肠通透性改变的研究初报

本文用气相色谱测定烧伤猪和羊的ＭＳＯＦ动物模型尿内乳果糖和甘露醇分泌比率（Ｌ／Ｍ）。烧伤猪治疗组７２小时Ｌ／Ｍ分泌比率为０．０２４±０．００７,而对照组为０．０２６±０．００３,伤前Ｌ／Ｍ分泌比率为０．００７±０．００５,与伤前比较,无显著性差异。这说明Ｌ／Ｍ分泌比率升高与烧伤前期治疗及伤后天数无关。而在羊的ＭＳＯＦ的动物模型内Ｍ组中,输入内毒素４小时及伤后３天的Ｌ／Ｍ分泌比率为０．６２±０．２、１．７０±０．６０,对照组为０．２８±０．２５、０．２０±０．１０,伤前为０．０２９±０．０２,Ｌ／Ｍ分泌比率升高显著（Ｐ＜０．０５）,提示内毒素血症可能与肠通透性改变之间具有密切联系。     

建立肠通透性改变测定方法

我们采用国产的气相色谱（ＳＰ－３４００型）建立了肠通透性改变的测定方法,主要是测定乳果糖和甘露醇的浓度,以确定肠粘膜致密结构情况,如出现甘露醇含量不变,乳果糖吸收增加,说明肠通透性增加,测定标准液中甘露醇和乳果糖,含量为５０～５００ｎｍｏｌ和１０～２００ｎｍｏｌ时,随进样量改变而呈线性改变,与ｓｈｉｐｐｅｅ报告一致。此方法结果稳定,重复性好,可用于烧伤病人及动物模型的肠通透性改变的检测。     

高压液色谱用于肠通透性的测定

目的 建立一种测定肠通透性的可靠方法。方法 以甘露醇和乳果糖为探测剂,用带脉冲电化学检测器的高压液相色谱仪对２０例健康成人样品中的甘露醇和乳果糖进行检测。结果 ２０个样品中的甘露醇和乳果糖均被检测到,其最低质量浓度分别为０．２０２８ｍｇ／Ｌ和０．１９３０ｍｇ／Ｌ,回收率分别为９８．２％和１０４．３％。结论 用带脉冲电化学检测器的高压液相色谱仪测定尿中的甘露醇和乳果糖是一种灵敏度高,特异性强,操作简     

烧伤后免疫抑制引起肠道细菌易位和脓毒症的病理学研究

近年来,创伤后体内细菌易位造成的内源性感染逐渐受到重视,为了探讨细菌易位的条件和途径等机理。本文利用烧伤后注射大剂量地塞米松的大鼠动物模型,模拟烧伤后应激状态下体内肾上腺皮质激素分泌增多,免疫机能下降的条件,观察肠道和其它脏器的病理改变。观察结果证实,烧伤与大剂量皮质激素确有引起肠道细菌易位和内源性感染,造成脓毒症的叠加作用。其基本条件包括肠道菌群微生态平衡的改变,宿主免疫机能下降和肠粘膜屏障的破坏。易位的肠道细菌主要是通过肠粘膜的坏死灶,上皮细胞坏变缺损处进入肠粘膜淋巴管和小静脉的。超微结构改变包括肠上皮细胞表面的微绒毛倒伏、变形、脱落、上皮细胞顶端崩解破坏,细胞间隙泡状增宽。脓肿中多数细菌微细结构完好,粒细胞和吞噬细胞胞浆中有多量糖元颗粒聚集,提示糖皮质激素可能影响吞噬细胞的糖代谢而降低其吞噬功能,使易位的细菌得以定植形成脓肿感染。     

