Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes.

BACKGROUND Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. MATERIAL AND METHODS In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. RESULTS The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. CONCLUSIONS This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.

[Advancement in the research of early detection of bacterial nucleic acid in molecular diagnosis of sepsis].

Early diagnosis of sepsis helps make effective clinical decisions and improve the survival rate of patients with severe infection. However, the timely and accurate diagnosis of sepsis is still a great challenge in clinic. In order to settle the very problem, the scientists in the world have made a lot of exploration and research in the field of rapid molecular identification of pathogens. Nowadays, the nucleic acid detection of sepsis is mainly composed of 3 types of methodological strategies, either based on positive blood culture, single colonies, or directly on blood specimens. This paper presents a comprehensive overview of advances in the research of early detection of bacterial nucleic acid as molecular diagnosis of sepsis.

[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.

[Analysis of distribution characteristics and drug resistance of 2748 strains of pathogens isolated from burn patients].

OBJECTIVE: To provide epidemiological data of the distribution characteristics and drug resistance of the pathogens isolated from burn patients in recent years for guiding rational use of antibiotics in clinic. METHODS: Totally 2748 strains of pathogens were isolated from 1977 specimens (blood, catheter, wound excretion, etc.) collected from 478 patients hospitalized in Institute of Burn Research of Southwest Hospital from March 2003 to June 2011. After being identified by API strips, drug resistance of the 2748 isolated pathogens to 55 commonly-used antibiotics including gentamicin, tobramycin, piperacillin, amikacin, etc. was tested by K-B paper disk diffusion method. The WHONET 5.3 software was used to analyze the following subjects: the distribution of the pathogens with different types and different sources each year, the changes in drug-resistant rates of Gram negative bacilli, Gram positive cocci, and fungi to several antibiotics, and the changes in sensitive rates of Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Acinetobacter baumannii (AB), Candida albicans (CA) to several antibiotics. RESULTS: Among 2748 strains of pathogens, 1879 strains of Gram negative bacilli accounted for 68.38%, 628 strains of Gram positive cocci accounted for 22.85%, and 241 strains of fungi accounted for 8.77%. The isolation rate of strains from wound excretion ranked the first (1022 strains accounted for 37.19%), followed by those from respiratory tract (995 strains accounted for 36.21%) and blood (421 strains accounted for 15.32%). Strains isolated from other types of specimens were rare. Isolation rate of PA ranked the first (996 strains accounted for 36.24%), followed by SA (495 strains accounted for 18.01%) and AB (395 strains accounted for 14.37%). Isolation rate of AB showed a trend of increase year by year, but that of SA presented the opposite trend. Isolation rate of PA was quite stable. There were 484 strains of methicillin resistant SA among Staphylococci, accounting for 17.61%. Resistant rates of PA and AB to polymyxin B and polymyxin E were below 30.00%, and those of PA and AB to other antibiotics, such as the third generation cephalosporins, ß-lactams, aminoglycosides, and quinolones, were from 57.91% to 100.00%. Resistant rate of AB to minocycline was 39.68%. From 2004 to 2011, sensitive rate of PA to quinolone antibiotics showed an increasing trend year by year, but that of AB to minocycline, netilmicin, imipenem, meropenem, tobramycin, and cefoperazone/sulbactam presented the opposite trend. Resistant rates of Enterococcus faecalis, Enterococcus faecium, and SA to teicoplanin and linezolid were less than 10.00%. Resistant rate of SA, Staphylococcus epidermidis and Enterococcus faecium to vancomycin was 0. Resistant rates of SA to quinupristin/dalfopristin, minocycline, fusidic acid, and compound sulfamethoxazole were low, respectively 0.82%, 9.35%, 2.21%, and 31.85%. Sensitive rates of SA to erythromycin, clindamycin, compound sulfamethoxazole, tetracycline, and minocycline showed an increasing trend year by year. Both infection rate and resistant rate of fungi were low. The resistant rates of CA to 5 kinds of antibiotics were less than 15.00%. The sensitive rate of CA to 5-flucytosine declined slightly, and those of CA to the other 4 antibiotics showed an increasing trend year by year. CONCLUSIONS: The three dominant pathogens that cause infection in burn patients hospitalized in Institute of Burn Research of Southwest Hospital in recent years are PA, SA, and AB in order. PA and AB are outstandingly multidrug-resistant among the isolated strains. AB might replace PA as the main pathogenic bacterium that cause the death of burn patients with infection.

