuppressive microenvironment after CRA therapy. Notably, the wild-type PD-L1 Avelumab (Bavencio), compared to the mutant PD-L1 atezolizumab (Tecentriq), was better at inducing the ADCC effect to target PD-L1highCD11b+ myeloid cells. Collectively, our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.

Macrophages and kidney transplantation / 器官移植

Kidney transplantation is the optimal treatment for patients with end-stage renal disease, whereas long-term survival of renal allografts remains a challenging issue. Renal ischemia-reperfusion injury (IRI) and rejection of renal allografts are considered as important influencing factors of long-term survival of renal allografts, which are regulated by innate and adaptive immune cells. Macrophages are one type of innate immune cells that could assist initiating adaptive immunity and are divided into M1, M2 and regulatory macrophages. Previous studies have revealed that M1 macrophages may aggravate renal IRI and acute T cell-mediated rejection (TCMR). However, M2 macrophages may mitigate renal IRI and acute TCMR, whereas it is positively correlated with antibody-mediated rejection (AMR). Regulatory macrophages are a special subgroup of macrophages, which may induce immune tolerance in organ transplantation and have promising clinical application prospects and basic scientific research value. In this article, the relationship among macrophage typing, macrophages and renal IRI, rejection of renal allografts, regulatory macrophages and immune tolerance was reviewed, and the potential mechanism was analyzed, aiming to induce changes in macrophage subtypes or eliminate specific subtypes of macrophages, thereby improving clinical prognosis of the recipients and long-term survival of renal allografts.

SARS-CoV-2-specific antibody-dependent cell-mediated cytotoxicity positively correlated with antibody titer and neutralizing activity / 中华微生物学和免疫学杂志

Objective:To investigate the antibody-dependent cell-mediated cytotoxicity (ADCC) in plasma samples from patients with SARS-CoV-2 infection and to evaluate its correlation with antibody titer and neutralizing activity.Methods:A simple method for ADCC detection was established using HEK293T cells expressing SARS-CoV-2 spike (S) protein as target cells and FcγRⅢa-V158-expressing Jurkat cells as effector cells. It was used to analyze the ADCC activity in 38 plasma samples after the ratio of effector cells to target cells was optimized. Plasma-specific antibody was detected by capturing ELISA, which was to capture the C-terminal-tagged recombinant SARS-CoV-2 S protein with an anti-tag antibody. The neutralizing activity in plasma samples was detected using a pseudovirus neutralization assay. Mann-Whitney U test was used for comparison between different groups and non-parametric Spearman correlation test was performed for correlation analysis. Results:The seroconversion rates for antibodies specific for S protein, S1 protein and RBD were all 97.4% (37/38), and the dynamic changes in antibody titers with recovery time showed that antibody titers peaked at 3-4 weeks. Among the plasma samples with neutralizing activity, those with antibody titers >1∶320 had stronger neutralizing activity than the plasma samples with antibody titers <1∶320 [IC 50: 749.6 (396.5-3 772.0) vs 81.4 (11.6-228.4), P<0.01]. ADCC activity was detectable in 86.8% (33/38) of the plasma samples, and its dynamic change with recovery time were consistent with that of specific antibody titer with a peak at 3-4 weeks. Correlation analysis showed ADCC was positively correlated with the titers of antibodies specific for S protein, S1 protein and RBD ( r=0.686, 0.535 and 0.471, all P<0.01). A positive correlation was also found between ADCC and neutralizing activity ( r=0.573, P<0.01). Conclusions:This study established a simple method for the detection of ADCC. Results of this study suggested that SARS-CoV-2 could induce specific ADCC in plasma and the ADCC might be associated with non-neutralizing antibodies. Besides, the activity of ADCC peaked at 3-4 weeks. These findings would be of reference value for clinical treatment with convalescent plasma.

Development of the method for determinating ADCC biological activity of IVIG / 中国输血杂志

【Objective】 To establish a method for determinating the antigen-dependent cell-mediated cytotoxicity (ADCC) of human immunoglobulin (pH4)for intravenous injection (IVIG) on luciferase reporter gene-modified cell assay. 【Methods】 As effector cells, Jurkat-NFAT-Luc-CD16 cells were used in the assay, and PLC/PRF/5 cells were used as target cells. After incubation of effector cells and target cells with IVIG, the method for determinating ADCC biological activity of IVIG was established by detecting luciferase released by activated T nuclear factor after binding of IVIG Fc fragment to effector cells. Meanwhile, the experimental assay conditions were optimized, and the methodology was verified subsequently. 【Results】 IVIG had a dose-response relationship in this method, which was consistent with four parameter logistic model. And the PLC/PRF/5 cells were finally determined as the target cells. The initial dilution concentration of antibody was 20 mg/mL, and the ratio dilution was 1∶2, and the effector to target ratio was 1∶3, and co-incubation time of two cells and IVIG was 24 hours. Within-run and between-run analysis including three independent tests, initial working concentration relative light unit (RLU) and the relative standard deviation (RSD) of the concentration for 50% of maximal effect(EC50) were less than 11%. The relative titers of the recovery samples of the two different dilution groups were (23.50±1.69)% and (49.30±2.97)%, respectively, and the corresponding recovery rates were (93.50±6.30)% and (96.24±5.43)%, respectively, with RSD less than 11%. 【Conclusion】 The method for determinating ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established. It could be applied in determinating the ADCC biological activity of IVIG, and has the advantages of satisfactory linearity, accuracy, precision and specificity.

