MSC-Derived Small Extracellular Vesicles Alleviate Diabetic Retinopathy by Delivering miR-22-3p to Inhibit NLRP3 Inflammasome Activation.

PURPOSE: This study aimed to investigate the effect of mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) on diabetic retinopathy (DR) and its underlying mechanism. METHODS: In vivo, MSC-sEVs were injected intravitreally into diabetic rats to determine the therapeutic efficacy. In vitro, MSC-sEVs with/without miR-22-3p inhibition were cocultured with advanced glycation end-products (AGEs)-induced microglia with/without NLRP3 overexpression to explore the molecular mechanism. RESULTS: In vivo, MSC-sEVs inhibited NLRP3 inflammasome activation, suppressed microglial activation, decreased inflammatory cytokines levels in the retina, and alleviated DR as evidenced by improved histological morphology and blood-retinal barrier function. Based on miRNA sequencing of MSC-sEVs, bioinformatic software, and dual-luciferase reporter assay, miR-22-3p stood out as the critical molecule for the role of MSC-sEVs in regulating NLRP3 inflammasome activation. Diabetic rats had lower level of miR-22-3p in their retina than those of control and sEV-treated rats. Confocal microscopy revealed that sEV could be internalized by microglia both in vivo and in vitro. In vitro, compared with sEV, the anti-inflammation effect of sEVmiR-22-3p(-) on AGEs-induced microglia was compromised, as they gave a lower suppression of NLRP3 inflammasome activation and inflammatory cytokines. In addition, NLRP3 overexpression in microglia damped the anti-inflammatory effect of sEV. CONCLUSION: These results indicated that MSC-sEVs alleviated DR via delivering miR-22-3p to inhibit NLRP3 inflammasome activation. Our findings indicate that MSC-sEVs might be a potential therapeutic method for DR.

IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental Sjögren's syndrome.

Excessive NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation has an important function in the pathogenesis of Sjögren's syndrome (SS). Increased and dysfunctional myeloid-derived suppressor cells (MDSCs) promoted SS. However, NLRP3 inflammasome activation of MDSCs in SS and its regulated components are unclear. Splenic MDSCs were purified by immunomagnetic beads and cultured. Western blot was used to assess NLRP3 inflammasomes. Interleukin-1ß (IL-1ß) and IL-18 were measured using enzyme-linked immunosorbent assay. Here we showed that the NLRP3 inflammasome was activated in non-obese diabetic (NOD) mice with SS-like manifestations. We found that NLRP3 inflammasome activation was augmented in MDSCs of SS mice and NLRP3 inflammasome activation was suppressed in IL-27-deficient NOD mice. Consistent with findings of SS mice in vivo, we observed that NLRP3 inflammasome activation by adenosine triphosphate and lipopolysaccharide was remarkably intensified in MDSCs with IL-27 treatment in vitro. Collectively, our data highlighted that IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental SS.

Notch1 signalling controls the differentiation and function of myeloid-derived suppressor cells in systemic lupus erythematosus.

Emerging studies have reported the expansion of myeloid-derived suppressor cells (MDSCs) in some autoimmune disorders, such as systemic lupus erythematosus (SLE), but the detailed molecular mechanisms of the aberrant expansion in SLE are still unclear. In the present study, we confirmed that the increased MDSCs positively correlated with disease activity in SLE patients. The suppressive capacity of MDSCs from patients with high activity was lower than that of MDSCs from patients with low activity. Moreover, the potential precursors for MDSCs, common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs), were markedly increased in the bone marrow (BM) aspirates of SLE patients. As an important regulator of cell fate decisions, aberrant activation of Notch signalling was reported to participate in the pathogenesis of SLE. We found that the expression of Notch1 and its downstream target gene hairy and enhancer of split 1 (Hes-1) increased markedly in GMPs from SLE patients. Moreover, the Notch1 signalling inhibitor DAPT profoundly relieved disease progression and decreased the proportion of MDSCs in pristane-induced lupus mice. The frequency of GMPs was also decreased significantly in lupus mice after DAPT treatment. Furthermore, the inhibition of Notch1 signalling could limit the differentiation of MDSCs in vitro. The therapeutic effect of DAPT was also verified in Toll-like receptor 7 (TLR7) agonist-induced lupus mice. Taken together, our results demonstrated that Notch1 signalling played a crucial role in MDSC differentiation in SLE. These findings will provide a promising therapy for the treatment of SLE.

