Helper T Cell (CD4+) Targeted Tacrolimus Delivery Mediates Precise Suppression of Allogeneic Humoral Immunity.

Antibody-mediated rejection (ABMR) is a major cause of dysfunction and loss of transplanted kidney. The current treatments for ABMR involve nonspecific inhibition and clearance of T/B cells or plasma cells. However, the prognosis of patients following current treatment is poor. T follicular helper cells (Tfh) play an important role in allograft-specific antibodies secreting plasma cell (PC) development. Tfh cells are therefore considered to be important therapeutic targets for the treatment of antibody hypersecretion disorders, such as transplant rejection and autoimmune diseases. Tacrolimus (Tac), the primary immunosuppressant, prevents rejection by reducing T cell activation. However, its administration should be closely monitored to avoid serious side effects. In this study, we investigated whether Tac delivery to helper T (CD4+) cells using functionalized mesoporous nanoparticles can block Tfh cell differentiation after alloantigen exposure. Results showed that Tac delivery ameliorated humoral rejection injury in rodent kidney graft by suppressing Tfh cell development, PC, and donor-specific antibody (DSA) generation without causing severe side effects compared with delivery through the drug administration pathway. This study provides a promising therapeutic strategy for preventing humoral rejection in solid organ transplantation. The specific and controllable drug delivery avoids multiple disorder risks and side effects observed in currently used clinical approaches.

Brucellosis in kidney transplant donor: A case report.

Brucellosis is a rare systemic zoonotic disease in kidney transplantation that affects graft survival. Only 7 cases have been reported to date. Herein, we report one case of brucellosis in a kidney transplant donor, which is different from previously reported recipient cases.

Urinary donor-derived cell-free DNA as a non-invasive biomarker for BK polyomavirus-associated nephropathy.

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, P<0.001 and 68.4% vs. 55.3%, P=0.013, respectively). Urinary ddcfDNA fractions (not concentrations) were higher in the BKPyVAN-pure subgroup than in the BKPyVAN-rejection-like subgroup (81.30% vs. 56.64%, P=0.025). With a cut-off value of 7.81 ng/mL, urinary ddcfDNA concentrations distinguished proven BKPyVAN from type I TCMR (area under the curve (AUC)=0.848, 95% confidence interval (95% CI): 0.734 to 0.963). These findings suggest that urinary ddcfDNA is a non-invasive biomarker which can reliably differentiate BKPyVAN from type I TCMR.

NMDA receptor-mediated CaMKII/ERK activation contributes to renal fibrosis.

BACKGROUND: This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-ß administration. METHODS: Acute renal fibrosis (RF) was induced in mice by unilateral ureteral obstruction (UUO). Histological changes were observed using Masson's trichrome staining. The expression levels of NR1, which is the functional subunit of NMDAR, and fibrotic and epithelial-to-mesenchymal transition markers were measured by immunohistochemical and Western blot analysis. HK-2 cells were incubated with TGF-ß, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were administered for pathway determination. Chronic RF was introduced by sublethal ischemia-reperfusion injury in mice, and NMDAR inhibitor dextromethorphan hydrobromide (DXM) was administered orally. RESULTS: The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-ß1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury. CONCLUSIONS: NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.

Elevated serum IL-21 levels are associated with stable immune status in kidney transplant recipients and a mouse model of kidney transplantation.

Allograft rejection after renal transplantation remains a challenge to overcome. Interleukin (IL)-21, a cytokine with pleiotropic effects, maintains immune homeostasis post-transplantation. Here, we report higher levels of IL-21 in kidney transplant recipients with non-rejection (NR) than in recipients with T cell-mediated rejection (TCMR, P < 0.001) and antibody-mediated rejection (ABMR, P = 0.005). We observed a negative correlation between IL-21 and creatinine (Cr) levels (P = 0.016). The receiving operating characteristic (ROC) curve showed a promising diagnostic value of IL-21 to identify acute rejection with an area under the curve (AUC) of 0.822 (P < 0.001). In contrast, exogenous administration of IL-21 accelerated acute rejection in a comparative translational kidney transplant (KT) mouse model. Reduced IL-21 levels in the peripheral blood were observed in KT mice after IL-21 injection. Further analysis revealed that increased IL-21 levels in the spleen induced proliferation of CD4+ T cells and CD19+ B cells after IL-21 treatment. Our findings suggest a critical function of IL-21 in kidney transplantation and the potential involvement of the IL-21/IL-21R pathway in acute rejection management.

Long noncoding RNA GAS5 impairs the proliferation and invasion of endometrial carcinoma induced by high glucose via targeting miR-222-3p/p27.

Long noncoding RNAs (lncRNAs) have been identified to be critical functional regulator in the human tumors, while the deepgoing mechanism by which lncRNAs modulates the endometrial carcinoma is still elusive. In this work, we found that lncRNA GAS5 was under-expressed in the endometrial carcinoma tissue specimens, especially these samples with type 2 diabetes mellitus. Besides, the aberrant under-expression of GAS5 was correlated with the advanced tumor stage as well as poor prognosis outcome. In cellular experiments, GAS5 was decreased in the cells exposed to the high glucose. Enforced GAS5 expression repressed the tumor phenotype of endometrial carcinoma cells, including proliferation and invasion. Molecular mechanism study further demonstrated that GAS5 functioned as a sponge for miR-222-3p, abrogating its ability of inhibiting p27 protein expression. In conclusion, these results confirmed the vital regulation of GAS5/miR-222-3p/p27 axis in the endometrial carcinoma tumorigenesis.