[Analysis of resistance phenotype and homology of Klebsiella pneumoniae in burn patients].

OBJECTIVE: To study the resistance phenotype and homology of Klebsiella pneumoniae (KPN) in burn patients with infection. METHODS: Fifty-four strains of KPN were isolated from wound excretion, blood, sputum, venous catheter, feces, and oral cavity of patients hospitalized in Institute of Burn Research of Southwest Hospital (briefly called our institute) from January 2007 to June 2011. Drug resistance of the 54 strains of KPN to 18 antibiotics commonly used in clinic, including ampicillin, ticarcillin, etc, was tested by K-B paper disk diffusion method after being identified. Extended-spectrum ß-lactamase (ESBL)-producing KPN was screened based on the drug resistance result. The positive rates of drug-resistant genes SHV, TEM, and CTX-M of the ESBL-producing KPN were detected by polymerase chain reaction. The homology of the ESBL-producing KPN was analyzed by pulse field gel electrophoresis and clustering methodology. The homology of ESBL-producing KPN isolated in each year was analyzed too. RESULTS: (1) The sensitive rate of the 54 strains of KPN to imipenem, meropenem, and ertapenem was respectively 96.30%, 92.59%, and 81.48%, that of these strains to cefotetan and cefoxitin was respectively 70.37% and 64.81%, and that of these strains to ceftazidime was 57.41%. The sensitive rates of the 54 strains of KPN to the other antibiotics were all lower than 40.00%. (2) Twenty-six ESBL-producing KPN strains were screened and the positive rate of SHV, TEM, and CTX-M was 96.15% (25/26), 76.92% (20/26), and 57.69% (15/26), respectively. Detection rate of ESBL-producing KPN strains carrying three genes at the same time was 42.31% (11/26), that of these strains carrying both SHV and TEM was 34.62% (9/26), and those of these strains carrying only a single gene were all less than 10.00%. (3) The twenty-six ESBL-producing KPN were classified into 9 gene types, with 30.77% (8/26) in type A, 19.23% (5/26) in type B, 15.38% (4/26) in type C, 11.54% (3/26) in type D, 7.69% (2/26) in type E, and the rest four strains respectively in type F, G, H, I [3.85% (1/26)]. (4) The major gene type of ESBL-producing KPN in the year of 2007 and 2010 was type A, respectively accounting for 2/3 and 1/2, while that in the year of 2009 was type B, accounting for 1/2. The three strains in 2008 was respectively in type C, E, and F. The four strains in 2011 was respectively in type A, D, H, I. CONCLUSIONS: KPN in burn patients with infection in our institute are highly resistant to commonly used antibiotics in clinic, but carbapenems antibiotics can be used for the treatment. Most of the ESBL-producing KPN strains carry two or three drug-resistant genes, and the main gene type of them is type A.

[Effect of particulate allogeneic acellular dermal matrix (PADM) combined with autologous split-thickness skin (STS) on wound healing in rats].

