Zhi-yong Sheng

Dedifferentiation of epidermal cells to stem cells in vivo

The effects of growth factors on wound healing have been studied extensively; however epidermal regeneration is not fully understood. We treated eight patients with leg ulcers with recombinant human epidermal growth factor (rhEGF) and compared biopsies of regenerating epidermis with those of controls who did not receive rhEGF. We used immunohistochemistry to identify cells expressing keratin 19 and beta1 integrin in regenerated epidermis from patients and controls. Patients treated with rhEGF had stem cells in the spinous and granular layers of regenerated epidermis. Histological analysis showed that these stem cells had reverted from differentiated to undifferentiated stem cells. Our findings provide evidence for epidermal cell reversion.

Analysis of differentially expressed genes in keloids and normal skin with cDNA microarray

BACKGROUND Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation. MATERIALS AND METHODS The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. RESULTS Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method. CONCLUSIONS DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.

The potential etiologic role of tumor necrosis factor in mediating multiple organ dysfunction in rats following intestinal ischemia-reperfusion injury☆

Endogenous inflammatory cytokines may function as mediators in the development of remote organ damage in response to local ischemic insult. This study was designed to (a) explore the potential role of tumor necrosis factor (TNF) formation in the pathogenesis of systemic tissue injury, (b) determine the relationship between induction of TNF and gut-derived endotoxemia and/or bacterial translocation, and (c) evaluate the protective effect of anti-TNF monoclonal antibody (MoAb) for vital organs following intestinal ischemia-reperfusion in rats. Animals were subjected to superior mesenteric artery occlusion (SMAO) for 45 min. Systemic plasma TNF levels increased rapidly after the onset of reperfusion, reaching a peak value 2 h later (P < 0.01). TNF elevation was found to be associated with gut origin endotoxemia, where the maximal TNF levels occurred approximately 2 h after the initial appearance of endotoxin in portal vein. Prophylactic treatment with anti-TNF MoAb markedly blunted the elevation in plasma TNF levels and afforded protection from the development of hypotension, vital organs dysfunction, and metabolic acidosis. Significant improvement in 48-h survival rate was observed by administration of anti-TNF MoAb prior to inducing ischemia (P = 0.007). These findings suggest that intestinal ischemia-reperfusion could result in TNF production, which may play a key role in mediating subsequent septic response and systemic tissue injury. It seems likely that passage of endotoxin and bacteria from the gut can be responsible for the TNF formation

The significance of changes in high mobility group-1 protein mRNA expression in rats after thermal injury.

There has been a widespread impression that tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-1&bgr; (IL-1&bgr;) mediate the toxicity of high doses of lipopolysaccharide (LPS, endotoxin) and are key factors in septic shock. However, the clinical efficacy of treatment with antagonists of TNF-&agr; and IL-1&bgr; is still controversial, suggesting that mediators other than TNF-&agr; and IL-1&bgr; might contribute causally to endotoxin-induced death. Recent studies implicated high mobility group-1 (HMG-1) protein as a late mediator of endotoxin lethality in mice. However, the role of HMG-1 in mediating multiple organ damage-associating trauma has not been studied. This study was designed to investigate changes in HMG-1 gene expression in vital organs, and its potential role in mediating multiple organ damage following major burns. Wistar rats were subjected to a 35 percent full-thickness thermal injury, and randomly divided into three groups as follows: normal controls (n = 7), thermal injury (n = 24), and recombinant bactericidal/permeability-increasing protein (rBPI21) treatment (n = 12). Tissue samples from liver and lungs were collected to measure tissue endotoxin levels and HMG-1 mRNA expression. In addition, blood samples were obtained for measurement of organ function parameters. Our data demonstrated a significant increase in HMG-1 gene expression in tissues at 24 h postburn, which remained markedly elevated up to 72 h after thermal injury (P < 0.05–0.01). Treatment with rBPI21 could significantly decrease tissue HMG-1 mRNA expression in the liver and lung (P < 0.01). In addition, there were high positive correlations between hepatic HMG-1 mRNA and serum aminoleucine transferase (ALT) and aspartate aminotransferase (AST) levels, and also between pulmonary HMG-1 mRNA and myeloperoxidase activities (P < 0.05–0.01). Taken together, these findings indicate that thermal injury per se can markedly enhance HMG-1 gene expression in various organs. Up-regulation of HMG-1 expression may be involved in the pathogenesis of endogenous endotoxin-mediated multiple organ damage secondary to major burns.

Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Increases Suppressive Capacity of Naturally Occurring CD4+CD25+ Regulatory T Cells in Mice In Vitro

α7 Nicotinic acetylcholine receptor (α7 nAChR) has been found in several non-neuronal cells and is described as an important regulator of cellular function. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. The present study investigated whether naturally occurring Tregs expressed α7 nAChR and investigated the functionary role of this receptor in controlling suppressive activity of these cells. We found that CD4+CD25+ Tregs from naive C57BL/6J mice positively expressed α7 nAChR, and its activation by nicotine enhanced the suppressive capacity of Tregs. Nicotine stimulation up-regulated the expression of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on Tregs but had no effect on the production of interleukin (IL)-10 and transforming growth factor-β1 by Tregs. In the supernatants of CD4+CD25+ Tregs/CD4+CD25− T-cell cocultures, we observed a decrease in the concentration of IL-2 in nicotine-stimulated groups, but nicotine stimulation had no effect on the ratio of IL-4/interferon (IFN)-γ, which partially represented T-cell polarization. The above-mentioned effects of nicotine were reversed by a selective α7 nAChR antagonist, α-bungarotoxin. In addition, the ratio of IL-4/IFN-γ was increased by treatment with α-bungarotoxin. We conclude that nicotine might increase Treg-mediated immune suppression of lymphocytes via α7 nAChR. The effect is related to the up-regulation of CTLA-4 as well as Foxp3 expression and decreased IL-2 secretion in CD4+CD25+ Tregs/CD4+CD25− T-cell coculture supernatants. α7 nAChR seems to be a critical regulator for immunosuppressive function of CD4+CD25+ Tregs.

The effect of high-mobility group box 1 protein on activity of regulatory T cells after thermal injury in rats.

The aim of the present study was to investigate in vivo the effect of high-mobility group box 1 protein (HMGB1) on activity of regulatory T cells (Tregs) and the influence on T-cell-mediated immunity after thermal injury. Male Wistar rats were randomly divided into four groups as follows: sham burn group, burn group, burn with ethyl pyruvate treatment group, and burn with antireceptor for advanced glycation end products (RAGE) antibody treatment group, and they were killed on postburn days 1, 3, 5, and 7, respectively, with eight animals at each time point. Magnetic cell sorting microbeads were used to isolate splenic Tregs and a column of nylon wool to obtain T cells. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), RAGE, and IL-2R&agr;, were analyzed by flow cytometry. Levels of HMGB1, IL-10, IL-2, IL-4 and interferon &ggr; were determined by enzyme-linked immunosorbent assay kits, and real-time reverse transcription-polymerase chain reaction was performed to detect mRNA expression of IL-10, IL-2, and IL-2R&agr;. Serum HMGB1 levels were significantly elevated during postburn days 1 to 7. In the burn group, CTLA-4 and Foxp3 expression levels of Tregs were strongly enhanced in comparison to the sham-injured group, and the capacity of Tregs to produce IL-10 was markedly increased. Administration of ethyl pyruvate to inhibit HMGB1 or anti-RAGE antibody could significantly decrease expression levels of CTLA-4, Foxp3 on Tregs, and IL-10 production after burns. Simultaneously, proliferative activity and expression levels of IL-2 and IL-2R&agr; of T cell were restored. The excessively released HMGB1 might stimulate CD4+CD25+Treg activity via binding RAGE on the surface of Tregs and trigger a shift of TH1 to TH2 with suppression of T-lymphocyte immune function after burn injury.

