Role of Treg Cells in Pathogensis of Mouse ITP / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>The ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR.</p><p><b>RESULTS</b>The ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-β and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls.</p><p><b>CONCLUSION</b>The alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.</p>

Repair of damaged intestinal mucosa in a mouse model of sepsis / 世界急诊医学杂志（英文）

BACKGROUND:The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injuryin vivo in a mouse model of sepsis.METHODS:Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.RESULTS:Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3-4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.CONCLUSIONS:Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.

Analysis of CD4(+)CD25(+)CD127(low/-) Treg cells in mice / 中国实验血液学杂志

The quantitative identification and enrichment of viable regulatory T cells (Treg) requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of natural Treg, but it cannot be used to isolate cells for functional studies. CD127 is a new surface marker expressed in Treg cells. In this study, two populations of Treg, including CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+)T cells, and profiles of the Foxp3 expression in CD4(+)CD25(+)CD127(low/-) cells were compared to evaluate which population is better. The peripheral blood cells were collected and spleen suspension of BALB/C mice were prepared, and using triple staining CD4, CD25, CD127 and CD4, CD25, Foxp3. The profiles of Treg, including CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+) were detected by flow cytometry. The quadruple staining CD4, CD25, Foxp3 and CD127 were used to determine the CD127 expression in CD4(+)CD25(+)Foxp3(+) cells. The results showed that on T cell subset the median expression levels of CD4(+), CD4(+)CD25(+) were 39.02%, 5.35% in peripheral blood and 23.49%, 3.86% in spleen. On CD4(+) T cell subset, the median expression level of CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+)T cells were 7.13%, 3.97% in peripheral blood and 12.8%, 8.23% in spleen. The ratio of CD4(+)CD25(+)CD127(low/-) T cells was higher than that of CD4(+)CD25(+)Foxp3(+) cells in both peripheral blood and spleen cells (P < 0.01). The CD4(+)CD25(+)CD127(low/-) cells highly expressed Foxp3, while the CD4(+)CD25(+)Foxp3(+)T cells lowly expressed CD127. It is concluded that compared with the CD4(+)CD25(+)Foxp3(+) populations, CD4(+)CD25(+)CD127(low/-) T cells better fit the definition of naturally occurring regulatory T cells in peripheral blood cells and spleen of BALB/C mice. CD127(low/-) is a characteristic marker on surface of CD4(+)CD25(+) Treg cells, and has been confirmed to be more specific marker for quantitatively sorting Treg cells.

Changes of the immunological barrier of intestinal mucosa in rats with sepsis / 世界急诊医学杂志（英文）

BACKGROUND:Sepsis has become the greatest threat to in-patients, with a mortality of over 25％.The dysfunction of gut barrier, especially the immunological barrier, plays an important role in the development of sepsis. This dysfunction occurs after surgery, but the magnitude of change does not differentiate patients with sepsis from those without sepsis. Increased intestinal permeability before surgery is of no value in predicating sepsis. The present study aimed to observe the changes of intestinal mucosal immunologic barrier in rat models of sepsis induced by cecal ligation and puncture. METHODS:Sixty Sprague-Dawley rats were randomly divided into a sepsis group (n=45) and a control group (n=15). The rats in the sepsis group were subjected to cecal ligation and puncture (CLP), whereas the rats in the control group underwent a sham operation. The ileac mucosa and segments were harvested 3, 6 and 12 hours after CLP, and blood samples were collected. Pathological changes, protein levels of defensin-5 (RD-5) and trefoil factor-3 (TFF3) mRNA, and lymphocytes apoptosis in the intestinal mucosa were determined. In an additional experiment, the gut-origin bacterial DNA in blood was detected. RESULTS:The intestinal mucosa showed marked injury with loss of ileal villi, desquamation of epithelium, detachment of lamina propria, hemorrhage and ulceration in the sepsis group. The expression of TFF3 mRNA and level of RD-5 protein were decreased and the apoptosis of mucosal lymphocyte increased (P<0.05) in the sepsis group compared with the control group. Significant differences were observed in RD-5 and TFF3 mRNA 3 hours after CLP and they were progressively increased 6 and 12 hours after CLP in the sepsis group compared with the control group (P<0.05, RD-5 F=11.76, TFF3 F=16.86 and apoptosis F=122.52). In addition, the gut-origin bacterial DNA detected in plasma was positive in the sepsis group. CONCLUSION:The immunological function of the intestinal mucosa was impaired in septic rats and further deteriorated in the course of sepsis.

