Sensitive chemiluminescence immunoassay for the determination of rat serum alpha1-acid glycoprotein.

We present the establishment of a sensitive immunoassay for the determination of alpha1-acid glycoprotein (orosomucoid, AGP) in rat serum. The assay is based upon antigen capture by surface-immobilized specific polyclonal rabbit anti-AGP antibodies with biotinylated rat AGP (rAGP) as the tracer, and formatted as competitive enzyme immunoassay. Signaling is performed by streptavidin-conjugated horseradish peroxidase. Enzyme activity is quantified by an enhanced chemiluminometric method, allowing the sensitive detection of rAGP serum levels in small sample volumes.     

核因子kB活性在心肺脑复苏后的变化

目的观察心搏骤停患者经心肺脑复苏（CPCR）后核因子kB（NF-kB）活性和肿瘤坏死因子-α（TNF-α）水平的变化及意义。方法以电泳迁移率变动分析法（EMSA）检测外周血单个核细胞的NF-kB活性，放射免疫法测定血清TNF-α水平。结果NF-kB活性、TNF-α水平和APACHEⅡ评分在复苏后多脏器功能障碍综合征（PR-MODS）组和死亡组分别明显高于非PR-MODS组和存活组（P〈0．05）；NF-kB活性、TNF-α水平和APACHEII评分均呈正相关（P〈0．05）。结论心搏骤停和CPCR过程可以使NF-kB激活、TNF-α表达增加，这对PR-MODS的发生、病情轻重具有重要决定作用。     

核因子κB活性在心肺脑复苏后的变化

目的观察心搏骤停患者经心肺脑复苏(CPCR)后核因子κB(NFκB)活性和肿瘤坏死因子α(TNFα)水平的变化及意义。方法以电泳迁移率变动分析法(EMSA)检测外周血单个核细胞的NFκB活性,放射免疫法测定血清TNFα水平。结果NFκB活性、TNFα水平和APACHEⅡ评分在复苏后多脏器功能障碍综合征(PRMODS)组和死亡组分别明显高于非PRMODS组和存活组(P<0.05);NFκB活性、TNFα水平和APACHEⅡ评分均呈正相关(P<0.05)。结论心搏骤停和CPCR过程可以使NFκB激活、TNFα表达增加,这对PRMODS的发生、病情轻重具有重要决定作用。     

Immunoassay of alpha 1-acid glycoprotein in the Cobas Bio centrifugal analyzer

There is an increasing demand for quantification of serum alpha 1-acid glycoprotein (AAG, orosomucoid) in studies evaluating the protein binding of highly bound basic drugs. This paper describes an adaptation of an automated immunoturbidimetric assay for this protein to the Cobas Bio centrifugal analyzer. Replicate analyses of aliquots from six different solutions were used in determining precision. We also analyzed 367 patients' serum samples, in duplicate, to determine the distribution of AAG in hospitalized patients. The intra- and inter-run CVs ranged from 1.3% to 4.4% and from 0.6% to 6.6%, respectively. AAG concentrations in patients' samples ranged from 0.38 to 3.16 g/L. Results by this method correlate well with those by radial immunodiffusion, with no significant amount of bias between the two methods. This immunoturbidimetric procedure is faster and less expensive than currently used radial immunodiffusion techniques, and precision is acceptable.     

Decreased elimination of unbound prazosin in the presence of alpha 1-acid glycoprotein in the rat in vivo

The kinetics of unbound prazosin under constant i.v. infusion (800 ng/kg/min) and after i.v. bolus (160 micrograms/kg) were determined in control rats and in rats pretreated with human alpha 1-acid glycoprotein (AAG). Administration of 40 and 100 mg/kg of AAG decreased the unbound fraction in plasma of prazosin from 0.238 to 0.175 and 0.090, respectively. Administration of 40 mg/kg of AAG decreased the clearance of prazosin with respect to unbound drug (CLu) on the average by 40%. There was no statistically significant difference in the decrease of CLu between animals given constant infusions and i.v. bolus of prazosin. Increasing the administered dose of AAG from 40 to 100 mg/kg caused no further decrease in CLu. Because prazosin is a low-to-medium extraction ratio compound in the rat, this decrease in CLu indicates that AAG has an inhibitory effect on the elimination of prazosin that goes beyond a simple protein binding effect. The apparent volume of distribution of unbound drug, steady state, VUss, decreased from 12.4 to 7.7 liters/kg, and the red blood cell/plasma water concentration ratio decreased from 9.2 to 4.0 after administration of AAG. The possible mechanisms for the observed effects are discussed.     