OBJECTIVE: To evaluate the effects of mixed grafting of allogeneic PADM and autologous STS on wound healing of full-thickness defect in rats. METHODS: Full-thickness defects with size of 6 cm x 4 cm were produced on the back of 12 SD rats, and they were divided into E group (n = 6) and C group (n = 6) according to the random number table. The wounds in E group were grafted with a mix of allogeneic PADM (expansion rate 10: 5) and autologous STS with thickness of 0.2 mm, while those in C group were grafted with autologous STS in the same thickness. The wound healing rate, survival rate, contraction rate, and expansion rate of transplanted skin were observed at post operation week (POW) 2, 3, 4, 6, 8, 12, 20. Tissue samples form wounds and surrounding normal skin were harvested at POW 20 for histopathological observation as follows. The structure of collagen fiber bundle was observed by HE staining, the diameter and gap rate of collagen fiber bundle were also measured. The distribution of type I and III collagen was observed by sircus red staining, and the contents of type I, III collagen and their ratio were also examined. Data were processed with independent samples t test, Levene test, and t' test. RESULTS: Survival rate of transplanted skin in E group at POW 2 [(76.1 +/- 13.1)%] was obviously lower than that in C group [(94.5 +/- 1.3)%, t' = 3.440, P = 0.018]. Contraction rate of transplanted skin in E, C groups at POW 3 showed significant difference [(34 +/- 8)% vs. (16 +/- 12)%, t = -3.211, P = 0.009]. Compared with those in peri-wound normal skin, collagen fiber bundles in C group showed signs of homogenization, and collagen fibers were thin with irregular arrangement. Collagen fiber structure and arrangement of composite skin in E group were similar to those surrounding normal skin with incomplete degradation of PADM. Diameter of collagen fiber bundle [(9.6 +/- 0.8) microm], gap rate between collagen bundle [(24 +/- 5)%], content of type I collagen [(80.2 +/- 5.4)%] and the ratio of type I to type III collagen (4.3 +/- 1.2) in E group were all increased as compared with those in C group [(7.3 +/- 1.4) microm (t = -3.562, P = 0.005), (17 +/- 4)% (t = -2.760, P = 0.020), (68.1 +/- 8.4)% (t = -2.981, P = 0.014), 2.3 +/- 1.0 (t = -3.204, P = 0.009)], while content of type III collagen [(19.8 +/- 5.4)%] in E group was lower than that in C group [(32.0 +/- 8.4)%, t = 2. 981, P = 0.014]. Above-mentioned indexes of collagen in wound of E group were similar to those of normal skin surrounding the wound. CONCLUSIONS: Allogeneic PADM used as dermal regeneration template is beneficial in improving collagen fiber bundle structure in dermis layer of rats with full-thickness skin wounds when repaired with autologous STS, and it accelerates maturation of regenerative dermal tissue.

[Morphological observation of human keratinocytes tridimensionally cocultured with xenogenic acellular dermal matrix].

OBJECTIVE: To establish the tridimensional culture method for tissue-engineered skin to observe the histomorphological change in human immortal KC strain (HacaT)cocultured with xenogenic acellular dermal matrix (ADM). METHODS: The ADM was prepared from SD rats by a modified method. HaCaTs were cultured in defined KC-serum free medium. HaCaTs in log growth phase were inoculated on ADM at the cell density of 2 x 10(5)/cm(2). They were submergedly cultured for 5 days and then changed to air-liquid phase culture for another 5 days. ADM and growth of HaCaTs on day 1 and 5 after cocultured with ADM were observed with scanning electron microscope. The histological change in ADM and HaCaTs on day 1, 5, and 10 after cocultured with ADM were examined by HE staining. RESULTS: The gross appearance of ADM was white with smooth and soft texture, and intact collagen bundles without cellular residue. HaCaTs adhered and stretched out pseudopodia on the surface of the ADM on day 1 after combined culture, and a monolayer of cells was formed on day 5, growing into 3-6 layers of cells on day 10 with a tendency to grow into ADM. CONCLUSIONS: SD rats ADM is benefit for the adhesion of HaCaTs and the permeation of nutrient solution, from which an engineered multiple-layered human skin can be obtained within 10 days.

[Study on influencing factors in efficiency of introducing gene into human keratinocyte (KC)].

OBJECTIVE: To observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC. METHODS: Four plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated. RESULTS: (1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols. CONCLUSIONS: It is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses

[Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin].

OBJECTIVE: To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT). METHODS: The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively. RESULTS: The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly. CONCLUSIONS: The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.

[Researches in immunological responses after burn injury in China].