Sodium butyrate prevents lethality of severe sepsis in rats.

This study was performed to investigate a novel strategy to pharmacologically inhibit high-mobility group box 1 protein (HMGB1) expression with sodium butyrate, a short-chain fatty acid. Using a sepsis model induced by cecal ligation and puncture (CLP), 100 male Wistar rats were randomly divided into 4 groups as follows: control group (10 rats), sham operation group (10 rats), CLP group (further randomized into 2, 6, 12, 24, 48, and 72 h post-CLP subgroups, each 10 rats), and sodium butyrate treatment group (further randomized into 12 and 24 h post-CLP subgroups, each 10 rats). Animals of all groups were killed at designated time points, and blood and tissue samples from livers, lungs, kidneys, and small intestines were harvested to determine organ damage-related variables, and HMGB1 mRNA expression was assessed by the reverse-transcription-polymerase chain reaction. In addition, we observed the effect of treatment with sodium butyrate on survival rate in septic rats. The results showed that early treatment with sodium butyrate can markedly reduce serum alanine aminotransferase, creatinine levels at 12 h, and pulmonary myeloperoxidase activity at 24 h post-CLP, and significantly improve the 1- to 6-day survival rates in animals subjected to CLP (P < 0.05-0.01). These findings suggest that HMGB1 is excessively expressed and produced in sepsis. Sodium butyrate can markedly inhibit HMGB1 mRNA expression and may have protective effect on multiple organ damage in sepsis.

Influence of selective decontamination of the digestive tract on cell-mediated immune function and bacteria/endotoxin translocation in thermally injured rats

OBJECTIVE To determine the influence of pretreatment with selective decontamination of the digestive tract (SDD) on systemic immunosuppression, and the relationship between bacteria/endotoxin translocation and abnormalities of immune function in thermally injured rats. DESIGN, MATERIALS, AND METHODS Animals were subjected to a 40% full-thickness scald injury, and divided into SDD-treated and control groups. The treatment group received SDD (polymyxin E, tobramycin, and 5-flucytosine) by gavage twice daily for 3 days before the experiment and continued for 5 days after thermal injury. The control group was given the same amount of water. The parameters reflecting cell-mediated immunity, including splenocyte proliferation in response to mitogens, interleukin 2 (IL-2) production, and lymphocyte subpopulation, were measured before injury and 1 and 5 days after burn, respectively. MEASUREMENTS AND MAIN RESULTS Thermal injury resulted in marked reduction in splenocyte proliferative response to T-cell mitogens, IL-2 production, and T-helper/suppressor cells (CD4/CD8) ratio. Prophylactic treatment with SDD significantly decreased the incidences of bacterial translocation and endotoxemia, prevented suppressive mitogenic response and inadequate IL-2 production (p < 0.05-0.01) but did not affect the abnormal ratio of CD4 to CD8 T lymphocytes in blood (p > 0.05). CONCLUSIONS These results suggest that bacteria/endotoxin translocation from the gut appears to be involved in cell-mediated immune dysfunction as a consequence of thermal injury. Pretreatment with SDD might attenuate postburn immunosuppression by preventing translocation events.

Astragalus polysaccharides regulate T cell-mediated immunity via CD11chighCD45RBlow DCs in vitro

