Junyi Zhou

Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

Objective Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism. Methods The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed. Results It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively. Conclusion Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.

P311 promotes renal fibrosis via TGFβ1/Smad signaling

P311, a gene that was identified in 1993, has been found to have diverse biological functions in processes such as cell proliferation, migration and differentiation. However, its role in fibrosis is unknown. We previously observed that P311 is highly expressed in skin hypertrophic scars. In this study, P311 over-expression was detected in a subset of tubular epithelial cells in clinical biopsy specimens of renal fibrosis; this over-expression, was found concurrent with α-smooth muscle actin (α-SMA) and transforming growth factor beta1 (TGFβ1) expression. Subsequently, these results were verified in a mouse experimental renal fibrosis model induced by unilateral ureteral obstruction. The interstitial deposition of collagen, α-SMA and TGF-β1 expression, and macrophage infiltration were dramatically decreased when P311 was knocked out. Moreover, TGFβ/Smad signaling had a critical effect on the promotion of renal fibrosis by P311. In conclusion, this study demonstrate that P311 plays a key role in renal fibrosis via TGFβ1/Smad signaling, which could be a novel target for the management of renal fibrosis.

CD86 Is an Activation Receptor for NK Cell Cytotoxicity against Tumor Cells

CTLA4Ig has been successfully used in the clinic for suppression of T cell activation. However, patients treated with CTLA4Ig experienced reduced incidence of tumors than predicted, but the underlying mechanism remains unknown. In this paper, we showed that brief administration of CTLA4Ig significantly reduced tumor metastasis and prolonged the survival of host mice bearing B16 melanoma. Depletion of NK cells prior to CTLA4Ig administration eliminated the CTLA4Ig-mediated anti-tumor activity. CTLA4Ig enhanced NK cell cytotoxicity to tumor cells via up-regulation of NK cell effecter molecules CD107a and perforin in vivo. In addition, we demonstrated that, upon activation, NK cells could significantly increase the expression of CD86 both in vitro and in vivo, and ligation of CD86 with CTLA4Ig significantly increased the ability of NK cells to kill tumor cells. Furthermore, a human NK cell line that expressed high level of CD86 was directly activated by CTLA4Ig so that killing of tumor targets was enhanced; this enhanced killing could be inhibited by blocking CD86. Our findings uncover a novel function of CTLA4Ig in tumor immunity and suggest that CD86 on NK cells is an activating receptor and closely involved in the CTLA4Ig-mediated anti-tumor response.

Three-Dimensional Histological Structures of the Human Dermis.

Spatial information has been shown to be critical for cell differentiation and function. Therefore, a better understanding of skin microstructures is very important for biomimetic and bioengineered scaffolds of engineering skin. The purpose of the study was to generate collagen/elastin-based three-dimensional (3D) images of human dermis to further understand the microstructures of the skin, which is believed to be helpful in the fabrication of bionic engineered skin. Skin samples were fixed, embedded, serially sectioned, stained with aldehyde-fuchsin, and photographed as serial panoramas. Dermal subregions were divided according to dermal depth and distance to hair follicle. The porosity, pore diameters, and wall thickness of human acellular dermal matrix (ADM) were measured by microcomputed tomography (micro-CT). Three-dimensional reconstructed images of collagen and elastic fibers were generated. Our results showed that there were fewer elastic fibers in the subregions close to hair follicles than in the subregions far away from hair follicles (p<0.001), but the collagen fibers were evenly distributed. Both collagen and elastic fibers were found in fewer numbers in the layers either close to the epidermis or close to the hypodermis. The mean proportions of collagen fibers and elastic fibers in the whole dermis were 28.96%±14.63% and 8.06%±3.75%, respectively. The porosity of ADM calculated by micro-CT was 68.3%±5.8%. The mean pore diameter of ADM was 131.2±96.8 μm, and the wall thickness of pores was 207.2±251.7 μm. This study represents for the first time that 3D histological cutaneous structures have been presented, which may be helpful for the next generation of skin engineering.

Epidemiology and outcome analysis of 6325 burn patients: a five-year retrospective study in a major burn center in Southwest China

Burns are a major cause of injury worldwide. We investigated the epidemiology and outcomes of burn patients in a major burn center in southwest China between 2011 and 2015 to provide guidance for burn prevention. Of the 6,325 included burn patients, 66.8% were male and 34.7% were 0 ~ 6 years old. The incidence of burns peaked in autumn. Scald was the most common cause of burns, which was predominant in patients aged 0 ~ 6 years. The mean total body surface area (TBSA) of burns was 13.4%, and patients with burns ≤10% TBSA comprised 64.1% of all cases. Patients with full-thickness burns accounted for 40.1% of all patients and 81.0% of operated patients; these burns were primarily caused by flame (34.8%), scald (21.0%), and electricity (20.4%). Fifty-six deaths occurred (mortality 0.9%), and risk factors included full-thickness burns, larger TBSA and older age. The median length of stay was 17 days, and major risk factors included more operations, better outcomes and larger TBSA. Our data showed that closer attention should be paid to children under 6 years old, males, incidents in autumn and scald burns to prevent burn injuries. Furthermore, individualized burn prevention and treatment measures based on related risk factors should be adopted.

Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling.

The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro.

Mixed lymphocyte reaction induced by multiple alloantigens and the role for IL-10 in proliferation inhibition

The frequency of T cells that can respond to alloantigens is unusually high. It remains unclear how T cells would respond when stimulated by multiple major histocompatibility complex (MHC) disparate alloantigens in the same cultures. In this report, we examined potential interactions of T cell clones that were stimulated simultaneously by two sets of complete MHC disparate alloantigens using mixed lymphocyte reaction (MLR). In this assay, we observed that proliferation of B6 lymphocytes (H-2b) stimulated by both BALB/c (H-2d) and C3H (H-2k) allogeneic cells was not increased but rather reduced as compared to B6 cells stimulated with either BALB/c or C3H allogeneic cells. Interestingly, interleukin (IL)-10 expressions at both protein level and mRNA level was significantly increased in cultures stimulated with the two MHC alloantigens, while IL-2, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 production did not show any differences. In addition, Foxp3 mRNA expression was comparable amongst all groups. In conclusion, we observed an inhibitory effect in T cell proliferation in response to multiple MHC mismatched alloantigens in MLR, and this effect might be associated with the upregulation of IL-10 expression.

Identification of ITGB4BP as a new interaction protein of P311

AIMS P311 is an 8 kDa protein that has been shown to be of importance in the process of myofibroblast transformation, glioblastoma invasion and nerve regeneration. However, the interaction protein of P311 has yet to be found. The purpose of this study was to find the interactive protein of P311. MAIN METHODS The yeast two-hybrid system was used for screening the potential interaction proteins of P311. Joint expression of the potential interactive protein and P311 was immunohistochemically stained. The interaction between P311 and the selected protein was further confirmed by fluorescence resonance energy transfer (FRET) in pulmonary adenocarcinoma tissue sections, and by coimmunoprecipitation in HEK293. KEY FINDINGS Integrin β4 binding protein (ITGB4BP) was confirmed as the interaction protein of P311. Co-expression and interaction of ITGB4BP and P311 were demonstrated in pulmonary adenocarcinoma by both immunohistochemistry and FRET. Moreover the interaction between P311 and ITGB4BP was demonstrated by coimmunoprecipitation in HEK293. SIGNIFICANCE The interactions between P311 and ITGB4BP may be very important in the process of tumor cell differentiation and metastasis. ITGB4BP may provide a potential new target for the therapy of tumors.

Risk factors for central line-associated bloodstream infection in patients with major burns and the efficacy of the topical application of mupirocin at the central venous catheter exit site

AIM The aim of this study was to evaluate the efficacy of the topical administration of mupirocin and other practices in central venous catheter (CVC) care to prevent central line-associated bloodstream infections (CLABSI) in patients with major burns. METHODS Patients with major burns admitted to a burn ICU were divided into four groups and disinfected at the CVC exit site with single povidone iodine (PVP-I) or PVP-I plus topical mupirocin ointment three times a day or once a day, respectively. The bacterial colonization of the skin at the CVC exit site and CVC tips and the incidence of CLABSI were recorded, and the risk factors were analyzed. RESULTS Administering mupirocin (RR=0.316, p=0.001), increasing the frequency of insertion-site care (RR=0.604, p=0.008), and avoiding cannulation at the burn site (RR=0.148, p<0.001) reduced skin colonization at the CVC insertion site. Topical administration of mupirocin significantly reduces both the bacterial colonization rate at CVC tips (RR=0.316, p=0.001) and the incidence of CLABSI (5.3 vs. 29.1 per 1000 catheter days, p<0.001). CONCLUSIONS Mupirocin is effective in the prophylaxis of CLABSI. Other CVC care practices were also found to affect the level of bacterial colonization, but their efficacy in preventing CLABSI needs to be evaluated further.

Fundamental Immunology of Skin Transplantation and Key Strategies for Tolerance Induction

Transplantation of allogeneic or xenogeneic skin grafts can evoke strong immune responses that lead to acute rejection of the graft tissues. In this process, donor-derived dendritic cells play crucial roles in the triggering of such immune responses. Both the innate and acquired host immune systems participate in graft rejection. At present, the rejection of skin grafts cannot be well-controlled by ordinary systemic immunosuppression therapy. Although several strategies for the long-term survival of allogeneic or xenogeneic skin grafts have been demonstrated in animal models, the induction of long-term tolerance to skin grafts is still a great challenge in clinical settings. In this article, we review the progress in the understanding of immune responses to skin grafts and discuss the possible methods that can decrease the immunogenicity of graft tissues and improve the survival of skin grafts, especially those included in preoperative pre-treatments.