High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes.

BACKGROUND: Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM) on ClC-2 chloride channels and cell migration of keratinocytes. METHODS: Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies. RESULTS: We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM) medium, accompanied by the decline of volume-activated Cl(-) current (I Cl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including I Cl,vol and migration keratinocytes were inhibited. CONCLUSION: Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling.

Smac/DIABLO regulates the apoptosis of hypertrophic scar fibroblasts.

In abnormal skin wound healing, hypertrophic scars (HS) are characterized by excessive fibroblast hypercellularity and an overproduction of collagen, leading to atypical extracellular matrix (ECM) remodeling. Although the exact mechanisms of HS remain unclear, decreased HS fibroblast (HSFB) apoptosis and increased proliferation are evident in the development of HS. In this study, the contribution of the second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein (IAP)-binding protein with a low isoelectric point (pI) (Smac/DIABLO), an apoptosis-promoting protein released from the mitochondria, was investigated in human normal skin and HSFB cultures. The expression of Smac/DIABLO is usually decreased in many malignant tumors compared with normal tissues. Immunohistochemical analysis of skin tissues and the western blot analyses of fibroblasts revealed that the expression of Smac/DIABLO was lower in HS tissues compared with normal skin tissues. Of note, adenovirus-mediated Smac/DIABLO overexpression in the cultured HSFBs significantly reduced cell proliferation, as detected by the cell counting kit-8, and increased caspase-3 and -9 activity, as detected by spectrofluorimetry. In addition, it increased apoptosis, as detected by fluorescence-activated cell sorting (FACS). Furthermore, we found that the silencing of Smac with siRNA in the HSFBs induced a noticeable decrease in caspase-3 and -9 activity, leading to a significant reduction in apoptosis. In addition, the mRNA expression of type I and III pro-collagen detected in the HSFBs was significantly increased following the silencing of Smac with siRNA and was inhibited following Smac/DIABLO overexpression, as shown by real-time RT-PCR. In conclusion, Smac/DIABLO decreases the proliferation and increases the apoptosis of HSFBs. To our knowledge, the data from our study suggest for the first time that Smac/DIABLO is a novel therapeutic target for HS.

Yeast from burn patients at a major burn centre of China.

AIM: This study presents fungi and their characteristics identified from burn patients at a major Chinese burn centre. METHODS: All burns patients admitted to our Burn Research Institute from 2003 to 2006 inclusive were included in this study. Once fungal infection was suspected clinically, samples including wound tissue, blood, urine, stool and sputum were harvested for the culture of yeast. The sensitivities of the identified yeast were determined and the positive samples and cases were analysed. RESULTS: Out of a total of 3909 cases, 467 patients were clinically suspected of fungal infection, of which 1970 samples were taken for yeast culture. A total of 38 samples and 36 patients tested positive. The three most positive samples were urine, blood and catheter. Candida tropicalis was identified as the most common yeast type (42.1%), followed by Candida albicans (31.6%), Candida famata (T. Famata) (10.5%) and Candida glabrata (T. Glabrata) (7.9%). Except for C. galbrata, most of the yeast strains found in the study were sensitive to the routine antimycotic agents. There were eight deaths in the 36 positive patients. As much as 83.3% of the positive cases suffered burns of more than 50% total body surface area (TBSA) and half of the positive cases were greater than 80% TBSA. A total of 78.95% of the positive samples were taken from patients after 2 weeks post-burn injury. CONCLUSION: A profile of the fungi isolated from burn patients in a major Chinese burn centre is presented.

Promotion of cutaneous wound healing by local application of mesenchymal stem cells derived from human umbilical cord blood.

This study aimed to determine whether mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) would promote cutaneous wound healing. MSCs from human UCB were isolated and identified. The characteristics of the isolated MSCs' growth and proliferation were assayed in vitro. The MSCs labeled with 5-bromodeoxyuridine (BrdU) were applied on fresh cutaneous mice wounds. The healing rates were surveyed. The distribution and the differentiation into keratinocytes of the labeled MSCs in the wound tissue were checked by immunohistochemistry staining. The isolated MSCs could grow and proliferate well in vitro. The isolated MSCs from UCB could be labeled by 5-bromodeoxyuridine successfully. The MSCs derived from UCB could enhance the healing of mice skin defect wounds, and it was found that the implanted MSCs could differentiate into keratinocyte in the wound tissue. It was demonstrated that MSCs from UCB can be isolated and proliferated successfully. The local administration of MSCs derived from UCB improves skin defect wound healing in mice.

Embryonic porcine skin precursors can successfully develop into integrated skin without teratoma formation posttransplantation in nude mouse model.

How to improve the wound healing quality of severe burn patients is still a challenge due to lack of skin appendages and rete ridges, no matter how much progress has been made in the fields of either stem cell or tissue engineering. We thus systematically studied the growth potential and differentiation capacity of porcine embryonic skin precursors. Implantation of embryonic skin precursors (PESPs) of different gestational ages in nude mice can generate the integrity skin, including epidermis, dermis and skin appendages, such as sweat gland, hair follicle, sebaceous gland, etc.. PESPs of embryonic day 42 possess the maximal growth potential, while, the safe window time of PESPs transplantation for prevention of teratoma risk is E56 or later. In conclusion, PESPs can form the 3 dimensional structures of skin with all necessary skin appendages. Our data strongly indicate that porcine embryonic skin precursors harvested from E56 of minipig may provide new hope for high-quality healing of extensive burns and traumas.

Inhalation injury in southwest China--the evolution of care.

