Jianglin Tan

Investigating the role of P311 in the hypertrophic scar.

The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (α-SMA), TGF-β1 and α1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-β1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-β1 expression. P311 could be a novel target for the control of hypertrophic scar development.

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.

Nano-silver-decorated microfibrous eggshell membrane: processing, cytotoxicity assessment and optimization, antibacterial activity and wound healing

An ideal wound dressing can both promote wound healing and prevent bacterial infection. Here, we report a potential dressing prepared by incorporating an optimized concentration of silver nanoparticles (AgNPs) into the microfibers of a natural eggshell membrane (EM) using environmentally friendly and mussel-inspired dopamine. Briefly, acid-treated EM was used as a porous membrane for polydopamine-reduced AgNPs synthesis. To obtain the optimal cytocompatible silver concentration, cellular attachment and MTT assay were performed with different concentrations of AgNPs. The morphology of the EM and AgNPs was confirmed by scanning electronic microscopy, scanning transmission electronic microscopy and Fourier transform infrared spectroscopy. The synthesized EM/AgNPs exhibited steady and safe AgNPs release, which was further tested for antibacterial activity against Escherichia coli and Staphylococcus aureus by disc diffusion method and bacterial suspension assay. Finally, in a murine full-thickness skin wound model, we found that EM/AgNPs could promote re-epithelialization, granulation tissue formation and wound healing via enhancing cell proliferation, as demonstrated by the expression of proliferating cell nuclear antigen (PCNA), and controlling inflammation response, as demonstrated by the expression of interleukin-1β (IL-1β). These findings suggest that EM/AgNPs may have a promising application in wound management.

In-Situ-Generated Vasoactive Intestinal Peptide Loaded Microspheres in Mussel-Inspired Polycaprolactone Nanosheets Creating Spatiotemporal Releasing Microenvironment to Promote Wound Healing and Angiogenesis

Vasoactive intestinal peptide (VIP) was reported to promote angiogenesis. Electrospun nanofibers lead to idea wound dressing substrates. Here we report a convenient and novel method to produce VIP loaded microspheres in polycaprolactone (PCL) nanofibrous membrane without complicated processes. We first coated mussel-inspired dopamine (DA) to nanofibers, then used strong adhesive DA to absorb the functional peptide. PCL membrane was then immersed into acetone to generate microspheres with VIP loading. We employed high pressure liquid chromatography to record encapsulation efficiency of (31.8 ± 2.2)% and loading capacity of (1.71 ± 0.16)%. The release profile of VIP from nanosheets showed a prolonged release. The results of laser scanning confocal microscope, scanning electron microscope and cell counting kit-8 proliferation assays showed that cell adhesion and proliferation were promoted. In order to verify the efficacy on wound healing, in vivo implantation was applied in the full-thickness defect wounds of BALB/c mice. Results showed that the wound healing was significantly promoted via favoring the growth of granulation tissue and angiogenesis. However, we found wound re-epithelialization was not significantly improved. The resulting VIP-DA-coated PCL (PCL-DA-VIP) nanosheets with spatiotemporal delivery of VIP could be a potential application in wound treatment and vascular tissue engineering.

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.

A novel recombinant immunotoxin with the smallest ribosome-inactivating protein Luffin P1: T-cell cytotoxicity and prolongation of allograft survival

In the creation of stable tolerance to MHC‐incompatible allografts, reducing the large mass of donor‐reactive cells via apoptosis is often required. Apoptosis induction by immunotoxins targeting surface molecules specifically presented on donor‐reactive cytopathic T effector (Teff) cells is a promising strategy. Traditionally, the toxin moieties are bacterial exotoxins or plant‐derived ribosome‐inactivating proteins (RIPs) with large molecular size and strong immunogenicity, hence causing the problems of tissue penetration, host immune reaction and quick clearance. We have identified a novel class of small molecule RIPs (<10 kD) from the seeds of the plant Luffa cylindrica. The smallest member of this family, Luffin P1, has a molecular weight of 5226.8 Da, yet possessing a highly potent inhibitory activity on cell‐free protein synthesis with IC50 of 0.88 nM. We now report a recombinant hIL‐2‐Luffin P1 immunotoxin, which strongly inhibited T‐cell proliferation in mixed lymphocyte reaction and ConA response with IC50 of 1.8–10 nM. In vivo, hIL‐2‐Luffin P1 significantly prolonged the survival of major MHC‐mismatched skin and kidney allografts in animal models. Thus, we demonstrate for the first time the efficacy of the smallest immunotoxin that could be further combined with other pharmacological and immunological reagents for synergistic control of pathogenic lymphocytes in immune‐mediated diseases.

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.