Role of Natural Killer Cells in Antibody-mediated Rejection of Kidney Transplantation.

Objective: This study explicitly demonstrates the roles of natural killer (NK) cells in different types of kidney transplantation. Methods: We'd done the whole study from October 2022 to October 2023. To further explore the significance of NK cells during renal transplantation, we provide a theoretical basis for clinically overcoming immune rejection after renal transplantation by developing new anti-rejection drugs. We selected twelve male mice and divided them into three groups (Syngeneic transplant group allograft transplant group allograft transplant (priming) group) by random. Initially, the morphological and histopathological changes in the kidney transplantation graft model of mice in different groups are observed. Further, the DSA-IgG levels in peripheral blood and C3d and IgG deposition in mice are detected by ELISA and immunohistochemical staining. Then, the Banff 2015 score is recorded to screen a suitable AMR mouse model. Finally, the expression of NK cells in different rejection modes is detected by flow cytometry, and the expressions of various cytokines (INF-γ, perforin, granzyme B, TNF-α) in peripheral blood are detected by enzyme-linked immunosorbent assay (ELISA). Results: In the allogeneic transplantation (priming) group, peritubular capillary inflammatory cell infiltration, moderate endarteritis, and small arterial fibrinoid necrosis are evident. The Banff score showed that the allogeneic transplantation (pre-sensitized) group is significantly higher than the syngeneic and allogeneic transplantation groups. The C3H→C57BL/6 mice are pre-sensitized by skin transplantation, and then kidney transplantation is performed to establish the antibody-mediated rejection (AMR) model. After kidney transplantation, the expression levels of NK cells in the peripheral blood, spleen, and transplanted kidney tissue of mice in the pre-sensitized group are significantly higher than in the allogeneic transplantation and control groups. In the C3H→C57BL/6 mouse model of AMR, NK cells and the related cytokines in the peripheral blood are highly expressed after kidney transplantation, proving that NK cells play an essential role in the occurrence of AMR. Conclusion: Our study proved the significance of NK cells in the occurrence of AMR by systematically monitoring the expression of NK cell-related cytokines in different types, which provided some ideas for the clinical treatment of AMR.

Neutrophil depletion attenuates antibody-mediated rejection in a renal transplantation mouse model.

Antibody-mediated rejection (AMR) can cause graft failure following renal transplantation. Neutrophils play a key role in AMR progression, but the exact mechanism remains unclear. We investigated the effect of neutrophils on AMR in a mouse kidney transplantation model. The mice were divided into five groups: syngeneic transplantation (Syn), allograft transplantation (Allo), and three differently treated AMR groups. The AMR mouse model was established using skin grafts to pre-sensitize recipient mice. Based on the AMR model, Ly6G-specific monoclonal antibodies were administered to deplete neutrophils (NEUT-/- + AMR) and TACI-Fc was used to block B-cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) signaling (TACI-Fc + AMR). Pathological changes were assessed using hematoxylin-eosin and immunohistochemical staining. Banff values were evaluated using the Banff 2015 criteria. Donor-specific antibody (DSA) levels were assessed using flow cytometry, and BAFF and APRIL concentrations were measured using ELISA. Compared to the Syn and Allo groups, a significantly increased number of neutrophils and increased C4d and IgG deposition were observed in AMR mice, accompanied by elevated DSA levels. Neutrophil depletion inhibited inflammatory cell infiltration and reduced C4d and IgG deposition. Neutrophil depletion significantly decreased DSA levels after transplantation and suppressed BAFF and APRIL concentrations, suggesting a mechanism for attenuating AMR-induced graft damage. Similar results were obtained after blockading BAFF/APRIL using a TACI-Fc fusion protein. In summary, neutrophil infiltration increased in the AMR mouse renal transplantation model. Neutrophil depletion or blockading the BAFF/APRIL signaling pathway significantly alleviated AMR and may provide better options for the clinical treatment of AMR.

Rapid and Sensitive Detection of Hepatitis C Virus in Clinical Blood Samples Using Reverse Transcriptase Polymerase Spiral Reaction.

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

Expression profiles of transcription factors for special CD4+ T-cell subsets in peripheral blood mononuclear cells from patients with hepatitis B virus infection.

This study is to characterize the transcription factor expression profiles for the peripheral CD4 T-cell subsets, and analyze its associations with the clinical measures of the hepatitis B virus (HBV) infection.Totally 275 subjects were included. The expression levels of transcription factors (T-bet, GATA-3, Foxp3, RORγt, and Bcl-6) in the peripheral blood mononuclear cells (PBMCs) were determined by the real-time fluorimetry quantitative PCR (FQ-PCR).Lowest expression levels of all these transcription factors were observed for the HBsAb(-) group, which were higher in the HBsAb(+) and RHB groups. The T-bet/GATA-3 ratios in the CHB and RHB groups were significantly lower than the HBsAb(-) group, whereas the RORγt/Foxp3 ratios in the AHB and RHB groups were significantly higher than the CHB and HBsAb(+) groups. Furthermore, the RORγt mRNA expression levels were significantly different among groups with different disease severities or with different alanine aminotransferase (ALT) levels. The asymptomatic carrier (AsC) group and the group with ALT ≤ 40 had the highest express level. The mRNA expression levels of T-bet, GATA-3, Foxp3, and RORγt varied along with the aspartate aminotransferase (AST) levels, with AST ≤ 40 having the highest expression levels. In addition, significant differences were observed in the transcription factor expression levels between the group with the serum HBV DNA load of (1.000-9.999) × 10 copies/mL and other groups.Expression profile of critical transcription factors for peripheral CD4 T-cell subsets may indicate clinical outcomes of HBV infection.