双歧杆菌活菌制剂对烧伤鼠肠源性感染的预防作用

本文以３０％烫伤同时口服抗生素,喂饲双歧杆菌制剂的大鼠为模型,将Ｗｉｓｔａｒ大鼠分为烧伤治疗组,烧伤对照组及正常对照组。并于伤后２４、４８、７２、９６小时分别观察盲肠内容物菌群,粘蛋白,分泌型免疫球蛋白Ａ（ＳＩｇＡ）;肠道细菌移位率;腔静脉内毒素含量。结果发现烧伤后肠道菌群有不同程度的变化,念珠菌迅速过度生长、厌氧菌下降、肠内需厌氧菌比例严重失调,治疗组中粘蛋白含量,肠道细菌移位率均明显小于烧伤对照组;而ＳＩｇＡ含量则高于对照组,菌群改变较对照组轻。故认为双歧杆菌制剂有助于预防烧伤肠源性感染的发生。     

甘露醇对拟南芥基因组DNA甲基化的影响

以拟南芥（Arabidopsis thaliana）为材料,研究不同浓度甘露醇处理下拟南芥幼苗生长发育及其基因组DNA的甲基化水平和变化模式。结果表明,用50、100、150和200mmol·L―1甘露醇处理拟南芥种子会对拟南芥幼苗的形态特征和生长态势产生影响;甲基化敏感扩增多态性（methylation-sensitive amplification polymorphism,MSAP）分析表明,经50、100、150和200mmol·L―1甘露醇处理后,基因组DNA甲基化比率分别为17.75%、21.15%、15.49%和46.10%。甘露醇处理使拟南芥发生基于DNA甲基化水平和模式改变的表观遗传变异。与对照相比,在50、100、150和200mmol·L-1甘露醇处理下拟南芥幼苗基因组DNA的甲基化和去甲基化比率分别为5.78%、15.48%、10.71%、33.73%及10.98%、5.36%、8.33%、7.69%。由此推测,5-甲基胞嘧啶百分含量随着甘露醇胁迫的增强而发生不同程度的变化。     

甘露醇对拟南芥基因组DNA甲基化的影响

以拟南芥(Arabidopsis thaliana)为材料, 研究不同浓度甘露醇处理下拟南芥幼苗生长发育及其基因组DNA的甲基化水平和变化模式。结果表明, 用50、100、150和200 mmol·L^―1甘露醇处理拟南芥种子会对拟南芥幼苗的形态特征和生长态势产生影响; 甲基化敏感扩增多态性(methylation-sensitive amplification polymorphism, MSAP)分析表明, 经50、100、150和200 mmol·L^―1甘露醇处理后, 基因组DNA甲基化比率分别为17.75%、21.15%、15.49%和46.10%。甘露醇处理使拟南芥发生基于DNA甲基化水平和模式改变的表观遗传变异。与对照相比, 在50、100、150和200 mmol·L^–1甘露醇处理下拟南芥幼苗基因组DNA的甲基化和去甲基化比率分别为5.78%、15.48%、10.71%、33.73%及10.98%、5.36%、8.33%、7.69%。由此推测, 5-甲基胞嘧啶百分含量随着甘露醇胁迫的增强而发生不同程度的变化。     

烧伤患者肠道内与尿中免疫球蛋白A含量变化关系初步研究

为了解烧伤患者肠道内与尿中免疫球蛋白A(IgA)含量变化关系 ,采用ELISA方法测定了大面积烧伤患者尿和大便标本中IgA的含量。结果表明 ,烧伤后肠道内IgA含量明显减少 ,于伤后第三周达到最低点 ,而后回升。尿中IgA水平在伤后的变化与肠道相反 ,于伤后第一周即明显升高并达到高峰 ,而后逐渐降低 ,至伤后第四周已接近对照水平 ,两组均数呈负相关 (r=-0 .763 )。提示烧伤后肠道与尿中IgA含量的变化可能存在一定的关系 ,由于尿标本的留取方便、及时 ,测定尿中的IgA含量对于临床判断烧伤患者肠道屏障功能有一定的意义     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