Mesenchymal stem cells inhibit MRP-8/14 expression and neutrophil migration via TSG-6 in the treatment of lupus nephritis.

Abnormal infiltration and activation of neutrophils play a pathogenic role in the development of lupus nephritis (LN). Myeloid-related proteins (MRPs), MRP-8 and -14, also known as the damage-associated molecular patterns (DAMPs), are mainly secreted by activated neutrophils in systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) regulate a variety of immune cells to treat LN, but it is not clear whether MSCs can regulate neutrophils and the expression of MRP-8/14 in LN. Here, we demonstrated that neutrophil infiltration and MRP-8/14 expression were increased in the kidney of MRL/lpr mice and both decreased after MSCs transplantation. Further, the results showed that tumor necrosis factor- (TNF) stimulated gene-6 (TSG-6) in MSCs is necessary for MSCs to inhibit MRP-8/14 expression in neutrophils and neutrophil migration. In addition, small-molecule immunosuppressant had no significant effect on the expression of MRP-8/14 in neutrophils. Therefore, our results suggest that MSCs inhibited MRP-8/14 expression and neutrophil migration by secreting TSG-6 in the treatment of LN.

Total glucosides of paeony alleviates experimental Sjögren's syndrome through inhibiting NLRP3 inflammasome activation of submandibular gland cells.

OBJECTIVES: The mechanisms by which total glucosides of paeony (TGP) mitigates Sjögren's syndrome (SS) remains elusive. In the present study, we aim to explore the relationship between the therapeutic effects of TGP in the treatment of SS and NLRP3 inflammasome activation in submandibular gland (SG) cells. METHODS: Female non-obese diabetic (NOD) mice were selected as the model of SS. The mice were divided into PBS and TGP treatment group. For treatment, TGP (400mg·kg-1) was administered intragastrically every day for 4 weeks. The SS-like symptoms and pathological changes of the SG of mice were compared between the PBS and TGP group. The activation of NLRP3 inflammasome in SG was detected by RT-qPCR, immunohistochemistry and western blot. The SG cells stimulated by lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for activation of NLRP3 inflammasome were treated with or without TGP. Then, NLRP3 inflammasome activation was assessed. The IL-1ß and IL-18 in homogenate of SG, serum and supernatant were detected by ELISA. RESULTS: Compared with balb/c mice, NOD mice showed SS-like symptoms and lymphocyte infiltration in SG, and the expression of NLRP3 inflammasome in SG was significantly increased. The SS-like symptoms were alleviated, and lymphocyte infiltration in SG was reduced, and the level of NLRP3 inflammasome in SG mice was decreased after TGP treatment. TGP also significantly inhibit the activation of NLRP3 inflammasome of SG cells in vitro. CONCLUSIONS: Collectively, our results indicated that TGP alleviates SS through inhibition of the activation of NLRP3 inflammasome of SG. These findings clarified the mechanism underlying the therapeutic effects of TGP on SS, and provided new evidence for the further application of TGP in the treatment of SS.

Factors associated with 25-hydroxyvitamin D level in Chinese hospitalized patients with systemic lupus erythematosus: a retrospective cohort study.

We aimed to investigate the factors associated with vitamin D deficiency and changes in 25 (OH)D levels, as well as the impact of those changes on disease activity and renal function among SLE patients. This retrospective cohort study was based on the medical records of SLE patients hospitalized between 2010 and 2021. We collected relevant information from this patient population. Logistic regression analysis was employed to determine the factors associated with vitamin D deficiency and increased 25 (OH)D levels, and we calculated the odds ratios (ORs) and 95% confidence intervals (CIs) accordingly. At baseline, among the 1257 SLE patients, the median and interquartile range of 25 (OH)D levels were 14 (9, 20) ng/ml, with 953 (75.8%) patients exhibiting 25 (OH)D deficiency (< 20 ng/ml). The presence of 25 (OH)D deficiency was found to be associated with renal involvement and a high glucocorticoid (GC) maintenance dose. Among the 383 patients who were followed up for an average of 18 months, an increase of at least 100% in 25 (OH)D levels was positively associated with a decreased GC maintenance dose and vitamin D3 supplementation, with adjusted odds ratios(OR) (95% confidence interval [CI]) of 2.16 (1.02, 4.59) and 1300 (70, 22300), respectively. Furthermore, an increased level of 25 (OH)D was significantly associated with a decrease in the Disease Activity Index 2000 score and the urinary protein/creatinine ratio. Patients with SLE have low vitamin D levels, especially those with impaired kidney function. Increased 25 (OH)D levels can be achieved through supplementation with high doses of vitamin D3 and are associated with improvements in disease activity and the urinary protein/creatinine ratio.

Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation.

BACKGROUND: Lipocalin-2 (LCN2) is an indicator of the severity of lupus nephritis (LN) and plays a pivotal role in immune responses, but it is not known if its effect on LN pathogenesis derives from regulating the immune imbalance of T lymphocyte subsets. METHODS: The expression of LCN2 in T cells and kidneys was assessed in renal biopsies from patients with LN. We investigated the relationship between LCN2 levels and development of LN and systemic illness by injecting anti-LCN2 antibodies into MRL/lpr mice and analyzing pristane-treated LCN2-/- mice. RESULTS: LCN2 is highly expressed in CD4+ T cells and in renal tissues, and is associated with severe renal damage in patients with LN and in mice with experimental lupus. LCN2 promotes IFN-γ overexpression in CD4+ T cells through the IL-12/STAT4 pathway in an autocrine or paracrine manner. Both neutralization of LCN2 in MRL/lpr mice and genetic depletion of LCN2 in pristane-induced lupus mice greatly ameliorate nephritis. The frequency and number of splenic and renal Th1 cells decrease in proportion to LN disease activity. Conversely, administration of LCN2 exacerbates the disease with significantly higher renal activity scores and increased numbers of Th1 cells. CONCLUSIONS: LCN2 plays a crucial role in Th1 cell differentiation, and may present a potential therapeutic target for LN.

Mesenchymal stem cells prevent podocyte injury in lupus-prone B6.MRL-Faslpr mice via polarizing macrophage into an anti-inflammatory phenotype.

BACKGROUND: Podocyte injury plays a pathogenic role in the development of lupus nephritis (LN). Mesenchymal stem cells (MSCs) have shown promising therapeutic potential for LN. However, whether MSCs can prevent podocyte injury in LN remains unknown. METHODS: Human umbilical cord-derived MSCs (UC-MSCs) were infused into lupus-prone B6.MRL-Faslpr (B6.lpr) mice to investigate the influences of UC-MSCs on podocyte injury in LN. Podocytes and macrophages were co-cultured with UC-MSCs in vitro to study the mechanism by which UC-MSC protect podocytes. We further explored the effects of UC-MSCs on macrophage polarization. RESULTS: We found that UC-MSCs promoted the expression of podocyte-specific markers, podocin and synaptopodin, in lupus-prone B6.lpr mice, along with the improvement of lupus renal pathology in terms of reduced IgG and C3 deposition in glomeruli and decreased anti-dsDNA antibody level. Besides, UC-MSC treatment decreased podocyte foot process effacement, as UC-MSCs-treated macrophages led to less podocyte injury in vitro. Interestingly, we further found that UC-MSCs-treated macrophages exhibited an anti-inflammatory phenotype with higher expression of CD206, and lower expression of tumor necrosis factor-α and interleukin-1ß. Additionally, UC-MSCs-treated lupus mice showed reduced renal macrophage infiltration and elevated CD206 expression in kidney. CONCLUSIONS: Our results demonstrated that UC-MSCs ameliorated LN by preventing podocyte injury possibly through reducing macrophage infiltration and polarizing macrophage into an anti-inflammatory phenotype.

Infection with Opportunistic Bacteria Triggers Severe Pulmonary Inflammation in Lupus-Prone Mice.