Toll-like receptor 4 expression mediates the activation of platelets induced by LPS / 中国实验血液学杂志

The study was aimed to investigate the expression of Toll-like receptor 4 (TLR4) on platelets and to determine whether platelet TLR4 involves in its activation induced by lipopolysaccharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy individuals pretreated with a concentration of 0.2 microg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4, CD62P (P-select) and CD40L on platelets were detected by flow cytometry, and platelet TLR4 expression was further determined by Western blot analysis. The results indicated that the percentage of TLR4-positive platelets induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, p < 0.05). TLR4 expression on platelets treated with LPS was remarkably elevated in the presence or absence of thrombin. However, the expression level of the former was much higher than that of the latter and thrombin stimulation alone (p < 0.05). Moreover, the similar results were found in Western blot analysis. Synchronously, expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin (42.68% and 14.8%) and LPS respectively, and the increases of expression of CD62P and CD40L were more significant when stimulated with both LPS and thrombin (63.03% and 13.94%). Although anti-TLR4 antibody inhibited significantly the increase of TLR4, CD62P and CD40L on platelets induced by LPS, which did not affect their increase induced by thrombin. In conclusion, the evidence has been shown that functional TLR4 can be expressed on human platelets. It may involve in platelet activation as an important mediator of LPS-induced CD62P and CD40L expressions on platelets.

The effect of Ulinastatin on the delivery of cytokines in patients with septic shock / 中华急诊医学杂志

Objective To investigate the effect of Ulinastatin on the delivery of cytokines in patients with septic shock.Methods It was a prospective and controlled clinical study.Seventy-eight patients with septic shock were randomly divided into control group and treatment group and thirty-nine in every group.Patients in treatment group received Ulinastatin 200 000 units intravenous everyday for 3 days,while those in control group received equal volume of normal saline as placebo.At different time points (at 24 th,48 th,72 th hour after start of treatment),the levels of tumor necrosis factor-alpha (TNF-?),interleukin-1 (IL-1),interleukin-6 (IL-6 ),interleukin-8 (IL-8) and superoxide dismutase (SOD) in serum were assayed.Results In comparison with control group,the levels of TNF-?,IL-1,IL-6,IL- 8 of treatment group decreased markedly (P＜0.05,P＜0.01) at different time points,whereas the level of SOD was higher markedly (P＜0.05,P＜0.01) at various time points.Conclusion Ulinastatin has protective effect on patients with septic shock through decreasing the levels of TNF-?,IL-1,IL-6,IL-8 and increasing in the level of SOD.

Effect of goblet cell in rat intestine on the restitution process of the gut barrier after hemorrhagic shock / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the changes of the goblet cells in the intestine during the restitution process of the gut barrier after hemorrhagic shock.</p><p><b>METHODS</b>Forty-nine Sprague-Dawley rats with body weight of 250-300 g were divided into control group (n=7) and experimental group (n=42). Rats in the experimental group was further divided into 6 groups (n=7 each) according to different time point at 1, 3, 6, 12, 24, and 36 hours after hemorrhagic shock resuscitation. The specimens from ileum tissue were taken to observe the morphological chan ges of the intestinal mucosa. The number of goblet cells was determined by light microscope and/or electron microscope. The contents of trefoil factor family 3 (TFF3) of goblet cells were examined using GC-9A gas chromatographic instrument.</p><p><b>RESULTS</b>After hemorrhagic shock, mucosal epithelial injury was obvious in the small intestine. Tissue restitution was found after 3 hours, and mostly established after 12 hours. Following tissue restitution,the denuded mucosal surface was covered intensively by goblet cells. The number of goblet cells on the intestinal mucosa was reduced significantly from 243+/- 13 at 1 h to 157+/- 9 at 24 h (r=- 0.910, P< 0.01), and returned to normal level at 36 h. In the experimental group, the content of TFF3 in the intestinal mucosa increased significantly at 12 hours, decreased, but was still higher at 24 hours (t=3.24, P< 0.05).</p><p><b>CONCLUSIONS</b>The goblet cells play a key role in the restitution of intestinal mucosa. High expression of TFF3 may facilitate the intestinal mucosal restitution in the early phase.</p>