用重组痘苗病毒表达pGHcDNA的研究

构建了含有ｐＧＨｃＤＮＡ的重组痘苗病毒,用ＥＬＩＳＡ证明该重组病毒在被感染的ｈ１４３细胞中,可表达出猪生长激素并将之分泌到培养基中,表达量约为１．０５μｇ／１０￣６细胞（２４ｈ）。用定位免疫化学法进一步证明该病毒可感染小鼠并在小鼠体内表达ｐＧＨｃＤＮＡ。同时还构建了含双拷贝ｐＧＨｃＤＮＡ的重组痘苗病毒,并证明其ｐＧＨ表达量比单拷贝重组病毒有明显提高,约为１．５０μｇ／１０￣６细胞（２４ｈ）。     

Properties of Staphylococcus aureus in case of the unfavorable course of burn infection

Clinical and microbiological study of wound discharge from 35 patients demonstrated a relationship between biological properties of S. aureus, the causative agent of burn infection, and the course of the infected burn trauma. The prognostic importance of the antioxidant properties of these staphylococci was established: in cases of the unfavorable course of the burn process they showed essentially greater resistance to peroxinitrite and higher superoxide dismutase activity in comparison with the cultures isolated from patients with uncomplicated wound infection.     

Comparison of silver sulfadiazine and gentamicin for topical prophylaxis against burn wound sepsis.

Daily prophylactic application of either 1.0% silver sulfadiazine cream or 0.1% gentamicin cream was compared for effectiveness in preventing bacterial colonization of burn wounds and sepsis. Pseudomonas aeruginosa colonized the wounds of 37% of the 38 patients treated with silver sulfadiazine and 30% of the 33 patients treated with gentamicin; gentamicin-resistant P. aeruginosa colonized the wounds of 21% of the patients treated with gentamicin. Staphylococcus aureus colonization occurred in 55% of the patients treated with silver sulfadiazine, whereas colonization with Candida species occurred in 58% of the patients treated with gentamicin. Although gentamicin-resistant organisms caused no deaths their repeated appearance resulted in discontinuation of prophylaxiz with gentamicin cream. The next year P. aeruginosa strains resistant to gentamicin were isolated from burn wounds of only two patients who had not previously received parenteral therapy with gentamicin or tobramycin. Gentamicin cream should be reserved for treating patients with wounds infected by gentamicin-sensitive P. aeruginosa and those allergic to sulfa drugs. For most patients with burn wounds silver sulfadiazine is safe and effective as an antibacterial agent for topical prophylaxis.     

Silver-resistant Enterobacteriaceae from hospital patients

The inclusion of agar medium containing 0.5 mM AgNO3 in the hospital laboratory replicating system for routine antibiotic-susceptibility determinations resulted in identification of species of Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, and Citrobacter freundii) with silver resistance. Since the study began in October, 1975, 11 in-hospital patients receiving silver sulfadiazine for burn wound prophylaxis have yielded silver-resistant bacteria from their infected burns. During this treatment routine burn-site cultures from these patients yielded 230 isolates of Enterobacteriaceae, including 211 which were sulfonamide-resistant, 97 of which were also silver-resistant, and 38 of which were untested for silver resistance. Seven silver-resistant but sulfonamide-sensitive isolates were incidentally recovered from respiratory specimens from four nonburn patients with silver tracheostomy tubes, one silver-resistant sulfonamide-sensitive isolate was recovered from a small infected burn on the foot of an Emergency Room patient. Previous treatment of this burn was unknown. Representative AgNO3-resistant E. coli isolates from four patients were serologically untypable. Serotyping of representative isolates of K. pneumoniae showed a diversity of types except from two patients who had been in the same ward at the same time.     