Advances in methodologies for evaluating cell-mediated immune responses / 生物工程学报

Cell-mediated immune response is an important part of machinery in maintaining the body's homeostasis. After the innate immune system selectively activates the adaptive immune system, the cell-mediated immunity exerts its killing and clearance functions. Therefore, evaluating the level of cell-mediated immune response is crucial in the diagnosis and treatment of cancer, monitoring the immune status after organ transplantation, diagnosing and preventing viral diseases, and evaluating the effectiveness of vaccines and other areas. From the initial overall assessment of the immune effects in vivo to the precise detection of the number and function of multiple immune cells, the evaluation methods of cell-mediated immune response have greatly advanced. However, cell-mediated immune response involves multiple levels in the body, and it's difficult to choose the numerous detection methods available. The article systematically compares the evaluation methods of cell-mediated immune response at four different levels: the organism, the tissue and organ, the immune cells and the immune molecules, with the aim to facilitate the applications of related technologies.

Diagnostic criteria and research progress on pancreas allograft pathology / 器官移植

Pancreas transplantation and pancreas-kidney transplantation are the optimal treatment for renal failure caused by type 1 diabetes mellitus, partial type 2 diabetes mellitus and their complications. Pancreas transplantation mainly includes simultaneous pancreas-kidney transplantation (SPK), pancreas transplantation after kidney transplantation (PAK) and pancreas transplantation alone (PTA). Among all types of pancreas transplantation, biopsy of pancreas allograft remains the best method for definitively diagnosing rejection and differentiate it from other complications. In this article, biopsy methods of pancreas allograft and related research progress, diagnostic criteria and research progress on rejection of pancreas allograft biopsy, and main complications and pathological manifestations of pancreas allograft were illustrated, aiming to provide reference for guiding the clinical diagnosis of the above mentioned complications and ensuring the long-term survival of pancreas allografts and recipients.

Research highlights on kidney transplantation in 2021: voices from China / 器官移植

Over the past 70 years, kidney transplantation has become not only the most mature but also the highest-success-rate surgery among all organ transplantation surgeries. However, the long-term survival of kidney transplant recipients is still challenged by such key factors as ischemia-reperfusion injury related to kidney transplantation, rejection, chronic renal allograft dysfunction, renal allograft fibrosis, immunosuppressive therapy, infections and others. Relevant fundamental and clinical studies have emerged endlessly. At the same time, the research related to kidney transplantation also becomes a new hot spot accordingly in the context of the normalization of novel coronavirus pneumonia. This article reviewed the cutting-edge hot spots in relation to the fundamental and clinical aspects of kidney transplantation together with relevant new techniques and new visions. The studies included in this article focused on the reports published by Chinese teams that are more applicable to the current situation of kidney transplantation in China, for the purpose of providing new thoughts and strategies for the diagnosis and treatment of kidney transplantation related issues in China.

A method for measuring the ADCP potency of anti-CD38 monoclonal antibody / 药学学报

A method to measure the antibody-dependent cell-mediated phagocytosis (ADCP) potency of anti-CD38 mAb was developed based on design of experiment (DoE) with a Jurkat/NFAT/CD32a-FcεRIγ transgenic cell line as the effector cell, the Daudi cell line as the target cells, and luciferase as the detection system. The DoE method was used for optimization of experimental parameters and methodological validation. The results show that anti-CD38 mAb exhibits a dose-response relationship with the following four-parameter equation: y = (A - D) / [1 + (x / C)B] + D. Several experimental parameters were optimized by statistical experimental design and determined as follows: the working concentration of anti-CD38 mAb was 800-20.81 ng·mL-1, the density of the target cells was 7.5×104 per well, and the density of effector cells was 2.5×104 per well, with an induction time of 6 h. The method showed good specificity. The recovery rate for samples from 5 different groups showed that the relative potencies of anti-CD38 mAb were (59.97 ± 4.74) %, (82.44 ± 5.15) %, (110.69 ± 11.71) %, (129.23 ± 5.22)% and (162.15 ± 3.66) %. The recoveries ranged from 103% to 120% and the RSDs of the above results were all less than 11%. The linear detection range was 50%-150%. Based on DoE design, this method for measuring ADCP potency of anti-CD38 mAb was optimized and validated with good specificity, repeatability and accuracy. This method can be used for evaluation of ADCP biological activity of anti-CD38 mAbs.

