Objective To investigate the characteristics of immune exhaustion in sepsis and analyze its association with gut microbiota translocation.Methods A total of 130 mice were randomly divided into a cecal ligation and puncture(CLP)group(n=100)and a Sham group(n=30)Mouse model of sepsis was established with CLP procedure.Flow cytometry was used to analyze the proportions of peripheral blood CD4+T and CD8+T cells and programmed cell death protein 1(PD-1)positive T cell subsets in mice.Bacterial colonization in organs such as the heart,liver and kidneys was quantified by plating homogenates of the organs.Pathological changes in immune organs were observed with HE staining.The expression and localization of CD4?,CD8?,and PD-1?cells in immune organs were detected with immunohistochemical staining,and Image J software was employed for subsequent quantification of the number of the positive cells.Results HE staining demonstrated that immune organs exhibited varying degrees of pathological damages with disease progression.Compared with the Sham mice,the CLP mice exhibited significantly increased bacterial colonization in parenchymal organs and peripheral blood(P<0.05),notably in the liver,which showed the most severe infection.In the CLP group,the proportion of CD4+T lymphocytes in peripheral blood at days 1,3,and 5 postoperatively was decreased by 56%,70.57%,and 87.42%,respectively,when compared with the Sham group(P<0.001).The proportion of CD8+T lymphocytes was decreased by 48.33%relative to the Sham group only at day 5(P<0.001).In contrast,the proportion of CD4+T cell subsets expressing PD-1 was increased to 673.08,423.08,and 600 times that of the Sham group,respectively,at the same postoperative time points(P<0.001).Immunohistochemical results showed that,in the CLP group,the proportion of CD4+T cells in the thymus,spleen,and mesenteric lymph nodes was increased to 7.65,2.66,and 3.7 times that of the Sham group,respectively,at the early-stage peak(P<0.001),and then these proportions were decreased by 82.8%(P<0.001),41.9%(P<0.01),and 60.15%(P<0.001),respectively,at the late-stage trough when compared with the early-stage peak in the corresponding organs.The proportion of CD8+positive cells was increased in the early stage and then decreased insignificantly,while the proportion of PD-1+positive cells was increased continuously,and reached 6.24,13.9,and 20.96 times that of the Sham group at the peak in the thymus,spleen,and mesenteric lymph nodes respectively(P<0.001),with their expression regions showing a rough overlap with those of CD4+cells.Conclusion During sepsis,the inflammatory response can cause severe damage to immune organs and persistent exhaustion of CD4?T lymphocytes,leading to declined defenses against infection,which may be the main causes for exacerbated gut microbiota translocation and then systemic infection.

In order to establish a high efficient regeneration system of sunflower, we optimized the process of callus induction, differentiation and rooting by screening the optimum genotype, explant materials, hormone and cytokine concentration and additives. The results indicated that hybrid sunflowers were easier to regenerate than selfing ones; The best explant was four days cotyledon. The optimum induction medium was Murashige and Skoog (MS) + 2.0 mg/L 6-benzyladenine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 1.0 mg/L kinetin (KT). The maximum rate of callus induction was 100%. The optimum differentiation medium was MS +0.2 mg/L 6-BA + 0.5 mg/L NAA + 0.3 mg/L KT + 0.3 mg/L silver nitrate (AgNO3) + 0.2 g/L active carbon (AC), and the buds differentiation rate was up to 71%. The best rooting culture medium was 1/2 MS + 0.6 mg/L indolebutyric acid (IBA). The highest rooting rate was 77%. The analysis of variance showed that genotype, explants growth time, different kinds and concentration of hormone, AC concentration had a significant effect on sunflower regeneration.
Cotyledon ; drug effects ; growth & development ; Genotype ; Helianthus ; genetics ; physiology ; Hybridization, Genetic ; Plant Growth Regulators ; pharmacology ; Plant Leaves ; drug effects ; growth & development ; Plant Roots ; drug effects ; growth & development ; Regeneration ; drug effects ; physiology ; Seedlings ; drug effects ; growth & development ; Tissue Culture Techniques