Increased CD4+CD45RA-FoxP3low cells alter the balance between Treg and Th17 cells in colitis mice.

AIM: To investigate the role of regulatory T cell (Treg) subsets in the balance between Treg and T helper 17 (Th17) cells in various tissues from mice with dextran sulfate sodium-induced colitis. METHODS: Treg cells, Treg cell subsets, Th17 cells, and CD4+CD25+FoxP3+IL-17+ cells from the lamina propria of colon (LPC) and other ulcerative colitis (UC) mouse tissues were evaluated by flow cytometry. Forkhead box protein 3 (FoxP3), interleukin 17A (IL-17A), and RORC mRNA levels were assessed by real-time PCR, while interleukin-10 (IL-10) and IL-17A levels were detected with a Cytometric Beads Array. RESULTS: In peripheral blood monocytes (PBMC), mesenteric lymph node (MLN), lamina propria of jejunum (LPJ) and LPC from UC mice, Treg cell numbers were increased (P < 0.05), and FoxP3 and IL-10 mRNA levels were decreased. Th17 cell numbers were also increased in PBMC and LPC, as were IL-17A levels in PBMC, LPJ, and serum. The number of FrI subset cells (CD4+CD45RA+FoxP3low) was increased in the spleen, MLN, LPJ, and LPC. FrII subset cells (CD4+CD45RA-FoxP3high) were decreased among PBMC, MLN, LPJ, and LPC, but the number of FrIII cells (CD4+CD45RA-FoxP3low) and CD4+CD25+FoxP3+IL-17A+ cells was increased. FoxP3 mRNA levels in CD4+CD45RA-FoxP3low cells decreased in PBMC, MLN, LPJ, and LPC in UC mice, while IL-17A and RORC mRNA increased. In UC mice the distribution of Treg, Th17 cells, CD4+CD45RA-FoxP3high, and CD4+CD45RA-FoxP3low cells was higher in LPC relative to other tissues. CONCLUSION: Increased numbers of CD4+CD45RA-FoxP3low cells may cause an imbalance between Treg and Th17 cells that is mainly localized to the LPC rather than secondary lymphoid tissues.

[Effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar].

OBJECTIVE: To study the effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar and its mechanism. METHODS: Fibroblasts from human hypertrophic scar were isolated and cultured with DMEM medium containing 10% FBS, and then they were divided into control (C, added with ethanol), low concentration (LC, added with 1 × 10(-5) mmol/L melatonin), middle concentration (MC, added with 1 × 10(-3) mmol/L melatonin), and high concentration (HC, added with 1 mmol/L melatonin) groups according to the random number table. After being cultured for 24 hours, cell morphologic change was observed under microscope; XTT-PMS assay was used to examine cell proliferative activity; cell cycle and apoptosis were assessed with flow cytometry after double staining of FITC and PI, and the levels of cyclin E, p53, and Fas mRNA were determined with fluorescence quantitative RT-PCR. Data were processed with analysis of variance and LSD test. RESULTS: (1) Fibroblasts in C group were spindle-shaped with growth in colonies. Along with the increase in melatonin concentration, fibroblasts in LC, MC, and HC groups gradually dispersed, deformed and atrophied, with shrunk cellular membrane, and decrease in ratio of nucleus and cytoplasm. (2) Proliferative activity of fibroblasts in LC, MC, and HC groups decreased along with an increase in melatonin concentration (1.49 ± 0.15, 1.24 ± 0.20, and 0.92 ± 0.09), which were lower that in C group (1.79 ± 0.10, F = 67.61, P < 0.05). Cell ratios of S and G2/M phases in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were all lower than those in C group [(10.6 ± 1.1)%, (6.1 ± 1.2)%, (3.2 ± 0.8)% vs.(16.9 ± 1.3)%, F = 286.10, P < 0.05; (13.5 ± 1.1)%, (9.8 ± 1.0)%, (6.0 ± 0.7)% vs. (16.7 ± 1.6)%, F = 162.69, P < 0.05]. Apoptotic rates in early and late stages of LC, MC, and HC groups increased along with an increase in melatonin concentration, all higher than those in C group (with F value respectively 424.05, 236.44, P values all below 0.05). The expressions of cyclin E mRNA in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were lower than that in C group (1.58 ± 0.21, 0.90 ± 0.20, and 0.24 ± 0.12 vs. 2.90 ± 0.30, F = 266.79, P < 0.05), while the expressions of p53 mRNA and Fas mRNA showed opposite tendency (with F value respectively 10.11, 12.03, P values all below 0.05). CONCLUSIONS: Melatonin can inhibit proliferation and induce apoptosis of fibroblasts in hypertrophic scar through regulating the gene expressions of cyclin E, p53, and Fas.