Infection is a common cause of hospitalization and mortality in patients with systemic lupus erythematosus (SLE). How the underlying immune dysfunctions affect the antimicrobial immunity remains largely unknown. In the present study, employing the pulmonary infection model, we determined the antimicrobial defence of lupus-prone mice. After infecting with opportunistic bacterium Haemophilus influenzae (Hi), lupus-prone mice (B6/lpr) exhibited inefficient bacterial elimination and recovered slowly. They generated severer inflammation at the early stage of infection, as excessive accumulation of neutrophils and enhanced production of proinflammatory cytokines were observed in the lung. In addition, a large number of apoptotic cells were detected in the lungs of B6/lpr mice. For adaptive immune responses, B6/lpr mice were capable to generate enough protective Hi-specific Th17 cells. They evoked stronger Hi-specific γδ T17 response in both lungs and spleens. Unexpectedly, both CD4 and γδ T cells from lupus-prone mice showed deficiency in IFN-γ production. For humoral immune responses, compared with those of WT mice, the concentrations of Hi-specific IgA, IgM, and IgG, especially IgG, were significantly higher in the B6/lpr mice. Our findings suggest that lupus mice are capable to generate antibacterial immune responses; however, the overwhelming inflammation and overactivated immune responses increase the severity of infection.

Citrullinated fibrinogen impairs immunomodulatory function of bone marrow mesenchymal stem cells by triggering toll-like receptor.

Bone marrow mesenchymal stem cells (BMSC) have been shown to possess immunomodulatory activities, while its role in rheumatoid arthritis (RA) remains unknown. Citrullinated fibrinogen (cfb) has been considered as a specific autoantigen in RA pathogenesis. Our study aims to determine the role of cfb on immunomodulatory function of BMSC. We demonstrated the specific role of toll-like receptor 4 (TLR4)-NFκB pathway in the pro-inflammatory response of BMSC to cfb with increased production of interleukin (IL)-6, IL-8 and chemokine CC motif ligand 2 (CCL2). Moreover, cfb impaired BMSC-mediated suppression of peripheral blood mononuclear cells (PBMC) proliferation and reduced the production of the key immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO) in BMSC. We have uncovered a previously unrecognized role of cfb in interfering BMSC-mediated immunoregulation in RA. Cfb could act as a damage-associated molecule pattern (DAMP) for BMSC and thereby contribute to the propagation of inflammation in RA.

Mesenchymal stem cells induced CD4+ T cell apoptosis in treatment of lupus mice.

Umbilical cord-derived mesenchymal stem cell transplantation (UCMSCT) has been used to treat human autoimmune diseases like lupus for example, but little is known about its effect on cell apoptosis. Here we evaluated the efficacy of UCMSCT for lupus treatment and explored the mechanism by which mesenchymal stem cells (MSCs) modulate T cell apoptosis in lupus mice. 1 × 106 human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were injected into B6.MRL-Faslpr (B6.lpr) mice via tail vein. 6 h, 24 h or 4 weeks later, the mice were sacrificed and the apoptosis of lymphocytes in peripheral blood and spleen were detected by flow cytometry. The immune cell subpopulations in spleen were also measured at 6 h and 24 h, respectively. The therapeutic effects were assessed after 4 weeks. The frequency of peripheral blood CD4+ T cell apoptosis was reduced in lupus-prone B6.lpr mice. UCMSCT alleviated the disease phenotypes in B6.lpr mice, decreased the ratio of Th1 as well as Th2 cells, and increased percentages of apoptotic CD4+ T cells in vivo and vitro. Collectively, our findings unravel that UCMSCT alleviate lupus disease and reverse immune imbalance possibly by promoting T cell apoptosis in B6.lpr mice.

Mesenchymal stem cells enhance autophagy of human intrahepatic biliary epithelial cells in vitro.

Dysfunctional autophagy in intrahepatic biliary epithelial cells (IBECs) is the main mechanism underlying the pathogenesis of bile duct lesions in primary biliary cholangitis. Autophagy may be a key pathogenesis for aetiology of primary biliary cholangitis. Immunoblotting and immunofluorescence analyses were used for the evaluation of autophagy in human intrahepatic biliary epithelial cells (HiBECs) at various time points. Glycochenodeoxycholate (GCDC) induced autophagy in HiBECs; the ratio of microtubule-associated protein light chain 3-II/microtubule-associated protein light chain 3-I (LC3-II/LC3-I) expression markedly increased at 48 hours, and then declined. However, compared with cells treated with GCDC alone, the expression of LC3-II increased and the clearance of autophagosome enhanced in GCDC-treated cells cocultured with mesenchymal stem cells (MSCs). Furthermore, the level of phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) decreased in HiBECs cocultured with MSCs relative to those cultured without MSCs. Following STAT3 silencing, decreased expression of phosphorylated eukaryotic initiation factor 2α was consistently observed. The present data suggest that mesenchymal stem cells may enhance autophagic flux of HiBECs through the inhibition of STAT3 activity. SIGNIFICANCE PARAGRAPH: The present findings constitute the first report that human umbilical cord-derived MSCs enhance autophagic flux in HiBECs through a STAT3-dependent way: MSCs enhance the autophagic flux by increasing the formation of autophagosome and autolysosome in GCDC-treated HiBECs. MSCs decrease the STAT3 activity and the expression of eIF2α in GCDC-treated HiBECs; in addition, MSCs increase the expression of PKR. With STAT3 silencing, MSCs enhance neither the levels of LC3II nor the expression of PKR in GCDC-treated HiBECs.

Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China.

BACKGROUND: Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease or herpangina worldwide. This phase 3 trial was designed to evaluate the efficacy, safety, and immunogenicity of an EV71 vaccine. METHODS: We conducted a randomized, double-blind, placebo-controlled, multicenter trial in which 10,007 healthy infants and young children (6 to 35 months of age) were randomly assigned in a 1:1 ratio to receive two intramuscular doses of either EV71 vaccine or placebo, 28 days apart. The surveillance period was 12 months. The primary end point was the occurrence of EV71-associated hand, foot, and mouth disease or herpangina. RESULTS: During the 12-month surveillance period, EV71-associated disease was identified in 0.3% of vaccine recipients (13 of 5041 children) and 2.1% of placebo recipients (106 of 5028 children) in the intention-to-treat cohort. The vaccine efficacy against EV71-associated hand, foot, and mouth disease or herpangina was 94.8% (95% confidence interval [CI], 87.2 to 97.9; P<0.001) in this cohort. Vaccine efficacies against EV71-associated hospitalization (0 cases vs. 24 cases) and hand, foot, and mouth disease with neurologic complications (0 cases vs. 8 cases) were both 100% (95% CI, 83.7 to 100 and 42.6 to 100, respectively). Serious adverse events occurred in 111 of 5044 children in the vaccine group (2.2%) and 131 of 5033 children in the placebo group (2.6%). In the immunogenicity subgroup (1291 children), an anti-EV71 immune response was elicited by the two-dose vaccine series in 98.8% of participants at day 56. An anti-EV71 neutralizing antibody titer of 1:16 was associated with protection against EV71-associated hand, foot, and mouth disease or herpangina. CONCLUSIONS: The EV71 vaccine provided protection against EV71-associated hand, foot, and mouth disease or herpangina in infants and young children. (Funded by Sinovac Biotech; ClinicalTrials.gov number, NCT01507857.).

Comparable therapeutic potential of umbilical cord mesenchymal stem cells in collagen-induced arthritis to TNF inhibitor or anti-CD20 treatment.

OBJECTIVES: The effects of mesenchymal stem cell (MSC) transplantation on established collagen-induced arthritis (CIA) were evaluated and compared to biologic therapies. METHODS: CIA was induced with the immunisation of type II collagen (CII) in DBA/1 mice. Human umbilical cord MSC, anti-TNF antibody, rhTNFR:Fc fusion protein and anti-CD20 antibody were respectively injected intraperitoneally into CIA mice. Arthritis severity was assessed by clinical and histological scoring. The frequencies of lymphocytes in spleen were analysed, and serum concentrations of cytokines and autoantibody to CII were also measured. The ability of MSC to regulate the balance of T helper cell subsets in CII stimulated CIA CD4+ T cells was assessed in vitro. RESULTS: MSC treatment significantly decreased the severity of arthritis, which was comparable to biologic treatments. All the treatments down-regulated Th1 subset. Except anti-CD20 all the treatments decreased Th17 subset. MSC treatment enhanced the proportion of regulatory T (Treg) cells and inhibited the generation of T follicular helper (Tfh) cells. The decrease in autoantibody level was detectable in all the treated groups. In vitro MSC induced Foxp3+ T cells, and down-regulated IL-17+, IFNγ+ T cells and pathogenic IL-17+IFNγ+ or IL-17+Foxp3+ T cells. MSC also reduced the secretion of IL-1ß, IL-6, IL-17 and TNF-α among collagen-specific T cells. CONCLUSIONS: MSC show comparable effects to the known biologic treatments and correct immune imbalance in CIA. MSC might provide a promising approach for the treatment of rheumatoid arthritis.