For five decades it has been recognized that severe burn injury may precipitate in marked alterations in immune function, resulting in life-threatening systemic infections, sepsis, multiple organ failure, and even death. Extensive and deep burns exert widespread and profound impacts on various cells and molecules of the immune system. The general characteristics of abnormal immune responses following major burns are hyperinflammatory response and hypoimmune response of innate and adaptive immunity. These are recognized as postburn immune dysfunction (PID). The stress reaction, massive necrotic tissue, shock, infection, malnutrition and various therapeutic procedures after burns alter the microenvironment of the immune cells and molecules in which they reside, and consequently result in the changes in immune cells and their secretions in quantity and/or activity, and also aberrant signal transduction in different immune cells. These events constitute the cellular and molecular bases in the pathogenesis of PID. The main clinical consequences of PID include tissue damages and increased susceptibility to opportunistic pathogens caused by refractory inflammation and suppressed adaptive immunity. In order to decrease the morbidity of these lethal complications, efforts to improve the immune dysfunction after burn injury have been made not only at the integral level of etiological factors, but also at the cellular and molecular levels of its mechanisms. In this review, all these above-mentioned aspects of PID are comprehensively discussed.

[The ectopic expression and quantitative analysis of Integrin beta1 during wound healing process after mixed microskin transplantation].

OBJECTIVE: To study the mechanism of promoting wound healing with mixed grafting of autologous and allogeneic microskin in rats. METHODS: Fifteen male Wistar rats served as alloskin donor. Forty-five female SD rats with full-thickness skin defect served as recipients in the study. In part one experiment, 27 SD rats were randomly divided into group I (n = 9, without allogeneic microskin), group II (n = 9, with mixed grafting of allogeneic microskin at area expansion rate of 10:1); group III (n = 9, with mixed grafting of allogeneic microskin at area expansion rate of 10:3) with grafting with the same amount of autologous microskin at area expansion rate of 10:1. In part two experiment, 18 SD rats were also divided into group I (n = 6, with autologous microskin only); group II (n = 6, with mixed grafting of autologous and allogeneic microskin with area expansion rate of 20:1 and 20:3 respectively); group III (n = 6, with mixed grafting of autologous and allogeneic microskin with area expansion rate of 20:1 and 20:6, respectively). Biopsy samples were obtained from healed wound area of SD rats in each group at different time points after operation. The histological changes, epidermal thickness, and immunohistochemical staining of Integrin beta1 were observed. RESULTS: (1) HE staining showed the thickness of epidermis in each group increased obviously, and various amounts of mononuclear cell infiltration and different degrees of vasodilation appeared in the dermal layer during 2 - 4 weeks. (2) Epidermal thickness in group II and III of part one experiment were significantly thicker than that in group I during 2 - 4 weeks after operation (P < 0.05), and the similar result was also seen in part two experiment on 3 and 4 weeks after operation (P < 0.05). (3) A positive staining pattern for Integrin beta1 was seen in the suprabasal layers (especially in the spinous and granular layers) in all groups. In part one experiment, the expression of Integrin beta1 in group II and III were obviously higher than that in group I on 2 week after operation (P < 0.01), and the expression of Integrin beta1 in group II (10 982 +/- 2169) was also higher than that in group III (4240 +/- 512, P < 0.01); the expression of Integrin beta1 in group II was still higher than that in group I and III (P < 0.01) 3 and 4 weeks after operation. In part two experiment, the expression of Integrin beta1 in group III (1618 +/- 171) was higher than that in group I 3 weeks after operation (1060 +/- 146, P < 0.05). CONCLUSION: The ectopic and increased expression of Integrin beta1 was closely associated with the proliferation and differentiation of epidermal cells, wound reepithelialization and thickened epidermis in mixed grafting of autologous and allogeneic microskin. Integrin beta1 may be responsible in promoting wound healing.

[The present and future of mixed skin grafting].

Skin grafting is one of the two major surgical procedures to repair losses of skin tissue. For severely burned patients, the autologous donor skin is not enough to cover extensive wounds. Therefore, several types of mixed skin grafting have been developed in the past fifty years. Two of them, the intermingled skin grafting and microskin grafting overlaid by a sheet of allogeneic skin, have been widely applied in the treatment of major deep burn patients, resulting in a and significant decreased of the mortality. Two other methods, i. e, mixed grafting of autologous and allogeneic microskin or keratinocytes are still under investigation. In this review, we summarize the evolution of mixed skin grafting, introduce the classification of mixed skin grafting, analyze their merits and demerits, and distinguish it with composite skin grafting or transplantation. The perspective of mixed skin grafting will be focused on three aspects, i. e, prolonging the survival of allograft by induction of donor-specific immune tolerance, accelerating the wound healing by strengthening the interactions between the keratinocytes and fibroblasts, and decreasing the wound scarring and contraction by optimizing the amounts of cellular or acellular allogeneic dermis.

