Xin Xie

Increase ICAM-1 and LFA-1 expression by cerebrospinal fluid of subarachnoid hemorrhage patients: Involvement of TNF-α

Subarachnoid hemorrhage (SAH) is a frequent occurrence in cerebrovascular accidents, and inflammation occurs in the subarachnoid space after SAH. Arachnoid cells have the capability to present antigens and active T-lymphocytes after stimulation by cerebrospinal fluid (CSF). However, the effect of CSF on T-lymphocytes and arachnoid cell adhesion was not clearly understood. In this study, we used ELISA to detected tumor necrosis factor-α (TNF-α) content in CSF of SAH patients. CSF or recombinant TNF-α were applied on arachnoid cells and T-lymphoctes, and RT-PCR and western blotting were performed to determine the expression of intercellular adhesion molecule-1 (ICAM-1) in arachnoid cells and Lymphocyte Function-Associated Antigen-1 (LFA-1) in T-lymphocytes, respectively. Meanwhile, the Matrix Metal Proteinase-9 (MMP-9) expression in these cells was also determined. We found that the content of TNF-α in the CSF was significantly increased in the CSF of SAH patients (from 22 ± 8 pg/mL of healthy people to 436-450 pg/mL of SAH patients). Treatement with CSF could increase the expression of ICAM-1 in arachnoid cells and that of LFA-1 in T-lymphocytes, mainly through the increased levels of TNF-α. We also found that the co-culture of arachnoid cells and T-lymphocytes increased the expression of MMP-9 in both cells through the interaction of ICAM-1 of and LFA-1. All of these results suggested that arachnoid cells are involved in the T-lymphocytes invasion in the subarachnoid space after SAH.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

Induction of Matrix Metalloproteinase-3 (MMP-3) Expression in the Microglia by Lipopolysaccharide (LPS) via Upregulation of Glycoprotein Nonmetastatic Melanoma B (GPNMB) Expression

Substantial evidence suggests that inflammation is an important contributor to many neurodegenerative disorders. Activated microglial cells play an important role in releasing pro-inflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) for inducing inflammation. Recently, some reports have suggested that glycoprotein nonmetastatic melanoma B (GPNMB) is highly expressed in microglia after LPS treatment. However, the role of GPNMB in activated microglia is not clearly understood. In this study, we used RT-PCR and Western blotting to detect GPNMB and matrix metalloproteinase-3 (MMP-3) expressions in activated microglia. GPNMB small interfering RNA (siRNA) or MMP-3 inhibitor was applied on microglial BV2 cells, and ELISA was performed to measure the expressions of TNF-α and IL-1β in BV2 cells. Levels of iNOS and NO in BV2 cells were also determined. We found that the levels of GPNMB and MMP-3 were significantly increased in BV2 cells after LPS treatment. Moreover, we found that GPNMB significantly upregulated the expression of MMP-3 in BV2 cells, and high expression of MMP-3 was dependent on the level of GPNMB. Inhibition of GPNMB or MMP-3 expression by GPNMB siRNA or MMP-3 inhibitor dramatically suppressed the expressions of TNF-α, IL-1β, iNOS, and NO in activated microglia. All of these results suggest that GPNMB is involved in the inflammatory responses of microglia.

The Four Reprogramming Factors and Embryonic Development in Mice

The transcription factors (Oct4, Sox2, c-Myc, and Klf4) play an important role in the generation of induced pluripotent stem cells. These factors are expressed in metaphase II oocytes and embryonic stem cells (ESCs). The mechanisms responsible for the reprogramming of ooplasm during nuclear transfer are expected to be associated with the four factors. Here, we show that different paternal genetic backgrounds are able to influence the in vitro development of parthenogenetic and cloned embryos. Using real- time polymerase chain reaction (PCR) we found that the expression level of Oct4 in oocytes was less than that of ESCs, whereas oocytes from KMu2009xu2009C3H females showed the highest expression level of Sox2 than the other strains tested or in G1 ESCs. c-Myc mRNA levels in oocytes from KM mice were greater than those found in ESCs or oocytes of KM xu2009C3H mice. These data demonstrate that the expression of the four transcription factors was different among the oocytes, which may be a contributing factor for the different efficiencies of parthenogenesis and the development of cloned embryos in vitro.

Expression of FGF4 mRNA is mediated by mating behaviours in mice

In mammals, breeding is preceded by species‐specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16u2009h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5u2009µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c‐Myc) in oocytes, apoptosis‐related genes (i.e. Bax, Bcl2 and c‐Myc) in cumulus cells and pluripotency‐related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (pu2009<u20090.05). Embryo development was primarily blocked at the eight‐cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25u2009ng/ml FGF4 to the medium at the four‐cell stage (pu2009>u20090.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression. Copyright

Development and evaluation of a sandwich ELISA method for the detection of human CD306.