Procoagulant role of neutrophil extracellular traps in patients with gastric cancer.

BACKGROUND: Patients with gastric cancer (GC) commonly exhibit a hypercoagulable state that results in significant morbidity and mortality. Recent studies have shown that neutrophil extracellular traps (NETs) trigger coagulation through an intrinsic pathway and contribute to thrombus initiation and progression. In this study, we aimed to determine the procoagulant activity (PCA) of NETs in patients with GC. METHODS: NET formation and their PCAs were assessed in 48 patients with GC and 36 healthy controls using immunofluorescence microscopy of neutrophil markers and extracellular DNA as well as a modified capture ELISA technique, and thrombin-antithrombin complex and clot (fibrin) spectroscopic detection, respectively. RESULTS: Here we showed that neutrophils isolated from patients with GC displayed significantly enhanced NET formation compared with those from healthy controls; furthermore, plasma or platelets obtained from patients with GC induced control neutrophils to release NETs. In addition, NETs released by GC neutrophils significantly increased the potency of control plasma to generate thrombin and fibrin. Notably, these procoagulant effects were dramatically attenuated by application of DNase I. We further found that spontaneous NET formation in patients with GC was significantly higher than that in controls, increased with tumor- node-metastasis stage elevation, and positively correlated with thrombin-antithrombin complex levels and D-dimers. Additionally, the effect of DNase I on cell-free plasma generation of fibrin was dependent on the concentration of NET formation. CONCLUSION: These results suggest that GC creates a systemic environment that primes neutrophils to release procoagulant NETs. Thus, targeting NETs might improve the coagulopathy of patients with GC.

The influence of HLA alleles and HBV subgenotyes on the outcomes of HBV infections in Northeast China.

Hepatitis B virus (HBV) infection has a wide variety of clinical outcomes, it could be spontaneouly recovered and also could develop fulminant liver failure or cirrhosis with hepatocellular carcinoma. Human leukocyte antigen (HLA) polymorphism and HBV (sub)genotypes have been speculated to associate with the outcome of HBV infection because the data obtained from various populations who bear different HLA alleles have shown a HLA polymorphism associated outcome of HBV infection. However, as the most important viral and host genetic factors, the impact of HBV (sub)genotypes in combination with HLA polymorphism on the clinical outcomes of HBV infections remains unclear. To demonstrate the association of HLA allele polymorphism in combination with HBV subgenotypes with the outcome of HBV infection in Northeastern Han Chinese population, a total of 230 HBV-infected individuals (Infection group) were compared to 210 random selected controls (Control group) who are negative for HBV infection for their HLA alleles frequency as well as the associations with the virus infection, clearance and persistence in combination with HBV subgenotypes. Of the 230 HBV-infected subjects, 54 were acute self-limited hepatitis (ASH) with HBV subgenotype C2 (ASH-C2), 144 were chronic hepatitis (CH) with HBV subgenotype C2 and B2 (CH-C2 and CH-B2), and 32 were spontaneously recovered (SR) without subgenotype results. When two groups are compared, the results suggest that B*48, B*51 and DRB1*12 carrier may have a high risk for HBV infection, but B*51 is likely association with spontaneous recovery and DRB1*07, 12 may be implied in viral persistence. HLA-B*15, DRB1*11 and 14 associated with viral clearance in the cases of HBV-C2 infection; HLA-B*54 carriers in chronic group are more sensitive to with the infection of HBV subgenotype B2; HLA-B*07 and DRB1*13 may protect subjects from HBV infection. The data presented a link between HLA polymorphism and HBV pathogenesis and suggested potential therapeutic targets for hepatitis B.

Association of human leukocyte antigen polymorphism with hepatitis B virus infection and genotypes.

Associations were studied between the polymorphism of northern Han Chinese leukocyte antigen (HLA) alleles and the outcomes of hepatitis B virus (HBV) infection and HBV genotypes. HLA-A, B, and DRB1 alleles in peripheral blood mononuclear cells (PBMCs) were detected by polymerase chain reaction (PCR) with sequence-specific primers. The PBMCs were collected from 61 persons who tested positive for hepatitis B surface antigen (HBsAg) for more than 6 months (Persistent group), 32 persons who tested negative for both HBsAg and HBV DNA but positive for both anti-HBc and anti-HBs (Recovered group), and 40 persons who tested negative for all serologic markers of HBV infection (Uninfected group). HBV genotypes in serum specimens from 56 of 61 patients with persistent HBV infection were determined by nested PCR with 6 pairs of HBV genotype-specific primers (A to F). The frequency of HLA-DRB1*12 was significantly higher in the Persistent group than in the Recovered group (P=0.004). HLA-A*02 was significantly higher in the Recovered group than in the Persistent group (P=0.044). HLA-DRB1*15 was significantly higher in the HBV genotype B group than in the C group (P=0.013). These findings suggested that there were associations not only between HLA polymorphisms and outcomes of HBV infection but also between HLA polymorphisms and the infected HBV genotypes.