[Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms].

Objective: To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms. Methods: OFs from patients with TAO were isolated and cultured in DMEM. Cells were divided into four groups and treated with 0, 250, 500 and 1 000 µg/ml pirfenidone for 24, 48 or 72 hours, respectively. Cell proliferation was detected by tetramethyl azo salt (MTT) assay, and cell viability was determined by trypan blue. Transforming growth factor (TGF) ß1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Type Ⅰ and type Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA). Results: (1) The primary cultured OFs had typical fibroblast spindle-like morphology. (2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P<0.05) with the proliferation rates of pirfenidone treated groups of -15.31%, -24.92%, -48.53% from 250, 500, 1 000 µg/ml after 72 h, respectively, in which the inhibition effect of 1 000 µg/ml pirfenidone was significantly different from the other two treated groups (P<0.05). There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h, 48 h and 72 h (P>0.05). Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250, 500, 1 000 µg/ml at 72 h were 78.37%, 79.21%, 78.24% and 76.28%, respectively. There were no significant differences between each drug treated and the control group (P>0.05). (3) RT-qPCR results showed that the mRNA expression levels of TGFß1 at 250, 500, 1 000 µg/ml pirfenidone treated groups at 72 h were 0.760±0.010, 0.440±0.006, and 0.290±0.002, respectively. Compared with the control group (0.950±0.014), the differences were statistically significant (all P<0.05). Moreover, TGFß1 mRNA expression level in 1 000 µg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P<0.05). The secretion of type Ⅰ collagen (0.633±0.006, 0.527±0.003 and 0.402±0.008) and type Ⅲ collagen (0.511±0.003, 0.439±0.007 and 0.223±0.006) in 250, 500 and 1 000 µg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005, 0.527±0.007, all P<0.05). Type Ⅰ and type Ⅲ collagen secretion in 1 000 µg/ml pirfenidone treated group were significantly lower than those in the other two groups (P<0.05). Conclusions: Pirfenidone inhibits the cell proliferation, TGFß1 expression and collagen secretion of OFs, which may contribute to the anti-fibrotic effect of pirfenidone.

[Effects of ApoE gene polymorphism on the efficacy of atorvastatin in the treatment of hyperlipidemia].

Objective: To study the role of ApoE gene polymorphism on efficacy of atorvastatin in lowering the lipid and its clinical significance. Methods: A total of 962 patients with hypercholesterolemia were selected between January 1 st and December 31 st 2014. The ApoE genepolymorphism in patients with hyperlipidemia was performed by using polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) method in translational medicine center of Huaihe Hospital. Patients with ApoE genotype E3/3 and E3/4 were selected and treated with atorvastatin 10 mg/d for 4 weeks. Before and after treatment, triglycerides (TG) and total cholesterol (TC) was detected by enzyme colorimetry method. High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were performed by Clearance method. Lipoprotein(a) (Lp(a)) was performed by turbidimetric inhibition immunoassay. ApoE gene expression was performed by real-time PCR. Results: In the 6 gene types, the frequencies of E3/4 and E3/3 were 30.6% (294 cases) and 59.1% (569 cases) respectively. After treatment with atorvastatin, the change percent of TC, LDL-C, HDL-C, TG, Lp(a) in E3/4 and E3/3 group were -(23.0±4.7)% vs -(12.0±3.1)% (P<0.001), -(33.0±4.8)% vs -(20.0±3.9)% (P<0.001), (18.0±3.8)% vs (6.0±2.6)% (P<0.001), -(23.0±3.9)% vs -(13.0±2.7)% (P<0.001), -(21.5±4.5)% vs -(20.9±4.0)% (P=0.054), respectively. ApoE gene expression in E3/3 and E3/4 groups were down-regulated in both groups, and the change in E3/3 group was obvious than that of E3/4 group. Conclusion: After treatment with atorvastatin, levels of lipids and ApoE gene expression in ApoE genotype E3/3 patients decreased, which were more evident than E3/4 patients.

Isotype switching increases efficacy of antibody protection against Cryptococcus neoformans infection in mice.

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.

T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice.

Cryptococcus neoformans is an encapsulated fungus that is a major cause of meningitis in patients with AIDS. In immunocompetent mice, administration of IgG1 mAb protects against cryptococcal infection, whereas administration of IgG3 is not protective and can accelerate the infection. In beige mice with impaired natural killer cell function, the effects of IgG1 and IgG3 are similar to those observed in immunocompetent mice, suggesting that natural killer cells are not crucial for antibody-mediated modulation of cryptococcal infection. In mice lacking CD4+ T cells, IgG1 is not protective and IgG3 accelerates infection, indicating that CD4+ T cells are required for antibody-mediated protection. In mice lacking CD8+ T cells, both IgG1 and IgG3 antibodies prolong survival, indicating that acceleration of the disease process by IgG3 involves CD8+ T cells. Both IgG1-mediated protection and IgG3-mediated acceleration of infection require interferon gamma. These results reveal a functional dependence of passively administered antibody on cellular immunity in cryptococcal infection in mice and have implications for antibody-based therapies in humans in the setting of CD4+ lymphopenia.