BL02, a phage against carbapenem- and polymyxin-B resistant Klebsiella pneumoniae, isolated from sewage: A preclinical study.

The emergence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a threat to public health. Polymyxin-B is generally considered a last-resort antibiotic. In this study, we isolated a carbapenem- and polymyxin-B resistant K. pneumoniae phage BL02 for the first time in Southwestern China and evaluated its biological characteristics and whole-genome sequence. Polymyxin-B resistant K. pneumoniae, (CK02), was isolated from the blood of a male with severe septic shock, and phage BL02 was screened and purified from the hospital sewage. BL02 could lyse 40 out of 46 CRKP isolates (86.96%) and has high activity in the pH range of 6-10 and the temperature range of 4-55 °C. The latency period of BL02 was about 10 min and the lysis period was about 50 min. The genome results showed that BL02 was a linear dsDNA with a total length of 175,595 bp and a GC content of 41.83%. A total of 275 ORFs were predicted and no tRNA, rRNA, drug resistance genes, or virulence genes were found in the genome. Phylogenetic analysis showed that BL02 belongs to the family Straboviridae. Treatment of infected mice with two antibiotics (tigecycline or ceftazidime/avibactam) resulted in 7-day survival rates of 28.57% and 42.86%, respectively. In contrast, the survival rate of mice in the single-dose BL02-treated group was 71.43%. In summary, this preclinical study isolated a phage capable of lysing polymyxin-B resistant K. pneumoniae and validated its safety and efficacy in an in vivo model, which provides a reference for further research on controlling MDR pathogens.

Early application of continuous high-volume haemofiltration can reduce sepsis and improve the prognosis of patients with severe burns.

BACKGROUND: In the early stage of severe burn, patients often exhibit a high level of inflammatory mediators in blood and are likely to develop sepsis. High-volume haemofiltration (HVHF) can eliminate these inflammatory mediators. We hypothesised that early application of HVHF may be beneficial in reducing sepsis and improving the prognosis of patients with severe burns. METHODS: Adults patients with burns ≥ 50% total burn surface area (TBSA) and in whom the sum of deep partial and full-thickness burn areas was ≥ 30% were enrolled in this randomised prospective study, and they were divided into control (41 cases) and HVHF (41 cases) groups. Patients in the control group received standard management for major burns, whereas the HVHF group additionally received HVHF treatment (65 ml/kg/h for 3 consecutive days) within 3 days after burn. The incidence of sepsis and mortality, some laboratory data, levels of inflammatory cytokines in the blood, HLA-DR expression on CD14+ peripheral blood monocytes, the proportion of CD25+Foxp3+ in CD4+ T lymphocytes, and the counts of CD3+, CD4+ and CD8+ T lymphocytes were recorded within 28 days post-burn. RESULTS: The incidence of sepsis, septic shock and duration of vasopressor treatment were decreased significantly in the HVHF group. In addition, in the subgroup of patients with burns ≥ 80% TBSA, the 90-day mortality showed significant decreases in the HVHF group. The ratio of arterial oxygen partial pressure to the fraction of inspiration oxygen was improved after HVHF treatment. In the patients who received HVHF treatment, the blood levels of inflammatory cytokines, including tumour necrosis factor-α, interleukin (IL)-1ß, IL-6 and IL-8, as well as the blood level of procalcitonin were found to be lower than in the control group. Moreover, higher HLA-DR expression on CD14+ monocytes and a lower proportion of CD25+Foxp3+ in CD4+ T lymphocytes were observed in the patients in the HVHF group. CONCLUSIONS: Early application of HVHF benefits patients with severe burns, especially for those with a greater burn area (≥ 80% TBSA), decreasing the incidence of sepsis and mortality. This effect may be attributed to its early clearance of inflammatory mediators and the recovery of the patient's immune status. TRIAL REGISTRATION: Chinese Clinical Trial Register, ChiCTR-TRC-12002616 . Registered on 24 October 2012.

Glutamine with probiotics attenuates intestinal inflammation and oxidative stress in a rat burn injury model through altered iNOS gene aberrant methylation.

