Aging-associated changes in CD47 arrangement and interaction with thrombospondin-1 on red blood cells visualized by super-resolution imaging.

CD47 serves as a ligand for signaling regulatory protein α (SIRPα) and as a receptor for thrombospondin-1 (TSP-1). Although CD47, TSP-1, and SIRPα are thought to be involved in the clearance of aged red blood cells (RBCs), aging-associated changes in the expression and interaction of these molecules on RBCs have been elusive. Using direct stochastic optical reconstruction microscopy (dSTORM)-based imaging and quantitative analysis, we can report that CD47 molecules on young RBCs reside as nanoclusters with little binding to TSP-1, suggesting a minimal role for TSP-1/CD47 signaling in normal RBCs. On aged RBCs, CD47 molecules decreased in number but formed bigger and denser clusters, with increased ability to bind TSP-1. Exposure of aged RBCs to TSP-1 resulted in a further increase in the size of CD47 clusters via a lipid raft-dependent mechanism. Furthermore, CD47 cluster formation was dramatically inhibited on thbs1-/- mouse RBCs and associated with a significantly prolonged RBC lifespan. These results indicate that the strength of CD47 binding to its ligand TSP-1 is predominantly determined by the distribution pattern and not the amount of CD47 molecules on RBCs, and offer direct evidence for the role of TSP-1 in phagocytosis of aged RBCs. This study provides clear nanoscale pictures of aging-associated changes in CD47 distribution and TSP-1/CD47 interaction on the cell surface, and insights into the molecular basis for how these molecules coordinate to remove aged RBCs.

Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

NK cells are important in regulating hepatic fibrosis via their cytotoxic killing of hepatic stellate cells (HSCs). NK cells are activated by both cytokines such as IL-12 and IL-18, and innate immune stimuli such as ligation of TLRs. The secretion of IL-18 depends upon activation of the inflammasome, whereas TLRs are stimulated by microbial products. In the case of NK cells, IL-18 acts synergistically with stimulation of TLR3 to cause cell activation and cytotoxic function. In the present study, we activated NK cells to kill HSCs via IL-18 and TLR3 ligand stimulation, and dissected the signaling pathways or molecules critical for such activation or killing. We find that such activation depends on signaling via the p38/PI3K/AKT pathway, and that the activated NK cells mediate HSC death in a TRAIL-involved mechanism. As liver fibrosis is a major global health problem with no good solution, these results emphasize that the p38/PI3K/AKT pathway in NK cells may be a novel drug target to promote fibrosis regression.

Inhibition of pregnancy-associated granulocytic myeloid-derived suppressor cell expansion and arginase-1 production in preeclampsia.

Myeloid-derived suppressor cells (MDSCs) expand in maternal peripheral blood and cord blood during normal pregnancy to maintain maternal-fetal tolerance. Here we investigated the expansion and function of MDSCs in preeclampsia (PE) patients. Maternal peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were sampled from healthy pregnant women and PE patients, and analyzed for the frequencies and phenotypes of MDSCs and T cells. Serum levels of key human MDSC effector enzymes were measured using appropriate detection kits. Peripheral blood samples of healthy non-pregnant women were used as controls. We found that normal pregnancy is associated with a significant increase of immunosuppressive MDSCs and regulatory T (Treg) cells. There was no significant difference in the frequency of Treg cells between normal pregnancies and PE patients, but the pregnancy-associated increase of granulocytic MDSCs (G-MDSCs), but not monocytic MDSCs (M-MDSCs), in both PBMCs and CBMCs was markedly inhibited in PE patients. Furthermore, serum levels of Arg-1, an important effector molecule for G-MDSC were significantly reduced in PE patients compared to healthy pregnant women. In conclusion, the lack of G-MDSC expansion is a most notable feature of PE-associated immune-cell alterations, suggesting that restoring G-MDSCs may have the potential to treat PE.

Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging.

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

Studying the mechanism of CD47-SIRPα interactions on red blood cells by single molecule force spectroscopy.

The interaction forces and binding kinetics between SIRPα and CD47 were investigated by single-molecule force spectroscopy (SMFS) on both fresh and experimentally aged human red blood cells (hRBCs). We found that CD47 experienced a conformation change after oxidation, which influenced the interaction force and the position of the energy barrier between SIRPα and CD47. Our results are significant for understanding the mechanism of phagocytosis of red blood cells at the single molecule level.

High resolution human leukocyte antigen class I allele frequencies and HIV-1 infection associations in Chinese Han and Uyghur cohorts.

BACKGROUND: Host immunogenetic factors such as HLA class I polymorphism are important to HIV-1 infection risk and AIDS progression. Previous studies using high-resolution HLA class I profile data of Chinese populations appeared insufficient to provide information for HIV-1 vaccine development and clinical trial design. Here we reported HLA class I association with HIV-1 susceptibility in a Chinese Han and a Chinese Uyghur cohort. METHODOLOGY/PRINCIPAL FINDINGS: Our cohort included 327 Han and 161 Uyghur ethnic individuals. Each cohort included HIV-1 seropositive and HIV-1 seronegative subjects. Four-digit HLA class I typing was performed by sequencing-based typing and high-resolution PCR-sequence specific primer. We compared the HLA class I allele and inferred haplotype frequencies between HIV-1 seropositive and seronegative groups. A neighbor-joining tree between our cohorts and other populations was constructed based on allele frequencies of HLA-A and HLA-B loci. We identified 58 HLA-A, 75 HLA-B, and 32 HLA-Cw distinct alleles from our cohort and no novel alleles. The frequency of HLA-B*5201 and A*0301 was significantly higher in the Han HIV-1 negative group. The frequency of HLA-B*5101 was significantly higher in the Uyghur HIV-1 negative group. We observed statistically significant increases in expectation-maximization (EM) algorithm predicted haplotype frequencies of HLA-A*0201-B*5101 in the Uyghur HIV-1 negative group, and of Cw*0304-B*4001 in the Han HIV-1 negative group. The B62s supertype frequency was found to be significantly higher in the Han HIV-1 negative group than in the Han HIV-1 positive group. CONCLUSIONS: At the four-digit level, several HLA class I alleles and haplotypes were associated with lower HIV-1 susceptibility. Homogeneity of HLA class I and Bw4/Bw6 heterozygosity were not associated with HIV-1 susceptibility in our cohort. These observations contribute to the Chinese HLA database and could prove useful in the development of HIV-1 vaccine candidates.

