Osteopontin accelerates chondrocyte proliferation in osteoarthritis rats through the NF-κb signaling pathway.

OBJECTIVE: To explore the influence of osteopontin (OPN) on the chondrocyte proliferation in osteoarthritis (OA) rats. MATERIALS AND METHODS: A total of 30 Sprague-Dawley rats were divided in the control group (n=10), model group (n=10), and OPN knockdown group (n=10). No treatment was performed in the control group, while OA rats were administrated with control adenovirus in the model group and OPN knockdown adenovirus in the OPN knockdown group. After sampling, the degree of OA was evaluated via hematoxylin-eosin (HE) staining, and the mRNA expression of OPN was detected. Moreover, the expression of the proliferation-associated protein cyclin D1 was detected using immunohistochemistry. The chondrocytes were isolated from the normal rats, cultured, and transfected with OPN overexpression vector or si-OPN. Methyl thiazolyl tetrazolium (MTT) assay was adopted to determine the proliferative capacity of chondrocytes, and Caspase3 activity was measured to evaluate the changes in the apoptotic capacity of chondrocytes. Meanwhile, Western blotting was performed to verify the influences of OPN on the pathways on chondrocyte proliferation. RESULTS: After the OA model was established, the expression level of OPN significantly increased. According to HE staining results, OPN knockdown effectively inhibited the onset of OA. Compared with that in the control group, the expression level of cyclin D1 in the model group was raised. However, upregulated cyclin D1 in OA rats was repressed in OPN knockdown group. OPN overexpression promoted the proliferation of chondrocytes, but suppressed their apoptosis, while OPN knockdown had the opposite effects. Besides, OPN overexpression upregulated nuclear factor-κB (NF-κB), and NF-κB knockdown eliminated the regulatory effects of OPN on proliferation and apoptosis of chondrocytes. CONCLUSIONS: OPN promotes the expression of NF-κB signals to accelerate chondrocyte proliferation, thereby inducing OA in rats.

MicroRNA-378 attenuates myocardial fibrosis by inhibiting MAPK/ERK pathway.

OBJECTIVE: To elucidate the role of microRNA-378-containing microvesicles (MVs) in the process of myocardial fibrosis and its underlying mechanism. MATERIALS AND METHODS: In vivo chronic myocardial fibrosis (MF) model in rats was established by aortic coarctation method. MicroRNA-378 mimic or inhibitor was injected into the rat tail vein at day 3 after the aortic coarctation. Two weeks later, rats were sacrificed for collecting myocardium. MVs were isolated from rat cardiomyocytes and further verified by detecting the expression level of its marker CD63. Expression levels of fibrosis-related indicators and microRNA-378 in MVs were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. After induction with transforming growth factor-ß1 (TGF-ß1) in primary rat cardiomyocytes for different time points, expression levels of fibrosis-related indicators and microRNA-378 were also accessed. Changes in mitogen-activated protein kinase (MAPK) pathway were observed during the process of MF by qRT-PCR and Western blot. RESULTS: Expression levels of microRNA-378-containing MVs decreased, and the MAPK pathway was activated during the process of MF, which further aggravated MF. CONCLUSIONS: MicroRNA-378-containing MVs alleviate myocardial fibrosis through inhibiting the phosphorylation of MAPK.

MiR-98-5p regulates myocardial differentiation of mesenchymal stem cells by targeting TBX5.

OBJECTIVE: This study aims to investigate whether miR-98-5p can participate in the myocardial differentiation of bone marrow mesenchymal stem cells (MSCs) by regulating TBX5. MATERIALS AND METHODS: In this study, we first identified the MSCs that were isolated from rat bone marrow samples. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect mRNA expressions of cardiac-related genes, including brain natriuretic peptide (BNP), α-actinin, and Islet-1. The binding site of miR-98-5p and TBX5 was detected by dual-luciferase report gene assay. In addition, co-transfection of miR-98-5p mimics and TBX5 overexpression plasmids was conducted to assess whether miR-98-5p could regulate myocardial differentiation by targeting TBX5. RESULTS: Overexpression of TBX5 or knockdown of miR-98-5p promoted myocardial differentiation of BMSCs. The mRNA expressions of α-actinin and Islet-1 were significantly increased after the miR-98-5p knockdown. Dual-luciferase report gene assay showed that miR-98-5p could bind to TBX5, which was further verified by qRT-PCR. Additionally, TBX5 overexpression reversed the inhibitory effect of miR-98-5p on regulating the mRNA expressions of α-actinin and Islet-1. CONCLUSIONS: MiR-98-5p can inhibit the differentiation of rat MSCs into cardiomyocytes through targeting TBX5.

Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Shared epitopes of the HLA-DR10 molecule recognized by murine and human mAbs.