Severe burns may lead to intestinal inflammation and oxidative stress resulting in intestinal barrier damage and gut dysfunction. In the management of severe burns, therapies are needed to attenuate whole-body inflammatory responses and control the burden of oxidative stress. In this study, we evaluated the effects of oral glutamine (Gln) with probiotics on burn-induced intestinal inflammation and oxidative stress using a Wistar rat burn injury model. We then explored potential molecular mechanisms for the effects of glutamine and probiotics on intestinal tissue inflammation and oxidative stress. We found that glutamine and probiotics together significantly inhibited nitric oxide (NO) content; reduced levels of the inflammatory factors TNF-α, IL-6, and IL-8; and altered expression of oxidative stress factors including reactive oxygen species and superoxide dismutase. We found that the apoptotic proportion of intestinal epithelial cells in severely burned subjects was notably decreased following treatment with glutamine plus probiotics. We also found that glutamine and probiotics given together markedly reduced NO content by down-regulating the expression of iNOS in blood and intestinal tissue. These findings indicate that regulation of the iNOS gene plays a pivotal role in inflammation and oxidative stress in the response to severe burns in the Wistar rat. We then further investigated the mechanism by which combined therapy with glutamine and probiotics might reduce expression of iNOS and found that this treatment resulted in increased methylation of the iNOS gene. The methylation level of the iNOS gene was found to be regulated via differential expression of DNMT1 and Tet1. Collectively, our results suggest that combined therapy with glutamine and probiotics can markedly reduce the synthesis of NO, suppressing intestinal inflammation and oxidative stress in the Wistar rat burn injury model.

CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury.

The mechanism underlying pulmonary inflammation in thermal inhalation injury remains elusive. Cystic fibrosis, also hallmarked with pulmonary inflammation, is caused by mutations in CFTR, the expression of which is temperature-sensitive. We investigated whether CFTR is involved in heat-induced pulmonary inflammation. We applied heat-treatment in 16HBE14o- cells with CFTR knockdown or overexpression and heat-inhalation in rats in vivo. Heat-treatment caused significant reduction in CFTR and, reciprocally, increase in COX-2 at early stages both in vitro and in vivo. Activation of ERK/JNK, NF-κB and COX-2/PGE2 were detected in heat-treated cells, which were mimicked by knockdown, and reversed by overexpression of CFTR or VX-809, a reported CFTR mutation corrector. JNK/ERK inhibition reversed heat-/CFTR-knockdown-induced NF-κB activation, whereas NF-κB inhibitor showed no effect on JNK/ERK. IL-8 was augmented by heat-treatment or CFTR-knockdown, which was abolished by inhibition of NF-κB, JNK/ERK or COX-2. Moreover, in vitro or in vivo treatment with curcumin, a natural phenolic compound, significantly enhanced CFTR expression and reversed the heat-induced increases in COX-2/PGE2/IL-8, neutrophil infiltration and tissue damage in the airway. These results have revealed a CFTR-regulated MAPK/NF-κB pathway leading to COX-2/PGE2/IL-8 activation in thermal inhalation injury, and demonstrated therapeutic potential of curcumin for alleviating heat-induced pulmonary inflammation.

A randomized and controlled multicenter prospective study of the Chinese medicinal compound Fufang Xuelian Burn Ointment for the treatment of superficial and deep second-degree burn wounds.

The objective of this study was to evaluate the efficacy and safety of a traditional Chinese medicine, Fufang Xuelian Burn Ointment (FXBO), to treat superficial and deep second-degree burn wounds. A four-center, randomized, controlled, and prospective study was conducted. Overall, 240 patients with either superficial or deep second-degree burn wounds were enrolled consecutively in this study. Patients who were randomly assigned to the control group (superficial: 72, deep: 48) underwent common burn wound therapy, whereas those randomized to the treatment group (superficial: 72, deep: 48) received common burn wound therapy plus topical FXBO. The healing rate, healing time, effective rate, and safety data were compared between the two groups. The baseline characteristics were comparable for the two groups. The healing rate was 94.79(±7.50) in the control group and 98.60(±5.69) in the FXBO group after 14 days for patients with superficial second-degree burn wounds (P = 0.000), and 95.17(±9.68) versus 97.44(±9.81) at 28 for deep second-degree burn wounds (P = 0.025). The median healing time in the FXBO group were 9 and 21 days for superficial and deep second-degree burns, respectively, compared to 10.5 and 22.5 days, respectively, in control group (P(superficial) = 0.000 and P(deep) = 0.009). The results of the effective rate showed that comprehensive efficacy of the FXBO group was improved compared to the control group for either superficial or deep second-degree burns (P(superficial) = 0.035 and P deep = 0.003). There were no reported drug-related adverse events in both groups. Therefore, FXBO was well tolerated and more effective than control group for treating superficial and deep second-degree burn wounds.