[Ectopic expression of keratin 19 and integrin beta1 during wound healing process after microskin grafting in rats].

OBJECTIVE: To observe the characteristics of keratin 19 and integrin beta1 expressions in the wound after microskin grafting , and to investigate the healing mechanism. METHODS: Full layer skin defects were created in twenty Sprague-Dawley rats and they were divided into two groups, i.e., A group (with grafting of autologous microskin accounting 10% in weight of epidermis loss from skin defect), B group (with grafting of autologous microskin and allogeneic microskin, accounting 10% and 40% weight of epidermis loss respectively in skin defect). The wound healing rate and contraction rate were observed at 2,3,4 post-grafting week (PGW), and the expression and distribution of keratin 19 and integrin beta1 were observed at 2 and 4 PGW. RESULTS: The wound healing rate in the B group on 2 and 3 PGW was obviously higher than that in A group [(85 +/- 5)% vs. (53 +/- 10)%, (84 +/- 8)% vs. (65 +/- 9)%, P < 0.01]. No obvious difference in wound contraction rate between the two groups was observed on the 2, 3 and 4 PGW (P > 0.05). Cells with expression of keratin 19 and integrin beta1 were observed in the suprabasal layers of the epidermis in healing wound, but not in the basal membrane. Integrin beta1 positive expression cells were not observed in the suprabasal layers until 4 PGW. CONCLUSION: Mixed grafting with autogenous and allogenous microskin can improve wound healing. Ectopic expression of keratin 19 and integrin beta1 exists during wound healing process after microskin grafting.

[Prolongation of the survival of skin allografts by intravenous injection of neuraminidase-treated donor bone marrow cells in rats].

OBJECTIVE: To optimize the best concentration of neuraminidase (Neu) that enhances the migration of neuraminidase (Neu)-treated donor bone marrow cells (dBMCs) to the liver, and observe the influence of short-term cyclosporin A(CsA) application combined with intravenous injection (i.v.) of Ne treated dBMCs on the survival of skin allografts. METHODS: The experiment consisted of two parts. For selection of an appropriate concentration of Neu, 26 female Wistar rats were randomly divided into four groups. The dBMCs were prepared by routine method and treated with four concentrations (0, 0.5, 1.0, 2.0 U/ml) of Neu at 37 degrees C for 30 min. The untreated and Neu-treated dBMCs were labeled by 99mTc, and injected via the tail veins to female Wistar rats in each group, respectively. After five hours, the radioactivity of various organs collected from sacrificed rats was measured by a gamma counter, and the values were expressed as percentage of total radioactivity of all organs from the same rat. To observe the survival of skin allograft, 23 male Wistar rats were randomly divided into control group, untreated dBMCs group and Neu-treated dBMCs group. All rats in each group were grafted with skin allografts from male Sprague-Dawley (SD) rats. The dBMCs from the same donor without and with Neu treatment by the concentration selected from the above experiment were injected via the tail veins of female Wistar rats in untreated dBMCs group and Neu-treated dBMCs group, respectively. Rats in untreated dBMCs group and Neu-treated dBMCs group received CsA (10 mg/kg) through intraperitoneal injection (i.p.) at 2 and 5 days post-grafting. Neither dBMCs or CsA were given in the control group. The survival of allograft skin in each group was checked and photographed daily after 5 days post operation. RESULTS: When the concentration of Neu was 1.0 U/ml, the percentage of dBMCs in liver was (75.3 +/- 9.8) %, which was obviously higher than that in 0 U/ml group [(58.9 +/- 4.2%)], (P < 0.01), indicating that the optimal concentration of Neu was 1.0 U/ml. The survival time of skin allografts in rats of Neu-treated dBMCs group was prolonged significantly in comparison with that of the rats in dBMCs group without Neu treatment (P < 0.01). The survival time in both dBMCs group and Neu-treated dBMCs group was longer that of control group (P < 0.01), and it was prolonged in Neu-treated dBMCs group compared with that in dBMC group. CONCLUSION: Administration of proper concentration of Neu can increase the affinity of dBMCs to the liver, and promote the Neu-treated dBMCs to migrate to liver. The intravenous injection of Neu-treated dBMCs combined with short-term CsA administration can delay the rejection of skin allografts in rats.