Diagnostic criteria and research progress on Banff classification of allograft pathology in composite tissue allotransplantation / 器官移植

Composite tissue allotransplantation (CTA) is a novel transplantation discipline to treat functional tissue or limb defects. Since a majority of CTA grafts were vascularized grafts, it is also known as vascularized composite allotransplantation (VCA). The grafts of CTA/VCA consist of two or more types of allogeneic skin, subcutaneous tissue, bone, muscle, nerve and vessel, etc. Most of CTA/VCA grafts contain skin tissues, which possess the highest antigenicity. Acute rejection after transplantation is the primary obstacle leading to CTA/VCA graft failure and primary graft dysfunction. Hence, histopathological characteristics of skin rejection in CTA/VCA grafts have become the primary hotspot. In this article, pathological features of CTA/VCA rejection, Banff classification in 2007 and related research progress were reviewed, aiming to provide reference for the diagnosis and treatment of rejection and other complications of CTA/VCA.

Research progress and application of donor-derived cell-free DNA in diagnosis and treatment of kidney transplantation / 器官移植

Early diagnosis and treatment of rejection after kidney transplantation play a critical role in alleviating allograft injury. Detection of donor-derived cell-free DNA (dd-cfDNA) could be performed based on the next-generation sequencing and other techniques. The content of DNA fragments derived from necrotic and apoptotic donor kidney tissues in circulating body fluids could be determined by concentration and absolute quantitative methods, which has application potential in monitoring allograft injury in clinical practice. Compared with traditional serum creatinine and other indicators, dd-cfDNA detection may monitor allograft injury from several weeks to months in advance, providing a "time window" for clinical treatment and delaying graft failure. Along with deepening research of dd-cfDNA in recent years, dd-cfDNA has captivated widespread attention due to its non-invasiveness, high sensitivity and real-time evaluation of therapeutic effect. In this article, current study evidence and conclusions related to multidimensional application of dd-cfDNA detection in diagnosis and treatment of kidney transplantation were reviewed, and the future research and clinical application direction of dd-cfDNA were discussed, aiming to provide reference for widespread application of dd-cfDNA detection in clinical practice in China.

Diagnostic criteria and research progress on lung allograft pathology / 器官移植

In recent years, the quantity of lung transplantation has been gradually increased in China along with the accumulation of surgical techniques and postoperative management experience of lung transplantation. Multiple lung allograft complications may occur after lung transplantation, mainly including primary graft dysfunction (PGD) caused by ischemia-reperfusion injury (IRI) of the lung allograft, acute and chronic rejection, opportunistic infection or lymphoproliferative disorder of lymphoid tissues induced by the decrease of host immunity due to postoperative use of immunosuppressants, etc. The diagnosis of complications after lung transplantation mainly relies on biopsy of the lung allograft. In this article, the brief history of lung allograft pathology, main approaches and pathological processing techniques of lung allograft biopsy, major complications after lung transplantation and pathological diagnostic criteria were elucidated, aiming to provide reference for targeted management of these complications in clinical practice.

Urinary donor-derived cell-free DNA as a non-invasive biomarker for BK polyomavirus-associated nephropathy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

BK polyomavirus-associated nephropathy (BKPyVAN) is a common cause of allograft failure. However, differentiation between BKPyVAN and type I T cell-mediated rejection (TCMR) is challenging when simian virus 40 (SV40) staining is negative, because of the similarities in histopathology. This study investigated whether donor-derived cell-free DNA (ddcfDNA) can be used to differentiate BKPyVAN. Target region capture sequencing was applied to detect the ddcfDNAs of 12 recipients with stable graft function, 22 with type I TCMR, 21 with proven BKPyVAN, and 5 with possible PyVAN. We found that urinary ddcfDNA levels were upregulated in recipients with graft injury, whereas plasma ddcfDNA levels were comparable for all groups. The median urinary concentrations and fractions of ddcfDNA in proven BKPyVAN recipients were significantly higher than those in type I TCMR recipients (10.4 vs. 6.1 ng/mL,

Pathology of T cell-mediated rejection in renal allograft / 器官移植

T cell-mediated rejection (TCMR) is one of the main mechanisms of rejection in organ transplantation, which is also the most common type of acute rejection.Based on Banff classification on allograft pathology (Banff classification) in 2019, TCMR can be divided into acute TCMR (aTCMR) and chronic active TCMR (caTCMR) according to the characteristics of immune lesions.In this article, the basic definition of TCMR, the research progress on TCMR pathology according to Banff classification for renal allograft, and the basic pathological changes and diagnostic grading of TCMR were reviewed, aiming to provide evidence for early identification, diagnosis and treatment of TCMR and prevent the progression of TCMR into caTCMR, thereby guarantying the long-term survival of both the renal allograft and recipient.