[A propose to suspend the use of hydroxyethyl starch for fluid resuscitation in shock phase of severe burns].

Based on the result of randomized controlled trials and meta-analysis recently, the infusion of hydroxyethyl starch (HES) was not shown to over match routine crystalline solution in exerting resuscitation effect against hypovolemia of patients with burn shock, severe systematic infection, or other critical conditions, on the other hand, it may induce renal toxicity and other toxic and side effects. Since the pathological mechanism underlying hypovolemia during shock phase after burn is similar to that of severe systemic infection, we propose to suspend the use of HES for fluid resuscitation during the shock phase of severe burn until further elucidation.

[Clinical characteristics and diagnosis of sepsis in pediatric burn patients].

Pediatric burn patients account for more than 1/3 of the inpatients in the same period, and its incidence surpasses that of burn patients in other age groups. However, it brings about much difficulty to treat pediatric burn patients complicated by sepsis, which brings a significantly higher mortality than that of the adult. Moreover, the physiological characteristics, development of organs, drug metabolism, and body response to burn injury in children are obviously different from those of the adult. Therefore, it is clinically important to understand the clinical characteristics of sepsis in pediatric burn patients in order to improve the diagnosis and treatment of this ailment.

[Influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of DC 2.4 cells].

OBJECTIVE: To observe the influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of dendritic cell strain DC 2.4 cells. METHODS: DC 2.4 cells were routinely cultured. Lentiviruses carrying GFP and those with up-regulated CCR7 were constructed. DC 2.4 cells were divided into DC 2.4 group (without any treatment), GFP-DC 2.4 group (infected with GFP-carrying lentivirus), and CCR7-DC 2.4 group (infected with CCR7-carrying lentivirus labeled by GFP) according to the random number table. The expressions of surface molecules MHCII, CD80, CD86, and CCR7 were detected by flow cytometry, Western blotting, and confocal laser scanning microscope. The migration of cells was detected by chemotaxis assay in vitro. The immunogenicity of cells was detected with mixed lymphocyte reaction. LPS-DC 2.4 group was set up as positive control. Data were processed with one-way analysis of variance and t test. RESULTS: Lentiviruses carrying stably-expressing CCR7 were constructed, and the transfection rate of which into DC 2.4 cells was 87.4%. There was no statistically significant difference among DC 2.4, GFP-DC 2.4, and CCR7-DC 2.4 groups in the expressions of MHC II, CD80, and CD86 as showed by flow cytometry (with F values from 0.17 to 1.19, P values all above 0.05). The protein expression of CCR7 of cells in CCR7-DC 2.4 group (45.1 ± 2.1) was obviously higher than that in DC 2.4 and GFP-DC 2.4 groups (25.3 ± 1.4, 28.6 ± 0.9, F = 162.90, P < 0.01), while the difference of which between DC 2.4 group and GFP-DC 2.4 group was not statistically significant (t = 2.20,P > 0.05). The fluorescence intensity of CCR7 in CCR7-DC 2.4 group was obviously increased compared with that of DC 2.4 group. The chemotaxis migration rate of cells in CCR7-DC 2.4 group with the influence of CCL19 was (41.0 ± 2.0)%, which was significantly higher than that of DC 2.4 and GFP-DC 2.4 groups [(6.0 ± 0.5)%, (6.8 ± 0.3)%, F = 84.21, P < 0.01]. There was no statistically significant difference between DC 2.4 group and GFP-DC 2.4 group in the migration rate (t = 0.45, P > 0.05). The absorbance values in DC 2.4, GFP-DC 2.4, CCR7-DC 2.4, and LPS-DC 2.4 groups were respectively 1.6 ± 0.4, 1.9 ± 0.4, 1.7 ± 0.4, 3.8 ± 0.4, and the differences among the former three groups were not obvious (F = 1.56, P > 0.05). The absorbance value in LPS-DC 2.4 group was obviously higher than that of the other three groups (with t values from 1.53 to 1.82, P values all below 0.01). CONCLUSIONS: DC 2.4 cells infected with efficiently CCR7-expressing lentivirus showed high chemotaxis to CCL19, but without obvious change in immunogenicity.

