Therapeutic Role for TSP-2 Antibody in a Murine Asthma Model.

BACKGROUND: Specific immunotherapy, including agonists for Toll-like receptor 2 (TLR2), have been shown to protect from allergies and to have a high immunomodulatory capacity. METHODS: A new antibody, TSP-2, reactive against an epitope of the extracellular domain of TLR2, was identified. The effect of the antibody on dendritic cells was assessed by immunohistochemistry, Western blot, and flow cytometric analysis. The effect of TSP-2 in a murine asthma model induced with ovalbumin (OVA) was assessed. The model is a form of airway hyperresponsiveness (AHR) and was analyzed by whole-body plethysmography, the measurement of Th1/Th2 cytokines in bronchial alveolar lavage fluid (BALF) and serum by ELISA, and the CCK-8 assay for lymphocyte proliferation. The effect of TSP-2 on the maturation of bone marrow-derived dendritic cells (BMDCs) was assessed by flow cytometric analysis. RESULTS: TSP-2 promoted the maturation of dendritic cells and the proliferation of lymphocyte in vitro and in vivo. The effect of TSP-2 on T helper 1 (Th1)/Th2 cytokine secretion was slightly more powerful than that of Pam3CSK4. TSP-2 antibody reduced AHR and OVA-specific IgE levels in allergic asthma. TSP-2 antibody also reduced lung inflammation and decreased leukocyte numbers in an OVA-sensitized and challenged asthma model. TSP-2 antibody increased OVA-stimulated I-A, CD80, CD86, and MHC-II levels on BMDCs. CONCLUSIONS: This study identifies a novel therapeutic strategy for AHR, which uses antibodies reactive against TLR2. It also provides theoretical evidence for the control of allergic asthma by targeting TLR2.

S-Nitrosylating protein disulphide isomerase mediates α-synuclein aggregation caused by methamphetamine exposure in PC12 cells.

Methamphetamine (METH) belongs to Amphetamine-type stimulants, METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). However, there are still no effective treatments to METH-induced neurodegeneration because its mechanism remains unknown. In order to investigate METH's neurotoxic mechanism, we established an in vitro PD pathology model by exposing PC12 cells to METH. We found the expression of nitric oxide synthase (NOS), nitric oxide (NO) and α-synuclein (α-syn) was significantly increased after METH treatment for 24h, in addition, the aggregattion of α-syn and the S-nitrosylation of protein disulphideisomerase(PDI) were also obviously enhanced. When we exposed PC12 cells to the NOS inhibitor N-nitro-L-arginine(L-NNA) with METH together, the L-NNA obviously inhibited these changes induced by METH. While when we exposed PC12 cells to the precursor of NO L-Arginine together with METH, the L-Arginine resulted in the opposite effect compared to L-NNA. And when we knocked down the PDI gene, the L-NNA did not have this effect. Therefore, PDI plays a significant role in neurological disorders related to α-syn aggregation, and it suggests that PDI could be as a potential target to prevent METH-induced neurodegeneration.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on nuclear factor-kappa B and cytokine expression in the intestine of septic mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the expression of nuclear factor-kappa B (NF-κB) and cytokines in the intestinal tissue of septic mice. METHODS: Male BALB/c mice were randomly divided into 4 groups, namely the sham-operated group, model group, TSP-2 treatment group and rabbit IgG treatment group. Sepsis was induced by cecal ligation and puncture (CLP), and at 6, 12 or 24 h after the operation, the ileal tissues were harvested from the mice for HE staining. NF-κB expression was detected with immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expressions were detected with qRT-PCR and their protein expressions by ELISA. RESULTS: The NF-κB expression in the intestinal tissue significantly increased in the model group as compared with that in the sham- operated group, and decreased after TSP-2 treatment. The model group also showed significantly increased expression levels of TNF-α and IL-6 mRNA and protein in the intestinal tissue (P<0.05), which were lowered by TSP-2 (P<0.05) but not by rabbit IgG treatment (P>0.05). CONCLUSION: The TSP-2 antibody can protect the intestine and delay the development of sepsis by inhibiting NF-κB activation and down-regulating TNF-α and IL-6 expressions in mice.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis. METHODS: In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80. CONCLUSION: TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.