Rosette Inhibition Test in Severely Burned Patients

The rosette inhibition test has been advocated as a test of immune reactivity in patients given transplants. We have done the test in lymphocytes from patients with severe burns, non-burnt control patients, and normal subjects. There was no correlation with the period of immunosuppression which follows a severe burn, though both groups of patients showed a wider range of minimal inhibitory concentrations (M.I.C.) than the normal group. Wide day-to-day variation of M.I.C. in burn and control patients was noted; this seemed to correlate with the ratio of sodium to potassium excreted in the urine. In-vitro experiments suggest that the M.I.C. is correlated with a plasma constituent which may be similar in action to hydrocortisone and that the rosette inhibition test is not a direct measure of immune reactivity.     

Prognostic significance of erythroblasts in burns

Changes in hematopoiesis that occur in humans after a burn injury may have important effects on morbidity and mortality. In patients with a variety of severe diseases, the presence of erythroblasts in peripheral blood is known to be indicative of a poor prognosis. However, the prognostic significance of erythroblasts in peripheral blood of burn patients has not yet been estimated. This study included 464 consecutive burn patients, of whom 81 did not survive their injuries (17.5 percent). Together with erythroblasts in blood, data on age, sex, total burn surface area, third-degree burn, inhalation trauma, white blood cell count, C-reactive protein, and hemoglobin were studied. The mortality rate of patients with erythroblasts in peripheral blood (n = 53) amounted to 56.6 percent (n = 30; total burn surface area, 39 percent), which is significantly higher (p < 0.001) than the mortality rate of patients without erythroblasts (12.4 percent, n = 51; total burn surface area, 18.69 percent). None of the 10 patients with more than 1000 erythroblasts x 10/liter survived. The detection of erythroblasts in the peripheral blood of burn patients is highly predictive of death, with the odds ratio after adjustment for the other known prognostic factors being 8.3 (95 percent confidence interval, 4.5 to 15.3). Erythroblasts were detected for the first time on average 10 +/- 4 days (median, 6 days) after admission and 13 +/- 6 days (median, 7 days) before death. Detection of erythroblasts in burn patients is of high prognostic power with regard to in-hospital mortality, providing physicians with a strong prognostic method with which to identify seriously threatened patients. It seems attractive to think about an incorporation of erythroblasts into further refinements of burn scores.     

Serum procalcitonin as an index of inhalation injury in burns.

The molecular heterogeneity of serum immunoreactive calcitonin (iCT) was analyzed from a prospective study of 41 burn patients. Using different region-specific anticalcitonin antisera, the ratio of mid-region-recognizing to carboxyl terminal-region-recognizing iCT was found to increase acutely in those who subsequently died. The highest ratios occurred in those who died early of respiratory complications. Sephadex chromatography and reversed-phase HPLC demonstrated that the serum iCT circulated predominantly in the large molecular mass prohormone form (16 kDa). In comparison, iCT of normal human lung and of normal thyroid was shown to consist primarily of smaller monomeric mass forms. Furthermore, in 12 normal volunteers who were evaluated with a calcium-pentagastrin infusion, the ratio of iCT levels did not differ from the baseline ratio despite a 50% increase in serum iCT. These results suggest that in burns, the inhalational injury-associated hypercalcitonemia is characterized by a preferential release of procalcitonin; a form of constitutive secretion. The measurement of serum procalcitonin levels would appear to be a useful prognostic indicator of the severity of inhalational injury occurring in burn patients.     

烧伤患者创面与环境中病原微生物耐药性及相关性的研究

了解烧伤科病人创面感染、环境监测的主要致病菌及其耐药性,探讨二者的关系。运用Sceptor鉴定仪对检出菌进行鉴定,K-B法对主要致病菌进行药敏试验。结果表明,引起烧伤创面感染的主要致病菌为不动杆菌(24.6%)、MRSA(19.8%)、绿脓杆菌(16.7%),烧伤科病房环境分离出的主要细菌为不动杆菌(17.7%)、绿脓杆菌(17.7%)、MRSA(13.3%),创面分离的致病菌与环境检出的细菌具有一定的相关性。特别是MRSA、MRSE,从创面与环境二者分离出的菌株具有相似的抗菌谱,这说明环境因素特别是医陪护人员的手表、物表成为该菌在烧伤病房传播的重要环节。     

Copper Uptake by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Infected Burn Patients

Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram positive bacteria Bacillus thuringiensis strain Israelis as well as Gram negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.     