Relation between proteinuria and acute kidney injury in patients with severe burns.

INTRODUCTION: Proteinuria in burn patients is common, and may be associated with acute kidney injury (AKI) and adverse outcomes. We evaluated the incidences, outcomes, characteristics and determinants of proteinuria and its influence on AKI and outcomes in burn patients. METHODS: This retrospective study was carried out in a hospital's burn department. The study population consisted of patients with burn injuries admitted during a five-year period. Positive urine dipstick readings were defined as mild (± or 1+) or heavy (≥ 2+) proteinuria, and AKI was diagnosed and staged according to the Risk, Injury, Failure, Loss, End Stage (RIFLE) classification system. Patient characteristics, management and outcomes were evaluated for associations with proteinuria using nonparametric tests, chi-square (χ(2)) tests and binary logistic regression. RESULTS: Of the patients admitted to the burn unit during the study period (n = 2,497), 865 (34.64%) were classified as having proteinuria. In the patients whose total burn surface areas (TBSA) were > 30% (n = 396), 271 patients (68.43%) had proteinuria and 152 of these patients (56.09%) met AKI criteria. No patients without proteinuria developed AKI. Intensive care unit (ICU) mortality rates were 0.8%, 16.67% and 30.77% (P < 0.001) in the groups with no, mild and heavy proteinuria, respectively. Logistic regression analysis identified proteinuria (OR 4.48; 95% CI, 2.824 to 7.108; P < 0.001) and sequential organ failure assessment (OR 1.383; 95% CI, 1.267 to 1.509; P < 0.001) as risk factors for AKI. CONCLUSIONS: We observed a high prevalence of proteinuria in patients with severe burns (> 30% TBSA). Severely burned patients with proteinuria had a high risk of developing AKI and a poor prognosis for survival. This suggests that proteinuria should be used for identifying burn patients at risk of developing AKI.

[Comparative study on the effect of restrictive fluid management strategy on the early pulmonary function of patients with severe burn].

OBJECTIVE: To retrospectively analyze the effect of restrictive fluid management strategy (RFMS) on the early pulmonary function and the prognosis of patients with extremely severe and extensive burn. METHODS: Thirteen patients with extremely severe burn hospitalized from June 2010 to November 2011, being treated with RFMS in the fluid reabsorption stage, were enrolled as treatment group. Twenty-six patients with extremely severe burn hospitalized from March 2008 to November 2011, being treated with normal fluid therapy in the fluid reabsorption stage, were enrolled as control group. The match proportion between treatment group and control group was 1:2. Fluid intake, fluid output, fluid balance (the difference between fluid intake and output), and plasma albumin level from post burn day (PBD) 3 to 10, pulmonary oxygenation index on PBD 3, 5, 7, 10, and 14, occurrence of lung and blood stream infections from PBD 7 to 14, and occurrence of acute respiratory distress syndrome (ARDS), occurrence of other organ complications, and mortality within 2 weeks post burn (PBW) were recorded and compared. Measurement data were processed with t test and randomized blocks analysis of variance, enumeration data were processed with Fisher's exact test. RESULTS: Daily fluid intake of patients showed a tendency of decrease in both groups from PBD 3 to 10. Except for that of PBD 4, there was no statistically significant difference between two groups in fluid intake (with F values from 0.072 to 1.939, P values all above 0.05). Daily fluid output of patients showed a tendency of increase in both groups from PBD 3 to 10. It peaked on PBD 10 in control group and PBD 6 in treatment group. The mean daily fluid output was higher in treatment group than in control group from PBD 4 to 9, but without statistically significant difference (with F values from 0.001 to 3.026, P values all above 0.05). Fluid balance lowered in both groups, and it was the lowest on PBD 10 in control group and PBD 6 in treatment group. Fluid balance was lower in treatment group than in control group from PBD 3 to 7, and it showed statistically significant differences on PBD 4, 5, and 6 (with F values from 4.799 to 8.031, P values below 0.05). Plasma albumin level was higher in treatment group than in control group from PBD 3 to 10, with statistically significant differences observed on PBD 4, 9, and 10 (with F values from 5.691 to 10.551, P < 0.05 or P < 0.01). Pulmonary oxygenation index was higher in treatment group than in control group from PBD 3 to 14, with statistically significant differences observed on PBD 7 (respectively 372 ± 78 in treatment group and 291 ± 92 in control group, F = 5.184, P < 0.05) and 14 (respectively 354 ± 39 in treatment group and 283 ± 72 in control group, F = 8.683, P < 0.05). Lung infection and blood stream infection were respectively observed in 1 and 4 patient (s) in treatment group, and 9 and 11 patients in control group from PBD 7 to 14. Occurrence of ARDS, occurrence of other organ complications, and mortality were fewer in treatment group than in control group within PBW 2, though the differences were not statistically significant (P values all above 0.05). CONCLUSIONS: RFMS is a useful strategy in improving early pulmonary oxygenation of patients with extremely severe and extensive burn by promoting the process of fluid reabsorption and rebalance. This strategy may be also beneficial for the prevention of organ complications as well as a better prognosis in severely burned patients.