[Molecular mechanisms underlying DEL phenotype among ethnic Han individuals from Jiangsu].

OBJECTIVE: To explore the molecular mechanism underlying the DEL phenotype among RhD negative ethnic Han individuals from Jiangsu, China. METHODS: The DEL phenotype was determined by an adsorption elution test among 57 RhD negative blood donors. The Rh C, c, E, and e phenotypes were detected by a tube method. PCR with sequence-specific primering (PCR-SSP) assay was used to determine the RHCE genotypes. The RHD gene of the DEL individuals were amplified with polymerase chain reaction and subjected to Sanger sequencing analysis. RESULTS: Among the 57 RhD negative donors, 10 (17.54%) were determined as having the DEL phenotype. The major RhCE phenotypes for DEL and RhD negative cases were RhCcee (80.0%) and Rhccee (61.7%), respectively. All RHD gene sequences of the 10 individuals have harbored a G>A mutation at position 1227 of exon 9. CONCLUSION: A proportion of RhD negative individuals determined by routine serological method are actually DEL with RHD gene mutations. RHD *1227A is the most prevalent DEL genotype among ethnic Han Chinese from Jiangsu. Further research on the phenotype and underlying molecular mechanism of DEL is important for blood transfusion.

Mesenchymal stem cells promote CD206 expression and phagocytic activity of macrophages through IL-6 in systemic lupus erythematosus.

Human umbilical cord-derived mesenchymal stem cells (UCMSCs) show therapeutic effects on systemic lupus erythematosus (SLE). Deficiency in functional polarization and phagocytosis in macrophages has been suggested in the pathogenesis of SLE. We found that macrophages from B6.MRL-Fas(lpr) mice exhibited lower level of CD206, the marker for alternatively activated macrophage (AAM, also called M2). In addition, the phagocytic activity of B6.MRL-Fas(lpr) macrophages was also decreased. UCMSC transplantation improved the proportion of CD206(+) macrophages and their phagocytic activity in B6.MRL-Fas(lpr) mice. Importantly, macrophages from SLE patients also showed lower expression of CD206 and reduced phagocytic activity, which were corrected by being co-cultured with UCMSCs in vitro and in SLE patients receiving UCMSC transplantation. Mechanistically, we demonstrated that IL-6 was required for the up-regulation of CD206 expression and phagocytic activity of UCMSC-treated SLE macrophages. Our results indicate that UCMSCs alleviate SLE through promoting CD206 expression and phagocytic activity of macrophages in an IL-6 dependent manner.

MiR-223 downregulation promotes glomerular endothelial cell activation by upregulating importin α4 and α5 in IgA nephropathy.

Glomerular endothelial cells (GEnCs) contribute to renal injuries in IgA nephropathy (IgAN). Here we profiled microRNAs (miRNAs) in GEnCs treated with conditioned medium from human mesangial cells in vitro. Levels of miR-223 in GEnCs decreased after incubation with the medium prepared with pIgA from patients with glomerular endothelial proliferation and were also decreased in the glomerular tissues of patients with glomerular endothelial proliferation. Mesangial-derived IL-6 caused miR-223 levels to decrease. The addition of exogenous miR-223 inhibited cell proliferation, ICAM-1 expression, and monocyte adhesion. The NF-κB and STAT3 signaling pathways collaborate during the activation process. MiR-223 mimics inhibited the nuclear localization and DNA binding of p65 and STAT3 but had no effect on the expression of upstream molecules. Instead, importin α4 and α5 (multipurpose nuclear transport receptors), validated as targets of miR-223, were responsible for the nuclear transport of p65 and STAT3. Importin α4 and α5 siRNA inhibited the nuclear localization of p65 and STAT3 and prevented cell proliferation and monocyte adhesion. The level of miR-223 in circulating endothelial cells was decreased and related to the clinical and pathological parameters. Thus, miR-223 downregulation promotes glomerular endothelial cell activation by upregulating importin α4 and α5 in IgAN. Monitoring the level of miR-223 in circulating endothelial cells may provide a noninvasive method for evaluating the severity of IgAN.

Hypoxia inducible factor-1 alpha promotes mesangial cell proliferation in lupus nephritis.

