Protective effects of losartan in mice with chronic viral myocarditis induced by coxsackievirus B3.

AIM: To investigate whether losartan has protective effects in mice with chronic viral myocarditis induced by coxsackievirus B3 (CVB3). MAIN METHODS: Thirty two male Balb/c mice were intraperitoneally injected with CVB3 (10×TCID50) to induce chronic viral myocarditis (CVM). Losartan at 12.5mg/kg (n=16) or normal saline (n=16) were orally administered daily for 28 days to these mice. Uninfected mice (n=6) were used as controls. On day 29, all mice underwent anesthesia and echocardiography prior to sacrifice. Serum IL-17, IL-4, IFN-γ and TNF-α levels were measured by enzyme-linked immunosorbent assay, and cardiac tissues were histologically examined after hematoxylin & eosin staining. In addition, the effect of losartan on the virus titers in primary cultured neonatal rat cardiomyocytes infected with CVB3 was measured on Hep-2 cells at 72 h post infection. KEY FINDINGS: Mice infected with CBV3 had significantly increased mortality, heart/body weight ratios, necrosis and inflammatory scores and decreased cardiac ejection fractions, compared with the controls (all P<0.05). Losartan significantly decreased mortality from 40.0% to 12.5%, heart/body weight ratios from 7.08 ± 2.17 to 4.15 ± 0.99, and necrosis and inflammatory scores from 3.33 ± 0.50 to 2.50 ± 0.65 (all P<0.05), and increased ejection fractions from 55.80 ± 9.25 to 72.31 ± 12.15 (P<0.05). Losartan significantly enhanced IL-4, and decreased IFN-γ, TNF-α and IL-17 (all P<0.05). In the in vitro experiment, losartan had no influence on virus titers. SIGNIFICANCE: Losartan protects mice against CVB3-induced CVM, most likely through upregulating Th2 responses, and down-regulating Th1 and Th17 responses.

[Study on the stability of the anergic CD8(+);NKT cells in vitro].

AIM: To study the CD8(+);NKT cell stability function. METHODS: Splenic lymphocytes from C57BL/J mice were treated with Staphylococcal enterotoxin B (SEB) and then cultured in vitro. Day 10, 20, 30 and freezen effector cells were harvested and used. The cells were cultured in medium containing ConA and LPS for 72 hours and measured their response to mitogens. The inhibitory action of the effector cells were examined. The effector cells were co-cultured with normal lymphocytes and above mitogens for 72 hours. The cells proliferation was assessed with MTT method. The effector cells were cultured with heterogenic lymphocytes and were assessed with MTT method. The NKT cell subsets among these effector cells were analyzed by flow cytometry. RESULTS: The response of these effector cells to ConA and LPS was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.67 and 0.61 to 0.30 and 0.31, 0.28 and 0.20, 0.26 and 0.24, 0.22 and 0.23 (P<0.05, n=3). The inhibitory ability of effector cells aginst the response of normal lymphocytes to ConA and LPS were clearly observed. They inhibited the response of normal lymphocytes to above mitogens. And the A values of cell proliferation were decreased from 0.67 and 0.61 to 0.33 and 0.39, 0.30 and 0.43, 0.36 and 0.43, 0.26 and 0.29(P<0.05, n=3). The response of effector cells to heterogenic lymphocytes was significantly decreased compared with normal lymphocytes. The A values of cell proliferation were decreased from 0.70 to 0.42, 0.42 and 0.54 on day 20, 30 and freezen effector cells respectively(P<0.05, n=3). The CD8(+);NKT cell subsets among these effector cells with tolerance function were significantly increased(P<0.05, n=3). CONCLUSION: The anergic CD8(+);NKT cells could culture in vitro. And the anergic function of these cells were still existing.

[Identification of cell division of CD8+ NKT cells in vitro].

AIM: To measure the in vitro cell division ability of CD8(+);NKT cells by CFSE staining and flow cytometry(FCM). METHODS: Fresh spleen lymphocytes of C57BL/J mice were separated and stained with CFSE, and then stimulated by ConA and LPS for 3 d, and by SEB for 5 d and 10 d respectively. The stimulated cells were harvested and analyzed for CD69 expression on the cell surface and the ability of cell division using FCM. The SEB-activated effector cells for 10 d further stimulated with IL-2 for the consecutive 10 days, and were analyzed for their cell division ability, CD69 expression and NKT cell subsets by FCM. RESULTS: ConA, LPS and SEB stimulated the proliferation of spleen cells. ConA and LPS made the cells divide 3 times within 3 d, and increased CD69 expression up to 74.19% and 41.56% respectively. SEB made the cells divide 5 times within 5 d and 7 times within 10 d respectively, with increased CD69 expression of 32.09% and 48.66% respectively. Ten-day IL-2 stimulation of SEB-activated cells caused population expansion for 7 times with the CD8(+);NKT cell subsets significantly increased from 0.36% to 38.58% and CD69 expression significantly increased from 0.11% to 83.74%. CONCLUSION: The SEB-activated CD8(+);NKT cells proliferated in vitro and their cell division capability could be determined by CFSE staining and FCM.