Banff classification and progress on liver allograft pathology / 器官移植

The pathology of liver allograft biopsy is not only essential for the evaluation of liver donor, but also for the diagnosis and differential diagnosis of posttransplantation complications. With the development of liver transplantation in clinical practice, relevant studies of the pathological diagnosis of liver allograft complications have been deepened. Banff classification on liver allograft pathology have been gradually established within the international community. In China, pathological studies related to liver allograft pathology have been steadily carried out, and the pathological diagnostic basis of liver allograft pathology suitable for the clinical practice of liver transplantation in China has been gradually formed. This article reviews the history of Banff liver allograft pathology and major pathological lesions of liver allograft complications, aiming to provide reference for implementing pathological diagnosis of liver allograft pathology in China, assisting clinical diagnosis and targeted treatment of complications after liver transplantation, and further improving the survival of liver allograft and recipients.

The origin and development of Banff classification on allograft pathology and its effects in promoting organ transplantation / 器官移植

The Banff conference on allograft pathology (Banff conference) and the establishment of Banff classification on allograft pathology (Banff classification) are milestones in the development of international allograft pathology. At present, all organ transplantation centers around the world routinely perform pathological diagnosis by biopsy of the transplant kidney according to Banff classification. Subsequently, the consensus process and update mode of Banff classification for transplant kidney was quickly extended to transplant heart, lung, liver, pancreas, and small intestine, etc. The Banff conference has not only become a thematic meeting that includes the pathology study and discussion of various transplant organs, but also gradually developed unified diagnostic standard for the biopsy of each transplant organ, which better promoted the accurate diagnosis and treatment of complications after organ transplantation. This article summarized the history of international allograft pathology research, the Banff conference and Banff classification in promoting organ transplantation, which aimed to provide a reference for the smooth development of clinical organ transplantation.

Assessment and monitoring of immune risk of kidney transplantation rejection / 器官移植

Kidney transplantation is the most efficacious treatment for end-stage renal failure. Although the shortterm survival and functional recovery of the kidney graft have been significantly improved, the long-term survival of the kidney graft remains to be enhanced. Antibody-mediated rejection (AMR) and T cell-mediated rejection (TCMR) caused by immune factors are still the most critical causes of kidney graft failure. In this article, the immune risk assessment and monitoring of kidney transplant recipients during the awaiting period, before and after kidney transplantation were reviewed. Through the evaluation of preexisting human leukocyte antigen (HLA) antibodies and non-HLA antibodies, HLA matching, lymphocytotoxicity cross-matching and immune memory cells in the recipients before kidney transplantation, programmed biopsy of the kidney graft of the recipients after kidney transplantation and monitoring of HLA antibodies, non-HLA antibodies and donor-derived cell-free DNA (dd-cfDNA), individualized immunosuppressive treatment and monitoring regimes could be established, and the incidence of rejection could be prevented, timely detected and diagnosed. According to the immune monitoring results, ineffective treatment or over-treatment could be avoided, thereby improving the long-term survival of kidney graft.

Diagnostic criteria and research progress on heart allograft pathology / 器官移植

With the improvement of surgical technique of heart transplantation and clinical application of potent immunosuppressant, the quantity of heart transplantation and the survival time of heart allograft have been significantly improved. However, a series of complications, such as right ventricular failure, ischemia-reperfusion injury, acute rejection, "Quilty lesion", infection and chronic rejection characterized by transplant coronary artery disease (TCAD) may still occur at different stages after heart transplantation. The application of endomyocardial biopsy (EMB) makes it possible to observe and understand the pathological features of multiple complications of heart allograft including rejection, which has become the most accurate diagnostic tool for postoperative complications. In this article, the brief history of heart allograft pathology, main postoperative complications and pathological diagnostic criteria, and cutting edge research progress on diagnostic criteria of rejection were illustrated, aiming to bring clinical benefits to more recipients undergoing heart transplantation.

Immune mechanism of biliary epithelial cell injury in the small intrahepatic bile ducts in primary biliary cholangitis / 临床肝胆病杂志

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease caused by the combined effect of genetic and environmental factors and characterized by the apoptotic necrosis of biliary epithelial cells (BEC) in the small intrahepatic bile ducts. This article describes the effect of B cells, macrophages, natural killer cells, NKT, and T cells on the immune injury of BEC during PBC, so as to provide some guidance for the targeted immune therapy for PBC.

Expression and activity identification of bispecific antibody targeting PD-1/CD19 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity. Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19 BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml. ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upward trend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.