LONG-TERM ALTERATION OF DAILY MELATONIN, 6-SULFATOXYMELATONIN,CORTISOL, AND TEMPERATURE PROFILES IN BURN PATIENTS: A PRELIMINARY REPORT

Melatonin, which shows a robust nycthemeral rhythm, plays the role of an endogenous synchronizer, able to stabilize and reinforce circadian rhythms and maintain their mutual phase relationships. Additionally, melatonin is a potent antioxidant and displays immunological properties. Because free radical generation, immune dysfunction, and sleep and metabolic disorders are involved in the short- and long-term pathophysiology of the burn syndrome, we undertook the study of daily urine melatonin, 6-sulfatoxymelatonin (aMT6s, the main hepatic melatonin metabolite), and cortisol variations plus temperature profiles in burn patients using a non-invasive protocol. Eight patients (6 males, 2 females) were studied on three occasions after admission to the intensive care unit (early session: days 1 to 3; intermediate session: day 10; late session: days 20 to 30). Melatonin, aMT6s, and free cortisol levels were determined in urine samples collected at 4 h intervals over a continuous 24 h span. Core temperature was recorded daily. Controls consisted of healthy subjects in the same age range. Cosinor analysis of the data provided an evaluation of mesor, amplitude, and acrophase of circadian rhythms. Also, we calculated day (D), night (N), and 24 h hormone excretions, N/D ratio for melatonin and aMT6s, and D/N ratio for cortisol. These data were analyzed using Kruskal-Wallis test followed by multiple comparisons. Cosinor analysis did not detect a circadian rhythm in melatonin, aMT6s, or cortisol in any of the three sessions. D melatonin excretion displayed a major increase, resulting in a decreased N/D melatonin ratio, and the melatonin mesor (24 h mean) was increased in the early session, compared with controls. For aMT6s, only the early N/D ratio was decreased, and the mesor of the intermediate session increased. These results were not the consequence of hepatic and/or kidney alteration, as the patients' hepatic and renal parameters were in the normal range. The D and N melatonin/aMT6s ratios of controls and patients were similar, and the aMT6s profiles were superimposed on the melatonin ones, mainly during the day. The D, N, and 24 h cortisol values were increased in all sessions, except for the D level of the early session. The consistently increased mesors in the three sessions provided confirmation. The core temperature profiles were abnormal in all three sessions, mainly during the night, although there was a tendency toward normalization with time. The individual mesors were consistently increased compared with controls. Globally, the abnormalities we report could participate in the pathophysiology of short- and long-term alterations observed in burn syndrome, especially disturbances of sleep, metabolism, and immune function. (Author correspondence: bruno.claustrat@chu-lyon.fr).     

Study on the pathogenesis of pathophysiological changes of burn systemic infection

The present prospective study showed that incidence of systemic infection in severe burn patients was 30.9%. Toxic shock and multiple organ failure (MOF) developed in all patients with uncontrolled systemic infection. Both morbidity and mortality of MOF were 76.5%. In the infection group, plasma TXB(2) and TXB(2)/6-keto-PGF(1alpha) ratio increased markedly. Their changes were closely correlated with the clinical course and deterioration of systemic infection. Circulatory platelet aggregate ratio decreased significantly, while myocardiac enzyme spectrum greatly increased. Thrombi were observed in visceral tissues from patients dying of systemic infection. These suggested that TXA(2)/PGI(2). imbalance promoting microaggregate and thrombus formation may be one of the pathogenic effects of toxic shock and MOF in burn patients.