The purification of reduced ß2-glycoprotein I showed its native activity in vitro.

BACKGROUND: New evidence has shown that reduced ß2-glycoprotein I (ß2GPI) has anti-oxidative stress and anti-inflammatory activity. However, the details are still poorly understood. This study aims to prepare stable reduced ß2GPI with its native bioactivity in vitro. METHODS: Human ß2GPI was purified from plasma first with perchloric acid precipitation and then purified with a series of chromatography methods including Sephadex G-25 desalting, SP HP, AF-heparin HC-650 M, and Sephacryl S-200. The purified human ß2GPI was reduced with thioredoxin-1 (TRX-1) activated by DL-dithiothreitol (DTT). Glutathione (GSH) was selected to block the free thiols in reduced ß2GPI. LC/MS was used to verify the location of free thiols. Western blot analysis was used to detect ß2GPI immunoreactivity. MTS and flow cytometry were conducted to investigate its biological effect on oxidative-stress-induced death of human umbilical vein endothelial cells (HUVECs). The levels of tumour necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) interleukin-10 (IL-10),interleukin-12P70 (IL-12P70),interferon-gamma (IFN-γ) and monocyte chemoattractant protein -1(MCP-1) in mouse serum were quantified to assess its anti-inflammatory activity in lipopolysaccharide (LPS)-mediated systemic inflammation. RESULTS: We obtained approximately 10 mg ß2GPI (purity 98.7%) from 200 ml plasma. The protein yield was 0.05 mg/ml plasma. ß2GPI was then reduced by TRX-1/DTT in vitro; the free thiols were detected on Cys288 and Cys326 in domain V of ß2GPI. The GSH blockage stabilized the reduced ß2GPI in vitro. This reduced ß2GPI can be recognized by the anti-ß2GPI antibody, can significantly reduce the death of HUVECs after H2O2 treatment and can significantly decrease the levels of TNF-α, IL-6,IFN-γ and MCP-1 in mice upon LPS stimulation. CONCLUSION: Stable reduced ß2GPI can be obtained in vitro by TRX-1 deoxidation followed by the blockage of thiols with GSH. This reduced ß2GPI maintains the same immunological activity as oxidized ß2GPI and has the ability to counter the oxidative stress induced by H2O2 in HUVECs and inflammation in LPS-mediated inflammation in mice.

Microribonucleic acid-192 as a specific biomarker for the early diagnosis of diabetic kidney disease.

AIMS/INTRODUCTION: To evaluate the diagnostic value of microribonucleic acid (miR) as biomarkers in patients with diabetic kidney disease (DKD). MATERIALS AND METHODS: A total of 230 diabetes mellitus patients and 53 healthy participants were enrolled, and the diabetes mellitus group was further divided into normoalbuminuria, microalbuminuria and large amount of albuminuria group. MiRs of serum and urine were quantificated using real-time polymerase chain reaction. General clinical information was collected and analyzed for the risk factors. Cut-off values of diagnosis sensitivity were determined by receiver operating characteristic curves and the Youden Index. RESULTS: Compared with the healthy participants, the expression of miR-192 in serum decreased, whereas in urine it increased with the progression of DKD. The expression of both serum and urine miR-126 increased in the diabetes mellitus group, but no significant change was obtained among the DKD groups. The area under the curve receiver operating characteristic of both serum and urine miR-192 was higher than that of the albumin-to-creatinine ratio. Combined detection of urine and serum miR-192 has a higher specificity and lower misdiagnosis rate. CONCLUSIONS: Both serum and urinary miR-192 could be a potential biomarker of DKD, playing a crucial role in the prevention and treatment of DKD.

Effects of Insulin Plus Glucagon-Like Peptide-1 Receptor Agonists (GLP-1RAs) in Treating Type 1 Diabetes Mellitus: A Systematic Review and Meta-Analysis.

INTRODUCTION: Combination therapy with insulin and glucagon-like peptide-1 receptor agonists (GLP-1RAs) has already been proven an efficient treatment option for type 2 diabetes. This combination can effectively improve glycated hemoglobin levels, cause weight loss and reduce the dosage of insulin. In addition, it can also reduce the risk of hypoglycemia. Several randomized controlled trials have confirmed that this treatment may be just as effective for type 1 diabetes mellitus (T1DM) patients. The objective of this meta-analysis was to assess the effects and efficacy of the treatment on glycemic changes, weight loss and insulin dosage in type 1 diabetes mellitus patients. METHODS: We searched Embase, PubMed and Cochrane for randomized controlled trials (no time restrictions) that investigated combined insulin and GLP-1 treatment. The main endpoints were measurements of glycated hemoglobin and changes in the weight and the dosage of insulin. RESULTS: In total, 1093 were studies identified, and 7 studies were included in our meta-analysis. GLP-1 agonist and insulin combination therapy led to greater reductions in HbA1c levels [P = 0.03; mean difference -0.21; 95% confidence intervals (CI) (-0.40, 0.02)] and weight [P < 0.05; -3.53 (-4.86, 2.19)] compared to control treatments. The combination therapy did not significantly influence the daily weight-adjusted total insulin dose [P = 0.05; -0.11 (-0.23, 0)], but it did reduce the daily weight-adjusted bolus insulin dose [P = 0.001; -0.06 (-0.1, 0.02)]. CONCLUSION: Our meta-analysis supports the use of a combined therapeutic regimen of insulin and GLP-1RAs for treating patients with T1DM. Combination therapy with GLP-1 and insulin could achieve an ideal treatment effect on glycemic control, weight loss and bolus insulin dose in patients with T1DM.