Objective:To investigate the clinical efficacy of Tofacitinib combined with leflunomide in the treatment of rheumatoid arthritis (RA) and its effects on Janus Kinase (JAK)/signal transduction and activator of transcription (STAT) signaling pathway-related proteins and Matrix metalloproteinase levels in serum of patients with RA.Methods:This was a prospective case-control study. A total of 80 patients with RA in our hospital from March 2020 to September 2021 were included into the study. They were divided into observation group and control group by random number table method, with 40 cases in each group. The patients in observation group were treated with Tofacitinib combined with leflunomide, while the patients in control group were treated with leflunomide alone. After 12 weeks of continuous treatment, the curative effect of the two groups was observed. Typical clinical manifestation [including Visual analogue scale (VAS) score of joint pain, number of tenderness joints, number of swollen joints, time of morning stiffness], disease activity score uses 28 joint counts (DAS28) scores, the MOS item short from health survey (SF-36) total scores and serum JAK3, STAT3, interleukin (IL)-6, IL-17 and matrix metalloproteinase (MMPs) levels were compared between the two groups before and after treatment. The adverse effects of the two groups were also analyzed. Chi-square test, paired sample t test, independent sample t test and Fisher exact probability method were used for statistical analysis. Results:After 4, 8 and 12 weeks of treatment, the american college of rheumatology (ACR)20 compliance rates in the observation group were 37.5%(15/40), 62.5%(25/40) and 80.0%(32/40), respectively, which were significantly higher than those in the control group at the same period [17.5%(7/40), 37.5%(15/40), 57.5%(23/40); χ2=4.01, P=0.045; χ2=5.00, P=0.025; χ2=4.71, P=0.030]. After 8 and 12 weeks of treatment, the ACR50 compliance rates in the observation group were [35.0%(14/40) and 47.5%(19/40), respectively, which were significantly higher than those in the control group at the same period 15.0%(6/40) and 20.0%(8/40), χ2=4.27, P=0.039; χ2=6.76, P=0.009]. After treatment, the joint pain VAS score, number of tenderness joints, number of swollen joints, DAS28 scores and SF-36 total scores in the observation group were lower than those in the control group( t=5.55, P<0.001; t=9.98, P<0.001; t=11.77, P<0.001; t=4.50, P<0.001; t=5.28, P<0.001), and time of morning stiffness was shorter than that in the control group ( t=4.76, P<0.001). After treatment, The serum levels of JAK3, STAT3, IL-6, IL-17, MMP-3, MMP-9 and MMP-13 in the obser vation group were (2 354±476) pg/ml, (1.04±0.17) ng/ml, (12.4±2.8) pg/ml, (30±5) pg/mL, (65±14) μg/L, (76±12) μg/L, (11.5±1.8) μg/L, which were lower than those in control group [(2 715±584) pg/ml (1.22±0.29) ng/mL, (16.8±3.6) pg/ml, (40±7) pg/ml, (98±15) μg/L, (123±20) μg/L, (14.9±2.8) μg/L, t=3.03, P<0.05; t=3.39, P<0.05; t=6.10, P<0.05; t=7.35, P<0.05; t=10.17, P<0.05; t=12.74, P<0.05; t=6.46, P<0.05]. The adverse reaction rate of the observation group [35.0%(14/40)] had no statistically significant difference compared with that in the control group [27.5%(11/40)]( χ2=0.52, P=0.469). Conclusion:The overall effect of Tofacitinib com bined with leflunomide in the treatment of RA is satisfactory and it is a safe and effective way to improve the clinical manifestation, disease activity and quality of life of patients with RA. The effect may be related to the significant down-regulation of the expression levels of Serum JAK/STAT signaling pathway-related Proteins and MMPs.