[Study on the optimal ratio of autoskin and alloskin during mixed microskin grafting].

OBJECTIVE: To investigate the influence of different amount of allogeneic microskin in mixed grafting with certain quantity of autologous microskin on wound healing in rats. Methods Male Wistar rats served as alloskin donor rats. Forty female SD rats with full thickness skin defect were enrolled in the study, and they were randomly divided into four groups, i.e. group I (n=10, with allogeneic microskin graft at area expansion rate of 10:3); group II (n=10, with autologous microskin graft at area expansion rate of 10:1); group III (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1, respectively); group IV (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1 and 10:3, respectively). The wound healing rate, wound contraction rate and histological changes were observed at the 2, 3 and 4 post graft weeks (PGW). RESULTS: (1) In group I, there was mainly granulation tissue with some de novo epithelial cells appearing at the wound edge along with the rejection of grafted allogenous skin in the rat wound. In group II, there was still some granulation tissue remaining at 2 PGW due to insufficient amount of microskin. However, the wounds in the mixed grafting group appeared almost totally epithelialized. (2) Various amounts of mononuclear inflammatory cell infiltration and different degrees of angiectasis were observed in the dermal layer after the skin grafting in all groups, especially in group II and IV. There was thickening of the epithelial layer in all groups except group I. (3) The wound healing rate decreased obviously along with the development of rejection in group I at 2 to 4 PGWs. The wound healing rate was (55 +/- 26)% in group II, which was obviously lower than that in group III (88 +/- 6)% and in group IV (76 +/- 10)% at 3 PGWs (P < 0.01). (4) The contraction rate of the wound in group IV (69 +/- 7)% was much higher than that in group I (58 +/- 11)% at 3 PGWs (P < 0.05), and there was no difference among all the other groups. CONCLUSION: Wound healing can be obviously accelerated by mixing some autologous microskin with appropriate amount of alloskin. Moreover, certain amount of autologous microskin (expansion rate 10:1) mixed with the same proportion of allogeneic microskin seems to be more beneficial in promoting wound healing.

[Influence of signal transduction modulators on the secretory function of T lymphocytes in severely scalded mice and its mechanism].

OBJECTIVE: To investigate the influence of various signal transduction modulators on the splenic T lymphocytes secretion of IL-2 and IL-10 in severely scalded mice, and to explore its mechanism. METHODS: The mice were inflicted with 18% TBSA full-thickness scald by high-pressure heat vapour, and T lymphocytes were isolated from murine splenocytes through nylon wool column at 12 and 96 post-scald hours (PSH). Then the cells were divided into following groups: i. e. control, scald, scald and modulator [1 ml of 50 micromol/L PKC inhibitor ( H-7) , 30 micromol/L tetradecanoylphorbol-13-acetate (TPA) , 10micromol/L nonreceptor tyrosine protein kinase inhibitor (herbimycin) , 25 microg/ml of mitogen activated protein kinase kinase inhibitor (PD098059) , 100 nmol/L Calcium ionophore ( A23187) were added to the cells, respectively] groups. The scald group was subdivided into S1 (with scald at 12 PSH) and S2 (with scald at 96 PSH) groups. The modulator group was subdivided into modulator, S1 and modulator( the modulators were added into cells at 12 PSH) , and S2 and modulator( the modulators were added to cells at 96 PSH) groups. The influence of modulators to T lymphocyte secretion of IL-2 and IL-10 were observed. RESULTS: After the addition of H-7, the IL-2 and IL-10 levels in each group were obviously lower than that in controls( P <0. 05 or 0.01) , and that in S1 and H7 group, S2 and H7 group were obviously lower than that in scald group at corresponding time-points( P <0.01). The levels of IL-10, and especially IL-2 were elevated by TPA, but they were markedly lower than that in control group after PD098059 pretreatment. The secretion of IL-2 and IL-10 was significantly suppressed by herbimycin in S1 and herbimycin, and S2 and herbimycin groups, but those in Sl and A21387[ (2 417+/-39) pg/ml, (2 793+/-25)pg/ml] , S2 and A21387 [ (921+/-50) pg/ml, (2 633+/-35)pg/ml] groups were evidently higher than those in S1[ (1 542+/-40)pg/ml, (2 390+/-15)pg/ml] , S2 [(328+/-19)pg/ml, (1 618+/-21)pg/ml,( P <0.05 or <0.01)]groups. CONCLUSION: PKC, calcium, MAPKK and TPK play critical roles in the dysfunction of splenic T lymphocyte secretion of IL-2 and IL-10 in severely scalded mice, among which TPK and PKC are mainly targeted to IL-2 secretion, and MAPKK is targeted to IL-10 secretion. TPA and A23187 can markedly rectify the disturbance of IL-2/IL-10 secretion ratio by increasing the IL-2 secretion after scald.

