Pathogenic roles and therapeutic potential of the CCL8–CCR8 axis in a murine model of IgG4-related sialadenitis

Background Our previous studies reveal that CCL18-CCR8 chemokine axis is upregulated in patients of immunoglobulin G4-related disease (IgG4-RD), suggesting that the CCL18–CCR8 axis is implicated in the etiology of IgG4-RD, although whether this axis has a potential as a therapeutic target remains unclear. Our purpose was to clarify the pathogenic roles and therapeutic potential of the murine CCL8 (analog of human CCL18)–CCR8 axis by using an animal model of IgG4-RD (LAT Y136F knockin mice; LAT mice). Methods We compared the infiltration of inflammatory cells and the fibrosis of the salivary glands of 6-week-old LAT mice and littermate mice. The expressions of Ccl8 and Ccr8 were also compared. Next, we investigated the therapeutic effects of intravenous administration of anti-CCL8 neutralizing antibody in LAT mice against inflammation and fibrosis of the salivary glands. We also investigated the effects of stimulation with recombinant mouse CCL8 on the collagen production in a mouse fibroblast cell line (NIH/3 T3) in vitro. Results When compared with the littermates, the LAT mice showed apparent infiltration of inflammatory cells and fibrosis in the salivary glands. The focus and fibrosis score in the salivary glands were significantly higher in the LAT mice than in the littermates. The expression levels of Ccl8 in the spleen and of Ccr8 in the salivary glands were significantly higher in the LAT mice than in the littermates. Anti-CCL8 antibody significantly improved the focus and fibrosis score in the salivary glands of the LAT mice. In vitro, stimulation with recombinant mouse CCL8 significantly increased the expression of collagen and ERK1/2 phosphorylation in NIH/3 T3. Conclusion We clarified the overexpression and therapeutic potential of the mouse CCL8–CCR8 axis in LAT mice, which could play a crucial role in fibrosis via ERK1/2 phosphorylation, as well as the chemotaxis of inflammatory cells. The human CCL18–CCR8 axis might be a novel therapeutic target for IgG4-RD.

Pathogenic roles and therapeutic potential of the CCL8–CCR8 axis in a murine model of IgG4-related sialadenitis

Background Our previous studies reveal that CCL18-CCR8 chemokine axis is upregulated in patients of immunoglobulin G4-related disease (IgG4-RD), suggest that the CCL18–CCR8 axis is implicated in the etiology of IgG4-RD. Although, whether this axis has a potential as a therapeutic target remains unclear. Our purpose was to clarify the pathogenic roles and therapeutic potential of the murine CCL8 (analogue of human CCL18)–CCR8 axis by using an animal model of IgG4-RD (LAT Y136F knockin mice; LAT mice).Methods We compared the infiltration of inflammatory cells and the fibrosis of the salivary glands of 6-week-old LAT mice and littermate mice. The expressions of Ccl8 and Ccr8 were also compared. Next, we investigated the therapeutic effects of intravenous administration of anti-CCL8 neutralizing antibody in LAT mice against inflammation and fibrosis of the salivary glands. We also investigated the effects of stimulation with recombinant mouse CCL8 on the collagen production in a mouse fibroblast cell line (NIH/3T3) in vitro.Results When compared with the littermates, the LAT mice showed apparent infiltration of inflammatory cells, and fibrosis in the salivary glands. The focus and fibrosis score in the salivary glands were significantly higher in the LAT mice than in the littermates. The expression levels of Ccl8 in the spleen and of Ccr8 in the salivary glands were significantly higher in the LAT mice than in the littermates. Anti-CCL8 antibody significantly improved the focus and fibrosis score in the salivary glands of the LAT mice. In vitro, stimulation with recombinant mouse CCL8 significantly increased the expression of collagen and ERK1/2 phosphorylation in NIH/3T3.Conclusion We clarified the overexpression and therapeutic potential of the mouse CCL8–CCR8 axis in LAT mice, which could play a crucial role in fibrosis via ERK1/2 phosphorylation, as well as the chemotaxis of inflammatory cells. The human CCL18–CCR8 axis might be a novel therapeutic target for IgG4-RD.

Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ

Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes. Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method. Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting. Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.

PENGARUH PEMBERIAN INSULIN-LIKE GROWTH FACTOR-I (IGF-I) DARI SERUM KUDA CROSSBREED BUNTING TERHADAP KETEBALAN ENDOMETRIUM MENCIT (Mus musculus)

The purpose of the research was to know the effect of Insulin-Like Growth Factor-I (IGFI) derived from pregnant crossbreed mare serum (PMS) on endometrium thickness of mice (Mus musculus). The subject of this research were 35 female mice. The research was arranged by Completely Randomized Design (CRD) with seven treatment and five replications. The treatment were P0 = 10 ng/ml of physiological NaCl, P11 = 10 ng/ml of IGF-I PMS, P12 = 20 ng/ml of IGF-I PMS, P13 = 40 ng/ml of IGF-I PMS, P21 = 10 ng/ml of IGF-I recombinant mouse, P22 = 20 ng/ml of IGF-I recombinant mouse, and P23 = 40 ng/ml of IGF-I recombinant mouse. Observed variables include histopatological endometrium thickness of mice. The data were analyzed by Analysis of Variance (ANOVA), followed by HSD (Honestly Significant Difference) test. The data was also be analyzed using General Linear Model Univarieted to see the comparison between IGF-I PMS and recombinant mouse. The result showed that the addition of IGF-I PMS did not significantly affect (p>0,05) on endometrium thickness of mice . It showed that did not significantly difference (p>0,05) between the effect of IGF-I PMS and IGF-I recombinant mouse against the endometrium thickness of mice.

Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae ( P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia ( P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Optimization of Cloning Conditions for High-level Production of Recombinant Mouse Interleukin-2 in Escherichia coli

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