Objective To investigate the influences of IL-17 on regulatory T ( Treg) cells during Chlamydia trachomatis infection. Methods Wild-type ( WT) C57BL/6 mice and IL-17-/- mice were in-tranasally injected with 1×103inclusion forming units (IFU) of Chlamydia muridarum (Cm) to establish the mouse model of Chlamydia trachomatis respiratory tract infection. Mouse spleen and lung single cells were prepared. The percentages of CD4+CD25+T and CD4+CD25+Foxp3+T cells were detected by flow cytome-try. Expression of Foxp3 and TGF-β at mRNA level in lung was detected by RT-PCR. The levels of IL-10 in bronchoalveolar lavage fluid samples were detected by ELISA. Results Compared with the WT mice, the IL-17-/- mice had higher percentages of CD4+CD25+T and CD4+CD25+Foxp3+T cells in spleen and lung on the third day of Cm infection. Both of the expression of Foxp3 at mRNA level in lung and the secretion of IL-10 in bronchoalveolar lavage fluid were increased in IL-17-/- mice as compared with those in WT mice. No significant difference in the expression of TGF-β at mRNA level in lung tissues was found between the two groups. Conclusion IL-17 might inhibit the proliferation of Treg cells and the secretion of IL-10 in the very early stage of Cm respiratory tract infection.

Objective:To investigate the effects of Chlamydia muridarum ( Cm) respiratory tract infection on the infiltration and polarization of alveolar macrophages (AMs) and pulmonary interstitial macrophages (IMs). Methods:A C57BL/6 mouse model of Cm respiratory tract infection was established through nasal inhalation. Flow cytometry was used to detect AMs (CD45 + F4/80 + CD11c + ) and IMs (CD45 + F4/80 + CD11c -) in lung tissues at 0, 3, 7 and 14 d after Cm respiratory tract infection. The proportions of M1 (CD80 + , CD86 + , MHCⅡ + , iNOS + ) and M2 (CD206 + , Arg1 + ) macrophages in AMs and IMs were also detected. Results:(1) Cm respiratory tract infection induced the infiltration of AMs and IMs. Compared with the uninfected group (0 d), the proportions and the numbers of AMs and IMs of were significantly increased 3 d after infection ( P<0.05, P<0.01). The numbers of AMs and IMs reached the peak 7 d after infection ( P<0.001). (2) Compared with the uninfected group, the proportions of CD80 + and CD86 + cells in AMs were significantly up-regulated 3 d after infection ( P<0.05, P<0.01); the proportion of MHCⅡ + cells in AMs increased after infection and reached the peak at 14 d ( P<0.05), while the proportion of CD206 + cells decreased after infection ( P<0.05). (3) Compared with the uninfected group, the proportions of CD80 + and CD86 + cells in IMs were increased 3 d after infection ( P<0.05, P<0.001) and the proportion of MHCⅡ + cells was significantly increased 14 d after infection ( P<0.01), while there was no significant change in the proportion of CD206 + cells. (4) In AMs, the proportion of iNOS + cells increased continuously after infection ( P<0.01), while the proportion of Arg1 + cells decreased continuously after infection, especially at 7 d and 14 d ( P<0.05). In IMs, the proportion of iNOS + cells reached the peak at 7 d ( P<0.001), but the proportion of Arg1 + cells showed no significant change after infection. Conclusions:Cm respiratory tract infection induced the infiltration of AMs and IMs, stimulated the polarization of AMs and IMs towards the M1 phenotype and weakened the polarization of AMs to M2 macrophages, but had no significant influence on the polarization of IMs towards the M2 phenotype.

Objective:To investigate the infiltration and polarization of macrophages in mice during Chlamydia muridarum ( Cm) respiratory infection. Methods:C57BL/6 mice were intranasally infected with 1×10 3 inclusion-forming units (IFU) of Cm to establish the mouse model of Cm respiratory tract infection. The percentages of CD45 + F4/80 + cells and the macrophages expressing CD86, major histocompatibility complex Ⅱ (MHC), inducible nitric oxide synthase (iNOS) and CD206 were detected by flow cytometry. Expression of iNOS, CD206 and CCL2 at mRNA level was detected by real-time quantitative PCR. Results:Cm respiratory tract infection induced the increase of macrophages in mouse lung tissues. Compared with uninfected group, CD45 + F4/80 + macrophages were increased significantly from day 3 and reached the peak on day 7 after Cm infection. Moreover, the expression of CD86, MHCⅡ and CCL2 was increased, and the macrophages were polarized to M1 phenotype. However, the expression of M2 macrophage marker CD206 was decreased gradually. Further studies showed that iNOS expression, the indicator of M1 macrophage activation, was increased after Cm infection and reached to the top on day 7. Conclusions:Cm respiratory infection could induce the infiltration of macrophages in lung tissues and promote the polarization of macrophages to M1 phenotype.