CD306, also known as soluble leukocyte-associated immunoglobulin-like receptor-2 (LAIR-2), is a member of an immunoglobulin superfamily with the shared characteristic of an immunoglobulin-like C2-type domain. CD306 is speculated to be secretory and has 84% similarity with the extracellular domain of CD305, which binds to the same ligands as CD306. However, data on its distribution are absent due to the lack of an efficient method to detect it. In this study, we successfully cloned the cDNA of CD306 from the peripheral blood mononuclear cells (PBMCs) of patients with hemorrhagic fever with renal syndrome. The fusion proteins were expressed and purified, and three strains of monoclonal antibodies (mAbs) against CD306 were prepared and characterized. The sandwich ELISA for detecting CD306 was established and optimized with sensitivity up to 15 pg/ml, and the assay showed high specificity for the detection of CD306. With this method, a right skewed frequency distribution of CD306 in the sera of healthy subjects was determined. The concentrations of CD306 in sera and urine were detected in patients with different diseases. Aberrantly high levels of CD306 were found in the sera of pregnant women and patients with inflammation and rheumatic heart disease and in the urine of pregnant women. Meanwhile, there was a positive correlation between CD306 and soluble CD305 expression and secretion levels in sera and urine samples from patients, and both proteins inhibited CD305-mediated immunosuppressive functions. Our results demonstrate that CD306 represents a potentially useful predictor for disease diagnosis and that the method developed has potential for clinical application.

A Non-Stimulatory Monoclonal Antibody Against the Inhibitory Immunoreceptor LAIR-1

The human leukocyte-associated Ig-like receptor (LAIR) family contains two members: LAIR-1 (CD305) and LAIR-2 (CD306). Among them, LAIR-1 is a transmembrane glycoprotein bearing two intracellular immunoreceptor tyrosine-based inhibition motifs (ITIM) and LAIR-2 is soluble. Both molecules bind collagen and LAIR-2 has higher affinity than LAIR-1. LAIR-1 can mediate strong inhibitory signal but the functions of leukocytes expressing LAIR-1 are unclear because of the absence of an effective method to isolate them with resting status. In this study, we generated a monoclonal antibody (MAb) by immunizing BALB/c mice with the recombinant LAIR-2-GST fusion protein, which we termed 3G4. The subclass of 3G4 was identified as IgG1. Specificity analysis by Western blotting demonstrated 3G4 could react with both LAIR-1 and LAIR-2. Unlike another LAIR-1-specific MAb (9.1C3), 3G4 did not inhibit the lysis of target cells P815 by NK cells in a redirected cytotoxicity assay. Preincubation of LAIR-1-transfected K562 cells with 3G4 mildly prevented the binding of LAIR-1 to collagens I and III in a dose-dependent manner. Taken together, the novel MAb 3G4 provides a useful tool to isolate LAIR-1-positive cells without changing their resting state for further application.

H1N1 influenza virus epitopes classified by monoclonal antibodies

Epitopes serve an important role in influenza infection. It may be useful to screen universal influenza virus vaccines, analyzing the epitopes of multiple subtypes of the hemagglutinin (HA) protein. A total of 40 monoclonal antibodies (mAbs) previously obtained from flu virus HA antigens (development and characterization of 40 mAbs generated using H1N1 influenza virus split vaccines were previously published) were used to detect and classify mAbs into distinct flu virus sub-categories using the ELISA method. Following this, the common continuous amino acid sequences were identified by multiple sequence alignment analysis with the GenBank database and DNAMAN software, for use in predicting the epitopes of the HA protein. Synthesized peptides of these common sequences were prepared, and used to verify and determine the predicted linear epitopes through localization and distribution analyses. With these methods, nine HA linear epitopes distributed among different strains of influenza virus were identified, which included three from influenza A, four from 2009 H1N1 and seasonal influenza, and two from H1. The present study showed that considering a combination of the antigen-antibody reaction specificity, variation in the influenza virus HA protein and linear epitopes may present a useful approach for designing effective multi-epitope vaccines. Furthermore, the study aimed to clarify the cause and pathogenic mechanism of influenza virus HA-induced flu, and presents a novel idea for identifying the epitopes of other pathogenic microorganisms.

Silencing of PMEL attenuates melanization via activating lysosomes and degradation of tyrosinase by lysosomes

The functionally specialized melanosome is a membrane-enclosed lysosome-related organelle, which coexists with lysosomes in melanocytes. Pre-melanosomal protein (PMEL) initiates pre-melanosome morphogenesis and is the only cell-specific pigment protein required for the formation of fibrils on which melanin is deposited in melanosomes. But the effects of PMEL on melanin synthesis and lysosome activity remain unclear. In the study, PMEL was silenced in human epidermal melanocytes by siRNA transfection. Compared to the non-treated group, melanin content in the transfected cells was greatly reduced. Real-time qPCR, Western blotting and immunofluorescence analyses all showed that PMEL-siRNA transfection reduced protein level of tyrosinase, a key enzyme in melanogenesis, but it does not affect tyrosinase gene expression. Moreover, in the absence of PMEL, lysosomal activation was manifested by an increase in the number of lysosomes and activity of hydrolysis enzymes. The lysosome inhibitors restored tyrosinase expression after PMEL silencing, indicating that tyrosinase was degradated by lysosomes. The data collectively showed that silencing of PMEL suppressed melanization through activating lysosomes and degradation of tyrosinase by lysosomes. Our findings provide novel insight into the interaction between the melanosome and its related organelle, the lysosome, supplying a new idea for the pathogenesis and clinical treatment of pigmented diseases.

Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16

BackgroundSafe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3′-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination.ResultsThe fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65xa0°C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2u2009×u20092xa0mm bleeding wound in the mouse liver after 27.21u2009±u20091.92xa0s, 18.65u2009±u20091.97xa0s and 15.85u2009±u20091.21xa0s, respectively. The hemostasis time was 21.23u2009±u20091.84xa0s for rat-tail collagen and 14.44u2009±u20091.33xa0s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64u2009±u20091.34xa0s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze.ConclusionsThese results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.