[Reproduction of a rat model of burn with infection].

OBJECTIVE: To reproduce a reliable rat model of burn with infection for the study of prevention and treatment of infected wound. METHODS: (1) Electrical burn producing apparatus equipped with constant temperature (80°C) and pressure (0.5 kg) was used to reproduce burn injury (with area of 4.5 cm(2)) on both sides of the back in 50 SD rats for different duration (4, 6, 8, 10, 12 s), with 10 rats for each burn duration. On post burn day (PBD) 1, gross condition of wounds was observed with naked eyes. Wounds on the left side were used to observe healing time. The wounds on the right side were used for histological observation to determine the depth of injury, and they were classified into superficial and deep partial-thickness injury. (2) Another 36 SD rats were divided into A (inflicted with superficial partial-thickness burn, n = 18) and B (inflicted with deep partial-thickness burn, n = 18) groups according to the random number table. Rats in both groups were treated in accordance with method of preliminary experiment. Immediately after burn, 0.1 mL of liquid containing 1 × 10(9), 1 × 10(7), 1 × 10(5) CFU Pseudomonas aeruginosa (PA) ATCC 27853 was respectively inoculated to the wounds on one side (with 6 rats for each amount), while the wounds on the other side were treated with the same volume of normal saline as control. Inflammatory reaction of wounds was examined with HE staining on post inoculation day (PID) 1. On PID 1, 2, 3, 5, 7, and 14, the number of subeschar bacteria was respectively counted and the bacteria were identified with Gram stain and biochemical reaction. Wound healing time was recorded. Data were processed with t test. RESULTS: (1) Burn for 6, 8 s was respectively identified as injury time resulting in superficial or deep partial-thickness injury according to histological observation and wound healing time. (2) Obvious inflammatory cell infiltration was observed in the wounds in B group which were inoculated with 1 × 10(7), 1 × 10(9) CFU PA, and the infiltration was less marked in A group with inoculation of 1 × 10(9) CFU PA. (3) The bacteria isolated from wounds of A and B groups was identified as PA. The subeschar bacteria count within PID 14 in A group, in which different amount of PA was inoculated, was mostly less than 1 × 10(5) CFU/g of tissue, while that in B group in which 1 × 10(9) CFU PA was inoculated was more than 1 × 10(5) CFU/g of tissue. (4) There was no obvious difference in wound healing time between wounds inoculated with different amount of PA and wounds treated with normal saline in A group (with t value respectively 1.26, 0.29, 1.07, P values all above 0.05). Wound healing time of wounds in B group, in which 1 × 10(9) CFU PA was inoculated, was longer as compared with that treated with normal saline [(22.5 ± 1.0) d vs. (19.4 ± 1.6) d, t = 2.73, P < 0.05]. CONCLUSIONS: In rat, deep partial-thickness burn wound inoculated with 1 × 10(9) CFU PA ATCC 27853 is a reliable model with high reproducibility for the study of infection of burn wound.