AIM: This study aims to review the changes in management of inhalation injury and the associated reduction in mortality over the past 2 decades. METHODS: The records of burn patients with inhalation injury hospitalised in our institute from 1986 to 2005 were retrospectively analysed. The incidence of inhalation injury and the associated mortality were analysed. Meanwhile, the relationship of inhalation injury with age, total burn area, tracheostomy intubation and mechanical ventilation were studied. RESULTS: The incidence of inhalation injury was 8.01% in the total 10 608 hospitalised burn patients during the 20 years surveyed. Inhalation injury was always associated with large-sized burn and was more common in adults. The incidence of tracheostomy and mechanical ventilation increased from 39.46 and 30.28% in the period from 1986 to 1995 to 70.12 and 39.74% from 1996 to 2005, respectively. The overall mortality of inhalation-injured burn patients was 15.88% compared with 0.82% of the non-inhalation group. The mortality of the burn patients with inhalation injury dropped from 25.29% during the first 10 years to 11.71% during the second decade (p<0.01). Mortality secondary to inhalation injury as the lead cause decreased from 14.56 to 6.29% (p<0.01). CONCLUSION: The care of inhalation injury has made significant progress over the past 2 decades. The early diagnosis of inhalation injury, early airway control and pulmonary function assistance with mechanical ventilation contribute to the reduction of mortality.

[A clinical study of fungal infection in burn patients].

OBJECTIVE: To address the features of the fungal infection after burn injury in clinic. METHODS: Three thousand nine hundred and nine burn patients admitted to our institute from Jan. 2003 to Dec. 2006 were involved in this study. Two thousand two hundred and seventy-one samples were harvested for fungal detection by culture from 467 patients suspected to be infected by fungi based on their clinic manifestations. The collected samples included wound tissue, blood, urine, stool, sputum, catheters and others. The antibiotic sensitivity of the identified fungi were determined by routine method. When same kind of fungus was found from different samples taken from one patient, it was recorded as one positive sample. The samples were ranked in an ascending order as wound secretion, stool, urine, sputum and bronchial alveolar lavage fluid, arteriovenous catheter or urinary catheter, blood. Only the positive sample of the highest rank source was recorded as the positive strain of fungus from this particular patient. RESULTS: It was found 61 fungal positive samples from the 2271 samples collected. Out of 467 patients, 38 strains of fungi were detected from 36 burn patients during the investigated period, the incidence was 0.92% (36/3909). The most three commonest types among the identified 38 strains of fungi were Candida tropicalis (42.1%), Candida albicans (31.6%) and Candida famata (T. Famata, 10.5%). The drug sensitivity tests demonstrated that most of the strains detected in this investigation, with the exception of candida glabrata, were sensitive to most of the routine antimycotics agents such as Amphotericin B, Fluconazole, and Itraconazole etc. Among the 36 fungus positive patients, in 18 patients the burn area exceeded 80% TBSA, 12 patients with 50%-79% TBSA, 4 patients with 30%-49% TBSA, and in 2 patients the burn area was smaller than 30% TBSA. It was found most of the fungal infections (77.78%) occurred 2 weeks after burn injury, and 8 of the 36 fungus-infected patients died (the mortality was 22.22%). Conclusions Further examinations are necessary to confirm the diagnosis in burn patients suspected to have fungal infection. Once fungal infections are confirmed, antimycotic therapy must be started immediately.

Fluid resuscitation for major burn patients with the TMMU protocol.

BACKGROUND: Fluid resuscitation is one of the critical treatments for the major burn patient in the early phases after injury. We evaluated the practice of fluid resuscitation for severely burned patients with the Third Military Medical University (TMMU) protocol, which is most widely used in many regions of China. METHODS: Patients with major burns (>30% total body surface area (TBSA)) presenting to Southwest Hospital, Third Military Medical University, between January 2005 and October 2007, were included in this study. Fluid resuscitation was initiated by the TMMU protocol. RESULTS: A total of 71 patients were (46 adults and 25 children) included in this study. All patients survived the first 48 h after injury smoothly and none developed abdominal compartment syndrome or other recognised complications associated with fluid resuscitation. The average quantity of fluid infused was 3.3-61.33% more than that calculated based on the TMMU protocol in both adult and paediatric groups. The average urine output during the first 24h after injury was about 1.2 ml per kg body weight per hour in the two groups, but reached 1.2 ml and 1.7 ml during the second 24h in adult and pediatric groups, respectively. CONCLUSION: This study indicates that the TMMU protocol for fluid resuscitation is a feasible option for burn patients. Individualised resuscitation - guided by the physiological response to fluid administration - is still important as in other protocols.

[Clinical practice and evaluation of relative fluid resuscitation formula at burn shock stage].

OBJECTIVE: To evaluate the application of the Third Military Medical University (TMMU) formula for fluid resuscitation on the major burn patients during shock stage. METHODS: Seventy-one thermal injury patients (burn area more than 30% TBSA, without especial illness, hospitalization within 8 hour after burn) admitted from 2005 to 2007 were divided into adult group (n = 46), child group (n = 25). Fluid resuscitation was initiated as per the TMMU formula. RESULTS: All patients survived the first 48 hours post burn injury and none developed recognized complications associated with fluid resuscitation. The average infused fluid was 16% approximately 38% more than the calculated in both adult and child groups. The average urine output during the first 24 hours post burn injury was 1.1 approximately 1.2 mL x kg(-1) x h(-1) in the two groups, but reached 1.2 mL and 1.7 mL x kg(-1) x h(-1) during the second 24 hours in adult and child groups respectively. CONCLUSION: TMMU formula for fluid resuscitation is a feasible option for major burn patients. Individual fluid resuscitation, guided by the physiological response, is also important and necessary.