In order to produce mAbs directed specifically against HLA-DR10 molecule, transfected mouse L cells, expressing the DRB1*1001 allele, were used to immunize C3H mice over a period of 4 weeks. Two mAbs, 2C12 and 4B6, derived from this fusion were found to recognize, with different affinity, polymorphic epitopes of DR10 that are shared with DR1, 3, 7, and 9. These mAbs were screened on a large panel of homozygous B lymphoblastoid cell lines using microlymphocytotoxicity and the results were confirmed by flow cytometry. The reactive pattern of 2C12 and 4B6 was compared to that of MP10 human mAb also recognizing the DR10 specificity in addition to DR1, 2 and 9. Based on serologic specificity and cellular absorption experiments, we conclude that the epitopes the murine and human mAbs respectively recognize on the DR10 molecule, are probably different.

[The establishment of human anti-tetanus toxoid hybridomas with in vitro immunized human tonsil cells].

Human tonsil cells in vitro immunized with tetanus toxoid were fused with human-mouse heteromyeloma line RF to generate human-mouse hybridomas. Hybridoma 891112-50 was cloned and 2 subclones (891112-50-3 and -4) were obtained. The secreted antibodies from the subclones were antigen specific, since they did not cross react with three irrelevant antigens (OVA, TCS and F gamma G). The hybridomas were quite stable. After 13 passages in tissue culture flasks, they still retained their antibody secreting ability. From flow cytometry analysis the subclone 50-3 was more stable than the subclone 50-4. The human immunoglobulin contained in supernatant collected during regular passages was equivalent to 69.6 micrograms/ml.

Production and characterization of murine monoclonal antibodies recognizing HLA-DQ polymorphisms obtained by immunizing mice with transfected L cells.

Two polymorphic anti-HLA-DQB1 mAbs, TM 902 and TM 903, have been produced by immunizing F1 mice (Balb/C x C3H) with HLA-DQ-transfected mouse L cells. Cytotoxic analysis on a panel of HLA-typed cell lines has shown that TM 902 reacts with all the DQB1* alleles except DQB1*0501, *0502, and *0503, and DQB1*0601, *0602, *0603, and *0604, whereas TM 903 reacts with the DQB1*0501, *0502, and *0503, DQB1*0601, *0602, *0603, and *0604, and DQB1*0401 and *0402 alleles. The same reactivity pattern has been confirmed by cytofluorimetric analysis. Indirect immunofluorescence with various class-II-transfected cell lines showed no binding of both mAbs to the DR or DP products, suggesting their reactivity to the DQ products. The use of transfectants expressing HLA-DR/DQ heterodimers demonstrates that TM902 and TM903 mAbs are both specific for the DQ-beta chain. Comparison of the amino acid sequences of the DQ-beta chain suggests the involvement of residues 84-90 (QLELRTT) in the formation of TM902 epitope and of residues 54-55 (GR) in the formation of TM903 epitope.

[Cellular aspects of in vitro induction of antibody responses of human cells].

An in vitro system for induction of antibody responses of human cells has been established in our lab. B cell enriched fractions from excised human tonsils or trauma spleen were cultured for 7-14 days with tetanus toxoid or HBsAg vaccine with or without human T cell conditioned medium (C. M.) or a mixture of low concentrations of PWM and LPS (MTG). Positive antibody responses could be detected in cultures. Cells taken from different culture periods were subjected to FACS analysis in order to expound cellular changes during antibody induction periods so as to improve the in vitro antibody induction system. The results were described as follows: 1. Variations in total percentages of T cells during culturing periods seemed to be related its initial percentages. Cells with bigger initial percentages tended to decrease first and finally maintained at about 30%. While cells with smaller initial percentages tended to increase and finally also maintained at 30%. 2. CD4+ Th cells and CD8+ Ts cells from tonsils and spleen behaved somewhat differently. In tonsil cell cultures the percentages of CD4+ cells were often bigger than the percentages of CD8+ cells throughout the culture period. However, the inverted proportions of CD4+/CD8+ were shown in spleen cell cultures, especially in the culture with C. M. The possible relationships between the variations in CD4+/CD8+ proportions described as above and the intensities of antibody responses were discussed. Additionally, adding 1-Leucine-Methyl Ester showed no effects either on CD8+ or CD4+ cell percentages. 3. B cell (SIg+) percentages in both tonsil and spleen cultures were quite stable throughout the culture period, about 60% of total cells. CD19, a marker of B cell, was only present in part of the cultured SIg+ cells. The significance of the variations in CD19+, SIg+ cells was unclear. CD5+ B cells were known as cells secreting autoantibodies. Our results showed that these cells consistently maintained a relatively low percentage in the whole antibody induction period. 4. The reasonableness standard we used for "gating" in FACS analysis was discussed.