Leukocyte-endothelium interactions after hemorrhagic shock/reperfusion and cecal ligation/puncture: an intravital microscopic study in rat mesentery

Hemorrhagic shock/reperfusion (HS/R) followed by sepsis triggers systemic microcirculatory disturbances that may induce multiple organ failure. The present study evaluated the effects of HS/R and cecal ligation and puncture, followed by necrotic cecal resection/peritoneal lavage (REL) on leukocyte-endothelium interactions at the mesentery. Eighty-one anesthetized Wistar rats (200-250 g) were randomly assigned to a first injury: (1) control-HS-no hemorrhagic shock/no reperfusion group, (2) HS/blood-HS/R with 25% shed blood, and (3) HS/blood + LR-HS/R with 25% of the shed blood + lactated Ringer's solution, 3x shed blood volume. Twenty-four hours post-HS/R, animals were submitted to cecal ligation and puncture and, 24 h thereafter, to REL. Leukocyte-endothelium interactions were assessed by intravital microscopy and intercellular adhesion molecule (ICAM) 1 and P-selectin expression by immunohistochemistry. Lungs were observed for ICAM-1 expression and neutrophil infiltration. Single and double injury induced significant increases in rolling (approximately 2-fold), adherent (approximately 5-fold), and migrated leukocytes (approximately 7-fold); ICAM-1 expression (approximately 1/2-fold), and P-selectin expression (approximately 1/2-fold) at the mesentery compared with control-HS group. REL normalized leukocyte-endothelium interactions at the mesentery in single-injured animals. However, in double-injured rats, adherence and migration of leukocytes decreased but did not normalize. Similar results were observed on ICAM-1 expression and neutrophil infiltration in the lungs from these animals. In conclusion, the current in vivo observation of the mesenteric microcirculation after a double injury followed by REL is a suitable model for the systematic evaluation of the inflammatory reaction at local and distant sites. In addition, data presented herein emphasized the importance of surgical removal of the septic focus in controlling the otherwise lethal sepsis-induced multiple organ dysfunction syndrome.     

Interferon-gamma inhibition attenuates lethality after cecal ligation and puncture in rats: implication of high mobility group box-1

Interferon (IFN)-gamma is an important immunomodulatory agent that is stimulated during infection to aid in host defense. However, increased IFN-gamma levels have been implicated as a mediator in various models of tissue injury and endotoxemia. We have previously shown that inhibition of IFN-gamma decreased bacterial load by accelerating peritoneal fibrin deposition in the cecal ligation and puncture (CLP) model of peritonitis. In addition, circulating inflammatory mediators such as interleukin (IL)-6 were reduced by IFN-gamma inhibition. In the present study, we show that administration of IFN-gamma antibody (1.2 mg/kg, i.v.) attenuated mortality after CLP. Administration of this antibody was able to reduce mortality when given immediately after CLP or 24 h after CLP surgery. Mortality in sepsis has been closely associated with increased release of high mobility group box-1 (HMGB1). Furthermore, it has been reported that IFN-gamma stimulates the release of HMGB1 from macrophages. Our studies showed that inhibition of IFN-gamma activity in vivo reduced the levels of HMGB1 in peritoneal fluid and serum of CLP rats 24 h after surgery. In addition, the decrease in HMGB1 was associated with an increase in tissue repair as evidenced by histological analyses. These results suggest that the attenuation of mortality in IFN-gamma antibody-treated rats was associated with a decrease in HMGB1 release.     