HSP70 protects intestinal epithelial cells from hypoxia/reoxygenation injury via a mechanism that involves the mitochondrial pathways.

Though recent studies have reported the importance of several endogenous cytoprotective factors including heat shock protein 70 (HSP70) that protect intestinal epithelial cells (IECs) from the effects of stress and injury, the exact mechanism of HSP70 underlying cytoprotection against hypoxia/reoxygenation induced IEC injury remains unclear. The present study was designed to investigate the possible mechanisms by which HSP70 protected IECs against hypoxia/reoxygenation injury and focused on the effects of HSP70 on IEC apoptosis induced by hypoxia/reoxygenation injury. Recombinant adenoviruses (Ad-HSP70) were transfected into the intestinal epithelial cell line in vitro and then suffered from 90 min of hypoxia followed by 60 min of reoxygenation. The LDH leaking, apoptosis, and mitochondrial membrane potential (Psi(m)) were evaluated after hypoxia/reoxygenation. The expression of HSP70, cytochrome c and Bcl-2 protein was determined by Western blot or immunofluorescence analysis. The results show that HSP70 protein was highly expressed in the IECs at 48h following Ad-HSP70 transfection. HSP70 overexpression could reduce LDH leakage and cell apoptosis in IECs following hypoxia/reoxygenation injury. Furthermore, the overexpression of HSP70 significantly reversed the decrease of mitochondrial membrane potential and the release of mitochondrial cytochrome c in IECs during hypoxia/reoxygenation. HSP70 overexpression was also associated with the increasing expression of Bcl-2 protein in IECs during hypoxia/reoxygenation. We conclude that HSP70 protects IECs against hypoxia/reoxygenation induced apoptosis through increasing Bcl-2 expression, which in turn could inhibit the mitochondria-related apoptotic pathway that involves the disruption of the Psi(m) and release of cytochrome c from mitochondria.

[Clinical assessment of colistin in treating infections caused by multidrug-resistant gram-negative bacillus in patients with severe burn].

OBJECTIVE: To investigate the therapeutic effect and side effects of colistin in treating infections caused by multidrug-resistant (MDR) gram-negative bacillus in patients with severe burn in order to provide the basis for reasonable application of this antibiotic in clinic. METHODS: Nine burn patients suffered from infections caused by MDR gram-negative bacillus admitted to our institute from August 2005 to January 2009 were involved in this study. On the premises that isolated bacteria were only sensitive to colistin or not sensitive to other antibiotics, patients were treated with intravenous drip of colistin (100 x 10(4) - 150 x 10(4) U/d), or intravenous drip combined with administration of the drug into respiratory tract by atomization or instillation (50 x 10(4) - 100 x 10(4) U/d). The bacteriologic and therapeutic effects and side effects (including neurotoxicity and nephrotoxicity, rise in serum levels of creatinine, urea nitrogen and cystatin C were detected and compared before and after administration) of colistin were observed. RESULTS: Out of 9 patients, 7 patients were with bloodstream and pulmonary infections, 1 patient was with bloodstream, pulmonary, and invasive wound infections, and 1 patient was with bloodstream and urinary tract infections. The pathogenic bacteria were proved to be Pseudomonas aeruginosa, Acinetobacter baumannii and Pseudomonas maltophilia. After the administration of colistin, bacteria clearance rate of blood reached 92.3% in 9 patients; isolation rate of MDR gram-negative bacillus of sputum was significantly decreased in 7 patients with pulmonary infection (before treatment 58.2% v.s. after treatment 14.6%, P < 0.01); a complete MDR gram-negative bacillus clearance of urine was observed in 1 patient with urinary tract infection. Colistin was clinically effective in 8 patients but ineffective in 1 patient (effective rate 88.9%). Compared with those before administration, serum levels of creatinine and urea nitrogen were decreased after administration in all patients; no significant difference in serum level of cystatin C among 8 patients was detected, except an obvious elevation in serum level of cystatin C in 1 patient after colistin therapy, and it lowered 1 month after discontinuation. No neurotoxicity or other side effect was observed during medication and 5 days after discontinuation in all patients. CONCLUSIONS: Reasonable application of colistin is a good option for treating infections caused by MDR gram-negative bacillus in patients with severe burn, as no other more effective drug is found.