[Expression of secretions of hypothalamus-pituitary-adrenal axis in human hypertrophic scar].

OBJECTIVE: To explore the expression and significance of secretions of hypothalamus-pituitary-adrenal (HPA) axis in human hypertrophic scar. METHODS: Hypertrophic scar tissues obtained from 12 patients with deep-partial thickness burn or full-thickness burn and normal skin tissues from the same 7 patients with hypertrophic scar were harvested for determination of gene expression of corticotrophin-releasing hormone (CRH), CRH receptor 1 (CRH-R1), pro-opiomelanocortin (POMC), melanocortin receptor 2 (MC-2R), and glucocorticoid receptor α (GR-α) by real-time fluorescence quantitative PCR. After addition of corresponding antibodies, distribution differences of CRH, CRH-R1, adrenocorticotropic hormone (ATCH), MC-2R, and GR-α were observed with immunohistochemical staining. Data were processed with t test. RESULTS: The mRNA expression of CRH, CRH-R1, POMC, and GR-α in hypertrophic scar was respectively 3.1 ± 0.8, 0.05 ± 0.03, 0.020 ± 0.007, and 0.0030 ± 0.0010, which were significantly lower than those in normal skin (20.6 ± 4.7, 0.30 ± 0.12, 0.060 ± 0.020, and 0.0200 ± 0.0070, with t values from 2.10 to 4.75, P values all below 0.05). There was no statistical difference in MC-2R mRNA expression between hypertrophic scar and normal skin (t = 1.48, P = 0.15). Immunohistochemical observation showed CRH, CRH-R1, ACTH, MC-2R, and GR-α in hypertrophic scar were located in basal layer of epidermis, fibroblast of dermis, and tube wall of sweat gland. Expressions of these indexes could also be observed in sebaceous gland and hair follicle besides above-mentioned structures. CONCLUSIONS: Decreasing expression of active material of HPA axis may be related to formation of hypertrophic scar.

[The expression of melatonin receptor in human hypertrophic scar].

OBJECTIVE: To investigate the expression and its significance of melatonin receptor in human hypertrophic scarring. METHODS: The expression of melatonin receptor GPR50 was detected with immunohistochemistry and the melatonin receptors (MT1, MT2) mRNA were assessed with RT-PCR method in 10 cases of human hypertrophic scar and normal skin. The positive production was sequenced with auto sequencing instrument. RESULTS: Positive signals of melatonin receptor could be found in the cell membrane and cytoplasm. The melatonin receptor GPR50 was located in the epithelial basal cells,sweat gland cells and hair follicle in both hypertrophic scar and normal skin. The melatonin receptor GPR50 was extensively expressed in fibroblasts of hypertrophic scar, but not in fibroblasts in normal skin. RT-PCR showed that the expression of melatonin receptor (MT1, MT2) mRNA in hypertrophic scar was significantly higher than that in normal skin (P < 0.05). In normal skin and hypertrophic scar group, the expression of MT1 mRNA was higher than MT2 mRNA (P < 0.05). In normal skin and hypertrophic scar group, the expression of MT1 mRNA was 0.99081 +/- 0.26485 and 1.16584 +/- 0.21829 copy number/microl cDNA, respectively; the expression of MT2 mRNA was 0.77083 +/- 0.15927 and 0.99550 +/- 0.14624 copy number/ microl cDNA, respectively. Sequencing results indicated that the positive product coincided with cDNA of human melatonin receptor in GeneBank. CONCLUSIONS: Positive expression of melatonin receptor can be found in human hypertrophic scar and normal skin, but it is higher in scar. The over expression of melatonin receptor in hypertrophic scar may be related to the development of hypertrophic scar.

Management of early complications of the electrical injury and repair of the injured hands.

OBJECTIVE: To explore the management of early complications and repair of hand injuries in electrically injured patients. METHODS: Forty-five patients with electrical injuries admitted during 1995 and 2001 were analyzed retrospectively in terms of the incidences of early complications and the deformity of the injured hands repaired using different approaches. RESULTS: In the order of incidence, the complications arising in the early stages after electrical injuries included electric shock, hypovolemic shock, cardiac dysfunction, hemorrhage of the digestive tract, pulmonary infection, massive bleeding of the wound and renal dysfunction. The incidence of deformity of the hands was 79%. CONCLUSION: Proper management of early complications and repair of the injured hands are of great importance in cases of electrical surgery.