[Preliminary study on the release of DNA from Pseudomona aeruginosa induced by piperacillin/tazobactam in vitro].

OBJECTIVE: To observe the release of DNA from Pseudomonas aeruginosa (P. aeruginosa) induced by different concentrations of piperacillin/tazobactam (Piper) in vitro. METHODS: The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Piper against 1244 strain (ATCC 27317) of P. aeruginosa were determined, respectively. This strain of P. aeruginosa was separately cultured with Piper in different concentrations at 37 degrees C for 4 h and 24 h. The samples of cultural supernatant were filtered and electrophoresis was conducted in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: This strain of P. aeruginosa was sensitive to Piper. The bacterial DNA was not detected in 4-h cultured P. aeruginosa either with or without Piper by this method. The bacterial DNA molecules could be detected in 24 h samples in cultures without Piper, and they were displayed in two zones of molecular weight over 2000 base pairs (bp) and lower than 100 bp. Similar results were observed when the MIC of piper (0.002, 0.004 g/L) were under the MIC measured at the 3rd time (0.008 g/L), but there was much more bacterial DNA with molecular weight lower than 100 bp. When Piper concentration was higher than its MIC, only smaller quantities of bacterial DNA in the area with molecular weight lower than 400 bp could be detected in 24-h culture samples. CONCLUSION: A certain amount of bacterial DNA was released from P. aeruginosa under its natural growth circumstance. Different concentrations of Piper showed different effects on DNA release, in regard to its quantity and molecular weight, from P. aeruginosa cultures.

[Release of DNA from Pseudomonas aeruginosa in vitro during spontaneous growth and treatment with ciprofloxacin].

OBJECTIVE: To observe the DNA release from Pseudomonas aeruginosa (P. aeruginosa) during spontaneous growth and exposure to different concentrations of ciprofloxacin (Cipro) in vitro. METHODS: The P. aeruginosa 1244 strain (ATCC 27317) was selected because it was sensitive to Cipro in vitro. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Cipro against this strain were determined, respectively. Different concentrations of Cipro were cultured with this strain at 37 degrees C for 4 h and 24 h. The samples of culture supernatant were filtered and electrophoresed in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: The MIC and MBC of Cipro were 0.25 - 0.5 mg/L. The free bacterial DNA in 4 h culture samples with or without Cipro could not be detected by this method. The certain amount of free bacterial DNA molecules in 24 h culture samples without antibiotic appeared at the two zones whose molecular weights were more than 2000 bp and less than 100 bp. The large amount of free bacterial DNA molecules showed at three zones in 24 h culture samples with Cipro when its concentrations were much lower than its MIC. In terms of DNA molecular weight, the first two zones were above 2000 bp, and the third zone was below 100 bp. There was no detectable DNA release from bacteria in 24 h culture samples when Cipro was at or above its MIC. CONCLUSIONS: The certain amount of bacterial DNA were released from P. aeruginosa in the spontaneous growth. Different concentrations of Cipro had quite differential effects on the DNA release from P. aeruginosa in quantities and molecular weights in vitro.