[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.

In vitro maturation and migration of immature dendritic cells after chemokine receptor 7 transfection.

Dendritic cells are specialized antigen-presenting cells that regulate immunity and tolerance. Chemokine receptor 7 (CCR7), which is expressed by mature dendritic cells, mediates the migration of the cells to secondary lymphoid organs and thus regulates immune responses. It has been demonstrated that immature dendritic cells can induce immune tolerance, but they do not express CCR7 and cannot migrate to secondary lymphoid organs. We transfected immature dendritic cells with a recombinant adenovirus carrying the CCR7 gene to obtain immature dendritic cells with the ability to migrate. The maturity of the cells was monitored by scanning electron microscopy and flow cytometry. In addition, we assessed the ability of cells to migrate and the function of the cells using in vitro chemotactic and mixed leukocyte reaction assays. The results showed that immature dendritic cells became semi-mature, exhibiting a mild upregulation of co-stimulatory molecular expression and a few dendritic processes. Immunofluorescence assay and Western blotting indicated that CCR7 protein expression increased significantly in immature dendritic cells following CCR7 gene transfection. The in vitro chemotactic assay showed a significantly enhanced ability to migrate in response to CCL19 following CCR7 gene transfection. Moreover, transfected cells showed an enhanced ability to stimulate allogeneic T cell proliferation in vitro, but their ability was significantly weaker than that of mature dendritic cells. Interleukin-10 inhibited the differentiation and maturation of immature dendritic cells. It is concluded that, following CCR7 gene transfection, immature dendritic cells exhibit an enhanced ability to migrate and some of the characteristics of mature cells. Thus, these cells are of potential clinical significance in studies of immune tolerance induction during skin grafting after severe burns.

[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.

[Influence of HSP70 on function and energy metabolism of mitochondria in intestinal epithelial cells after hypoxia/reoxygenation].

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated heat shock protein 70 (HSP70) on energy metabolism of mitochondria in intestinal epithelial cells (IEC-6) after hypoxia/reoxygenation injury . METHODS: IEC-6 cells were transfected with HSP70 recombinant adenovirus vectors (Ad-HSP70) and empty adenovirus vectors. The expression of HSP70 protein was detected by Western blotting. Cultured IEC-6 cells were divided into: control group (without treatment), hypoxia/reoxygenation group (with challenge of hypoxia/reoxygenation) and Ad-HSP70 transfection group (with challenge of hypoxia/reoxygenation after Ad-HSP70 transfection). The activity of mitochondrial dehydrogenase was assessed by MTf method. The contents of cellular ATP, ADP , AMP and energy charge (EC)were determined by high-performance liquid chromatography (HPLC). RESULTS: The expression of HSP70 protein in IEC-6 cells was significantly upregulated after Ad-HSP70 transfection compared with empty adenovirus vector transfection. Compared with that in control group, the activity of mitochondrial dehydrogenase was significantly lowered in IEC-6 cells in hypoxia/reoxygenation group (P < 0.01). The activity of mitochondrial dehydrogenase in Ad-HSP70 transfection group was significantly greater than that in hypoxia/reoxygenation group (P < 0.01). Compared with those in control group,the content of cellular ATP was significantly decreased in hypoxia/reoxygenation group, the contents of cellular ADP and AMP were significantly increased. The above cell energy indices in Ad-HSP70 transfection group was similar to those in control group (P > 0.05), which were ameliorated compared with those in hypoxia/reoxygenation group (P < 0.050 or P < 0.01). The cellular EC in hypoxia/reoxygenation group (0.615 +/- 0.060) was significantly lower than that in control group (0.748 +/- 0.012, P < 0.01) and Ad-HSP70 transfection group (0.736 +/- 0.028, P < 0.01). CONCLUSION: Ad-HSP70 transfection in IEC-6 cells can upregulate the expression of HSP70, the content of cellular ATP and EC after hypoxia/reoxygenation, and protect mitochondrial function. Mitochondria may be one of main target organelles for HSP70 in protection of IEC against hypoxia/reoxygenation injury.

[Clinical study on the repair of extensive deep burn wounds with autogenous fat granules and autologous microskin grafts in mixed grafting].