Modifications of concanavalin A patterns of alpha 1-acid glycoprotein and alpha 2-HS glycoprotein in alcoholic liver disease

In order to test whether abnormalities in hepatocytes affect the glycoprotein carbohydrate moiety, crossed immunoaffinoelectrophoresis (CIAE) with Concanavalin A (Con A) was used to study serum alpha 1-acid glycoprotein (alpha 1-AGP) and alpha 2-HS glycoprotein (alpha 2-HS) obtained from alcoholic patients with biopsy-proven liver disease. Cirrhotic patients, placed in groups C1, C2 or C3, according to Pugh's classification, were compared to healthy donors (N) and to steatosic non-cirrhotic patients (S). Con A CIAE patterns revealed in group N three subpopulations for alpha 2-HS and four for alpha 1-AGP. Two main results emerged from this study: (1) in the alcoholic groups, the proportions of Con A-unreactive subpopulations of both glycoproteins increased. Moreover, group N could be separated from group S and group S from all the cirrhotic groups. (2) There was a good correlation between the relative amounts in Con A-unreactive subpopulations of alpha 1-AGP and alpha 2-HS. The increases observed in Con A-unreactive subpopulations are probably a general phenomenon related to alterations in glycosylation processing during liver cell damage.     

Increased survival after cecal ligation and puncture in mice consuming diets enriched with sesame seed oil

OBJECTIVES: Lignans that present in the nonfat portion of sesame seed oil (SSO) can inhibit delta-5 desaturase activity, resulting in an increase in the accumulation of dihomo-gamma-linolenic acid and, subsequently, decrease the production of proinflammatory dienoic eicosanoids with a concomitant increase in the secretion of less inflammatory monoenoic eicosanoids. DESIGN: Female Balb/c mice were fed diets supplemented with 5wt% SSO or a physical mixture of oils (control) whose fatty acid composition resembled that of SSO for 3 wks. MEASUREMENTS AND MAIN RESULTS: During a 4-day observation period after cecal ligation and puncture, only 20% of the controls and as many as 65% in the SSO group survived. Furthermore, the levels of cytokines and dienoic eicosanoids produced in response to an intraperitoneal injection of a nonlethal dose (50 microg/mouse) of endotoxin were measured in both groups. The interleukin (IL)-10 levels were markedly higher in mice fed SSO diets compared with the controls. However, the plasma concentrations of prostaglandin E1 + 2, tumor necrosis factor-alpha, IL-6, and IL-12 did not differ significantly between the two groups of mice. CONCLUSIONS: Because the fatty acid composition is almost similar between the two diets, sesamin, sesamol and other lignans in SSO appear to be responsible for an increase in survival after cecal ligation and puncture and also for an increase in the IL-10 levels in response to a nonlethal dose of endotoxin in mice.     

核因子κB及其抑制蛋白ⅠκBα在成年大鼠坐骨神经许旺细胞的分布

目的观察核因子κB及其抑制蛋白ⅠκBα在成年大鼠坐骨神经许旺细胞的表达,并分析二者之间的关系及意义.方法实验于2005-01/06在重庆工学院生物工程学院及第三军医大学大坪医院野战外科研究所完成.随机取成年雄性SD大鼠6只,体质量200～250 g.1%戊巴比妥钠(20～40 mg/kg)腹腔麻醉后打开胸腔,用4%多聚甲醛溶液经左心室灌注后,取左侧坐骨神经,采用p65和p50(由p65和p50两个亚基组成的异源二聚体在核因子κB组成中发挥主要功能)及抑制蛋白ⅠκBα的多克隆抗体进行免疫组织化学染色,检测核因子κB及其抑制蛋白ⅠκBα在正常成年大鼠坐骨神经许旺细胞的表达及分布.结果①p65和p50在坐骨神经许旺细胞胞浆中表达均很弱,胞核中则不表达.②ⅠκBα则在胞浆中有较强的表达,胞核中不表达.结论核因子κB及其抑制蛋白ⅠκBα在正常成年大鼠坐骨神许旺细胞经均有一定程度的表达,在胞浆中前者表达较弱,后者较强,二者在胞核中均不表达.     