ETHNOPHARMACOLOGICAL RELEVANCE Astragalus polysaccharides (APS) isolated from one of the Chinese herbs, Astragalus mongholicus, are known to have a variety of immunomodulatory activities. However, it is not yet clear whether APS can induce the activation and differentiation of dendritic cells (DCs) and subsequently activate T cells. AIM OF THE STUDY This study was carried out to investigate the effect of APS on the differentiation of splenic DCs and its influence on T cell-mediated immunity through interleukin (IL)-12-producing CD11c(high)CD45RB(low) DCs in vitro. METHODOLOGY MACS microbeads were used to isolate splenic DCs, CD11c(high)CD45RB(low) DCs, CD11c(low)CD45RB(high) DCs and CD4(+) T cells. Phenotypes were analyzed by flow cytometry, and cytokine levels were determined with cytometric bead array or ELISA. RESULT The percentage of CD11c(high)CD45RB(low) DCs was significantly increased after treatment with APS compared to their counterparts. The cytokine secretion pattern of CD11c(high)CD45RB(low) DCs and CD11c(low)CD45RB(high) DCs was detected, and it was found that unlike the stable IL-10 secretion pattern of CD11c(low)CD45RB(high) DCs induced by APS, CD11c(high)CD45RB(low) DCs showed a dose-dependent relationship between IL-12 production and APS stimulation. In order to verify whether the activation of CD4(+) T was associated with the differentiation of splenic DCs mediated by APS to CD11c(high)CD45RB(low) DCs, anti-IL-12 receptor (IL-12R) as well as anti-IL-10R monoclonal antibody was used to inhibit the effect of CD11c(high)CD45RB(low) DCs and CD11c(low)CD45RB(high) DCs in CD4(+) T mixed lymphocyte reaction culture. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4(+) T+CD11c(high)CD45RB(low) DCs or CD11c(low)CD45RB(high) DCs mixed lymphocyte reaction, the inductions of these DCs on T cells were inhibited dramatically. CONCLUSION APS might induce the differentiation of splenic DCs to CD11c(high)CD45RB(low) DCs followed by shifting of Th2 to Th1 with enhancement of T lymphocyte immune function in vitro. Also, the effect of APS on T-cell differentiation to Th1 was not associated with the inhibition of IL-10 production in CD11c(low)CD45RB(high) DCs.

Astragalus Polysaccharides Attenuate Postburn Sepsis via Inhibiting Negative Immunoregulation of CD4+CD25high T Cells

Backgroud Astragalus polysaccharides (APS) isolated from one of the Chinese herbs, Astragalus mongholicus, are known to have a variety of immunomodulatory activities. However, it is not yet clear whether APS can exert an effect on the immune functions of regulatory T cells (Tregs). This study was carried out to investigate the effect of APS on the immune function of peripheral blood Tregs in postburn sepsis. Methodology/Principal Findings BalB/c mice were randomly divided into six groups as follows: sham burn group, burn control(burn without infection animals) group, burn plus P. aeruginosa group, burn plus P. aeruginosa with APS (50 mg/kg) treatment group, burn plus P. aeruginosa with APS (100 mg/kg) treatment group, and burn plus P. aeruginosa with APS (200 mg/kg) treatment group, and they were sacrificed on postburn day 1, 3, 5, and 7, respectively, with seven animals at each time point. Magnetic microbeads were used to isolate peripheral blood Tregs and CD4+ T cells. Phenotypes were analyzed by flow cytometry, and cytokine levels were determined with ELISA. In the burn plus P. aeruginosa group, forkhead/winged helix transcription factor p3 (Foxp3) expression on CD4+CD25+Tregs were strongly enhanced in comparison to the sham group, and the capacity of CD4+CD25+Tregs to produce interleukin (IL)-10 was markedly increased. Administration of APS to inhibit CD4+CD25+Tregs could significantly decrease expression of Foxp3 on CD4+CD25+Tregs, and IL-10 production in burned mice with P. aeruginosa infection. At the same time, proliferative activity and expression of IL-2 and IL-2Rα on CD4+ T cells were restored. In contrast, anti-Toll-like receptor 4 (TLR4) antibody could block the effect of APS on Tregs immune function. Conclusion APS might suppress CD4+CD25+Treg activity, at least in part, via binding TLR4 on Tregs and trigger a shift of Th2 to Th1 with activation of CD4+ T cells in burned mice with P. aeruginosa infection.