BACKGROUND: Evidence has accumulated that hypoxia plays a significant role in the pathogenesis and progression of both acute renal injury and chronic renal disease. However, little was known about the effects of hypoxia on lupus nephritis (LN). In the current study, we investigated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in LN. METHODS: Renal biopsies from 22 LN patients and 20 patients with renal carcinoma were obtained. In situ HIF-1α expression was examined by immunohistochemical staining, and the relationship between HIF-1α and clinical/pathological features was analyzed. HIF-1α expression in kidney from both MRL/lpr and C57BL/6 mice was detected by immunohistochemical technology. Dimethyloxaloylglycine (DMOG), an inhibitor of HIF-degrading prolylhydroxylases, was utilized to prevent HIF-1α degradation in mouse mesangial cells (MCs). After DMOG treatment, the proliferation and apoptosis rates of mouse MCs were determined. RESULTS: LN patients showed larger amounts of HIF-1α in both glomerular and tubulointerstitial areas. The levels of intraglomerular HIF-1α were closely associated with renal pathology activity index and clinical manifestations in LN patients. In MRL/lpr mice, intraglomerular HIF-1α-positive cells were also significantly increased. Interestingly, the levels of HIF-1α positively correlated with cell density in glomerulus in both LN patients and MRL/lpr mice. Upon treatment with DMOG, the proliferation of MCs was upregulated, and apoptosis was downregulated. CONCLUSION: HIF-1α is highly expressed in both glomerular and tubulointerstitial tissues in LN, especially in proliferative LN. HIF-1α may promote MCs growth through the induction of proliferation and inhibition of apoptosis, and hence plays an important role in the pathogenesis of LN.

Molecular genetic analysis and structure model of a rare B(A)02 subgroup of the ABO blood group system.

BACKGROUND: Serological analysis of ABO blood group has been widely applied in transfusion medicine. However, ABO subgroups with different expression of blood group antigens sometimes cannot be determined by serological methods. Therefore, genotyping is useful to understand the variant ABO phenotypes. MATERIAL AND METHODS: Exon 6 to exon 7 and adjacent introns of the ABO gene from a donor with ABO typing discrepancy were amplified and sequenced. Cloning sequencing was also performed to identify the allele. To explore the effect of mutation, three dimensional model of mutant p.Pro234Ala was built and optimized. RESULTS: The variant B (c. 700C > G) allele expressed an AweakB phenotype with anti-A in his serum with a ABO*B(A)02/O02 heterozygote genotype. Cloning sequencing confirmed that the c.700C > G single nucleotide polymorphism was associated with a B101 allele. Three dimensional molecular modeling suggested that p.Pro234Ala might affect the conformation of His233, Met266 and Ala268, which were known as critical residues for donor recognition. CONCLUSION: ABO genotyping is needed for correct identification subgroups to improve accuracy evaluation of blood typing and increase the safety of blood transfusion. Alteration of DNA sequence in the ABO gene resulted in amino acid substitutions and led to a weak or missing expression of ABO antigens.

A case of dispermic chimerism with normal phenotype identified during ABO blood grouping.

BACKGROUND: Human chimerism with normal phenotype derived from the fusion of two different zygotes is a rare phenomenon. We describe a case of a phenotypically normal 17-year-old diagnosed with dispermic chimerism during routine ABO blood grouping. METHODS: ABO grouping, ABO genotyping, karyotyping, human leukocyte antigen (HLA) typing, and short tandem repeat (STR) analysis were performed. RESULTS: Forward typing with anti-A and anti-B sera resulted in mixed-field agglutination of red blood cells. The mother and father were blood group O and AB, respectively. The proposita had O1, A201 and B alleles in the ABO locus; O1 was a maternal allele, while A201 and B were the paternal alleles. The proposita karyotype was 46,XX/46,XY. HLA typing revealed that the proposita had three alleles (46, 51, 54) at the HLA-B locus, with the additional allele of paternal origin. STR analysis identified three alleles for five of the 15 markers (D2S1338, TPOX, D8S1179, D19S433, and D21S11) analyzed in the proposita's blood- and skin fibroblast-derived DNA. The additional alleles of TPOX, D8S1179, and D21S11 were of paternal origin. CONCLUSIONS: The genetic findings suggest that this proposita was produced by dispermic fertilization of two identical haploid ova formed by parthenogenetic activation.