HIV vaccine candidates generate in vitro T cell response to putative epitopes in Chinese-origin rhesus macaques.

The Indian rhesus macaque is the established animal model for HIV infection and vaccine research. Growing evidence suggests that the more readily available Chinese rhesus macaque may be a more relevant option. As increasing numbers of novel Chinese rhesus MHC alleles are reported, we decided to explore potential HIV vaccine epitopes in this model. We immunized forty Chinese rhesus macaques with three different HIV vaccine candidates either individually or following a prime/boost strategy. We used ELISPOT to measure immune response in vitro to HIV-1 p24C and HIV-1 gp160 peptide libraries. We identified five putative epitopes with associations to HLA-I alleles including HLA*B-2705 and HLA-B*5101 (associated with slow disease progression and low viral set point) and HLA-B*18 (associated with rapid disease progression and high viral set point). This suggests the possible use of Chinese rhesus macaques to model different disease progressions. We also explored the use of fusion proteins as stimulators in ELISPOT assays. While PBMCs from 6 monkeys responded to peptide stimulation, PBMCs from 28 monkeys responded to the anthrax lethal factor fusion proteins LFn p24C and/or LFn gp140C. Our results support the use of Chinese rhesus macaques in HIV vaccine studies.

HIV-1-specific cytotoxic T lymphocyte (CTL) responses against immunodominant optimal epitopes slow the progression of AIDS in China.

To assess the immunodominance patterns of HIV-1-specific cytotoxic T lymphocyte (CTL) responses and the contribution of these responses against the peptides scanning optimal epitopes in chronic infection, we test the HIV-1-specific CTL responses against a panel of 413 overlapping peptides spanning HIV-1 Asian B sequence, including 147 peptides corresponding to optimal clade B epitopes in 49 chronically HIV-1 infected individuals by interferon-gamma Elispot assay. A large variation in the recognition of peptides restricted by the same HLA class I allele is presented. Some epitopes are targeted frequently by individuals while other epitopes restricted by the same allele are rarely recognized in our research. HLA-B35 and HLA-A03 rather than other HLA alleles contribute greatly to total virus-specific CTL responses. Furthermore, there is a significant inverse correlation between the total contribution of HIV-1-specific CTL responses restricted by different HLA alleles to virus-specific immune responses and viral load in the individuals during advanced infection (P=0.002, r=-0.549). The peptides targeted by individuals have significantly lower entropy compared with those not targeted but restricted by the same HLA class I alleles (P<0.05) in 49 individuals infected by HIV-1, especially the advanced infection subgroup (P=0.044). These data demonstrate that the consistent immunodominance patterns of HIV-1-specific CTL responses of Chinese HIV-1 infected individuals and an inverse correlation between the relative contribution of responses restricted by HLA alleles and viral load, which indicates the important protective effect of optimal epitopes against slow disease progression even in advanced infection.

[Studies of promoter methylation status and protein expression of E-cadherin gene in associated progression stages of gastric cancer].

Gastric cancer, like all cancers, is considered to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumour suppressor loss. More recently, the role of epigenetic change as a distinct and crucial mechanism to silence a variety of methylated tissue-specific and imprinted genes has emerged in many cancer types. The study of DNA methylation changes in gastric cancer has now provided additional clues into the pathogenesis of the disease. E-cadherin as a metastases suppressor is mutationally inactivated in both familial and sporadic forms of gastric cancers. Evidence now suggests that the transcriptional silencing of E-cadherin gene by promotor methylation plays a crucial role in the development and progression of gastric malignancies. In order to further analyze the role of E-cadherin gene promotor methylation in gastric carcinogenesis and progression, we performed the studies of promoter methylation status and protein expression of E-cadherin gene in associated progression stages of gastric cancer. DNA were extracted from the paraffin embedded gastric specimens of dysplasia(23 cases), early cancer (20 cases) and advanced cancer (20 cases). Methylation specific PCR and immunohistochemistry were used to analyze the promoter methylation status and the protein expression level of E-cadherin gene. Our results showed that E-cadherin promoter methylation occurred in all stages of gastric precancerous lesion and carcinogenesis, which suggests E-cadherin promotor methylation is an important event during gastric carcinogenesis and progression. The positive rate of E-cadherin promotor methylation in dysplasia, early gastric cancer and advanced gastric cancer was 78.3%, 80% and 90% respectively. There were significant differences between experimental groups and control group(30%), P < 0.05, but no significant differences among experimental groups, P > 0.05. All of advanced gastric cancer examined were completely E-cadherin protein-negative by immunohistochemistry. Fourteen of 20 early gastric cancer were E-cadherin-negative. And 23 dysplasia were all E-cadherin-positive. Thirty-one of 34(91%) of the E-cadherin-negative tumours had promotor methylation. This result indicated the downregulation expression of E-cadherin was associated with promotor methylation in early and advanced gastric cancer (P < 0.01).