OBJECTIVE: To observe the effects of autologous fat granules in mixed grafting microskin grafts on repair of extensive deep burn wounds in patients. METHODS: Twenty patients hospitalized in our ward were enrolled for autogenous self-control test in wounds on both or symmetrical parts of wounds of the trunk, and they were randomly divided into experimental (E) trol (C) groups, the wounds in E group were repaired with autologous fat granules together with microskin in mixed grafting (volume ratio 1 : 1), and in C group only autologous microskin grafting was given. Wound healing rate was measured on 30th, 45th, and 60th day after operation. Wound specimens harvested for HE staining and PCNA immunohistochemistry examination on 7th, 14th, 21st, and after operation. RESULTS: (1) The mean wound healing rate on 30th, 45th, and 60th day after E group was (56.3 +/- 3.1)%, (76.4 +/-6.1)%, (96.2 +/- 1.5)%, which were respectively higher C group [(28.3 +/-2.0)%, (47.3 +/-4.8)%, (85.4 +/- 2.2)%, P < 0.01]. HE staining showed epithelization in E group was earlier than that in C group, with regular arrangement of collagen fibers. The quantity NA positive cells in E group were larger than that in C group, and PCNA was mainly expressed cells of basal layer . CONCLUSION: Autologous fat granules in mixed grafting with autologous microskin promote wound healing.

Induction of heat shock protein 70 by sodium arsenite attenuates burn-induced intestinal injury in severe burned rats.

The present study was designed to assess the effects of induced heat shock protein 70 (HSP70) on intestinal injury after severe burn. Wistar rats were randomly divided into four groups: control group, burn group (B group), sodium arsenite pretreatment group (SA group), and sodium arsenite+quercetin pretreatment group (SA+Qu group). Plasma endotoxin and d-lactic acid content were determined at 3, 6, 12, 24, and 48h after severe burn. Samples of small intestine were obtained for histologic assessment of intestinal mucosal injury and the expression of HSP70 was assayed by Western blot. Apoptosis of the intestinal epithelial cells was examined by the TUNEL method. Results showed that SA pretreatment significantly increased expression of HSP70 in the small intestine. SA pretreatment attenuated the burn-induced increase in plasma endotoxin and d-lactic acid content, intestinal injury scores and the percentage of apoptotic intestinal epithelial cells. Co-administration of quercetin with SA abolished the SA-induced HSP70 over-expression and the beneficial effects of SA. Our findings suggest increasing expression of HSP70 induced by SA pretreatment attenuates burn-induced intestinal injury apparently by preventing apoptosis.

[Influence of heat shock protein 90 on protein serine threonine kinases expression in hypoxic cardiomyocytes].

OBJECTIVE: To explore the effect of endogenous heat shock protein 90 (HSP90) on the AKT signaling pathway of hypoxic cardiomyocytes. METHODS: The hypoxia model of neonatal rat cardiomyocyte was established. The cells were randomly divided into normal control, hypoxia, Geldanamycin (GA, with hypoxia after Geldanamycin treatment) groups. The myocardial cell activity and the expression of endogenous HSP90 and AKT were determined with MTT and Western blotting, respectively at 1, 3, 6, 12, 24 and 48 post-hypoxia hours (PHH). The apoptotic index (AI) of cardiomyocytes were determined with TUNEL method at 24 PHH. RESULTS: (1) At 24 and 48 PHH, the activity of cardiomyocytes in hypoxia group and GA group were evidently lower than that in control group (P < 0.05). The activity of cardiomyocyte in GA group began to decrease at 12 PHH, and it was obviously lower than that in hypoxia group at 48 PHH (P < 0.05). (2) At 24 PHH, the AI in hypoxia group (10.7 +/- 1.2)% was obviously higher than that in normal control group [(1.9 +/- 0.3)%, P < 0.05], while it was obviously lower than that in GA group [(26.3 +/- 5.3)%, P < 0.01]. (3) The expression of endogenous HSP90 and AKT in hypoxia and GA groups were markedly increased compared with that of normal controls at 12 PHH, and it decreased at 24 PHH in hypoxia and GA groups, especially in the latter. CONCLUSION: Endogenous HSP90 plays important roles in maintaining the cardiomyocyte activity, and its level might affect the expression of AKT.