Objective:To investigate the role of dendritic cells (DC) in Chlamydia muridarum ( Cm) respiratory infection and their effect on adaptive immune response. Methods:C57BL/6 mice were exposed to 1×10 3 inclusion-forming units (IFU) of Cm through inhalation to establish the mouse model of Cm respiratory infection. The proportion of CD11c + MHCⅡ + DC and the expression of costimulatory molecules (CD40, CD80 and CD86) in spleen tissues were detected by flow cytometry on 0, 3 and 7 d after infection. The expression of IL-12p40, IL-10 and IL-6 at mRNA level in spleen tissues was detected by qPCR. Mouse splenic DC isolated on 7 d after Cm infection were sorted by magnetic beads and then transferred to recipient mice. Th1 response in the recipient mice was measured using intracellular cytokine staining 14 d after infection. Results:Cm respiratory infection induced massive infiltration of DC and promoted the expression of costimulatory molecules on splenic DC. The expression of IL-12 and IL-10 at mRNA level in splenic DC reached the peak on 3 d after infection. Transferring the splenic DC of Cm-infected mice into the recipient mice could alleviate the disease condition in the recipient mice after Cm infection with reduced Cm inclusion-forming units in lung tissues and significantly increased proportion of Th1 cells in lung and spleen tissues. Conclusions:Cm respiratory infection could induce the maturation and activation of DC, which promoted Th1 immune response. DC played an important role in Cm infection.

Objective:To investigate the role of IL-21/IL-21R-mediated CD4 + T cells in Chlamydia muridarum ( Cm) respiratory infection. Methods:C57BL/6 mice (WT mice) and IL-21R -/- mice were used to establish the models of Cm respiratory infection through intranasal inhalation of Cm. Flow cytometry was used to detect the proportion, number, activity and function of CD4 + T cells in lung and spleen tissues at 0, 3, 7 and 14 d after Cm respiratory tract infection. IFN-γ and IL-4 levels in spleen cell culture supernatants were detected by ELISA. Na?ve WT mice were transferred with CD4 + T cells in the spleen tissues of IL-21R -/- mice or WT mice on 7 d after infection and given Cm intranasally 2 h later. Then the mice were weighed daily and sacrificed on 14 d after infection. The bacterial load and pathological changes in lung were analyzed. Flow cytometry was performed to detect the proportions and numbers of neutrophils (CD45 + CD11b + Gr-1 high) and alveolar macrophages (CD45 + F4/80 + CD11c high)as well as the proportions of Th1 (IFN-γ + CD4 + ) and Th2 (IL-4 + CD4 + ) cells. ELISA was also performed to measure IFN-γ and IL-4 levels in spleen cell culture supernatants. Results:Compared with WT mice, IL-21R -/- mice showed elevated numbers and enhanced activation of CD4 + T cells, increased proportion of Th1 cells and decreased proportion of Th2 cells in spleen and lung tissues after Cm respiratory infection. Besides, IFN-γ levels increased, while IL-4 levels decreased in spleen cell culture supernatants of IL-21R -/- mice. After Cm infection, the na?ve WT transferred with CD4 + T cells from IL-21R -/- mice showed less body weight loss, reduced bacterial load and alleviated pathological changes in lung tissues, increased proportion of Th1 cells in lung tissue and higher IFN-γ level in spleen cell culture supernatants. Conclusions:IL-21/IL-21R-mediated CD4 + T cells could aggravate Cm respiratory infection by suppressing Th1 cell immune responses.

Autologous ozonized blood transfusion(AOBT) is a therapy of re-transfusion of 100-200 mL of autologous blood after shaking and agitation with appropriate amount of oxygen-ozone in vitro. The oxidation of blood through the strong oxidation of ozone can enhance the non-specific immune response of the body, regulate the internal environment and promote health. This therapy has been increasingly applied in clinical practice, while no unified standard for the operation process in terms of ozone concentration, treatment frequency and treatment course had been established. This operation process of AOBT is primarily explored in order to standardize the operation process and ensure its safety and efficacy.