Chemically modified tetracycline (COL-3) improves survival if given 12 but not 24 hours after cecal ligation and puncture

Sepsis can result in excessive and maladaptive inflammation that is responsible for more than 215,00 deaths per year in the United State alone. Current strategies for reducing the morbidity and mortality associated with sepsis rely on treatment of the syndrome rather than prophylaxis. We have been investigating a modified tetracycline, COL-3, which can be given prophylactically to patients at high risk for developing sepsis. Our group has shown that COL-3 is very effect at preventing the sequelae of sepsis if given before or immediately after injury in both rat and porcine sepsis models. In this study, we wanted to determine the "treatment window" for COL-3 after injury at which it remains protective. Sepsis was induced by cecal ligation and puncture (CLP). Rats were anesthetized and placed into five groups: CLP (n = 20) = CLP without COL-3, sham (n = 5) = surgery without CLP or COL-3, COL3@6h (n = 10) = COL-3 given by gavage 6 h after CLP, COL3@12h (n = 10) = COL-3 given by gavage 12 h after CLP, and COL3@24h (n = 20) = COL-3 given by gavage 24 h after CLP. COL-3 that was given at 6 and 12 h after CLP significantly improved survival as compared with the CLP and the CLP@24h groups. Improved survival was associated with a significant improvement in lung pathology assessed morphologically. These data suggest that COL-3 can be given up to 12 h after trauma and remain effective.     

Oxidative variables in the rat brain after sepsis induced by cecal ligation and perforation

OBJECTIVE: The underlying mechanisms of the changes in mental status, septic encephalopathy, and long-term cognitive symptoms in sepsis survivors have only been defined in part. The present study was undertaken to assess different variables of oxidative stress in several brain structures after cecal ligation and perforation in the rat. DESIGN: Prospective animal study. SETTING: Animal basic science laboratory. SUBJECTS: Male Wistar rats, weighing 250-350 g. INTERVENTIONS: Rats were subjected to cecal ligation and perforation (sepsis group) with saline resuscitation (at 50 mL/kg immediately and 12 hrs after cecal ligation and perforation) or sham operation (control group). MEASUREMENTS AND MAIN RESULTS: Oxidative damage, assessed by the thiobarbituric acid reactive species and the protein carbonyl assays, occurred early (after 6 hrs) in the course of sepsis development in the hippocampus, cerebellum, and cortex. At longer times after sepsis induction (12-96 hrs), there was no evidence of oxidative damage in all analyzed structures. Except for the striatum, earlier in sepsis development (6 hrs) we demonstrated an increase in superoxide dismutase activity without a proportional increase in catalase activity with a consequent increase in the relation of superoxide dismutase/catalase. The balance between these enzymes was restored in the studied structures 12-96 hrs after sepsis induction. CONCLUSIONS: The short-term oxidative damage demonstrated here could participate in the development of central nervous system symptoms during sepsis development, or even septic encephalopathy. The alterations in the superoxide dismutase/catalase relation were temporally related to the occurrence or not of oxidative damage in the central nervous system.     

Effect of sodium arsenite on iNOS expression and vascular hyporeactivity associated with cecal ligation and puncture in the rat

Induction of the heat shock response protects animals from either endotoxemia or peritonitis. In endotoxemia, heat shock protein (HSP) induction is associated with reversal of vascular hyporeactivity and inhibition of iNOS expression. Recent studies suggest differences in the inflammatory mechanisms during endotoxemia and peritonitis animal models and their response to therapeutic interventions. We therefore studied the effect of the HSP inducer sodium arsenite (SA) on vascular reactivity and iNOS expression in rats undergoing cecal ligation and puncture (CLP). CLP resulted in suppression of the pressor effect of norepinephrine (NE) in vivo (measured by changes in blood pressure in response to NE boluses) and ex vivo (changes in contraction force in isolated mesenteric arteries in response to NE concentrations), and in the expression of iNOS protein. Pretreatment of the rats with SA resulted in reversal of CLP-induced vascular hyporeactivity in vivo and ex vivo, and inhibition of iNOS expression after 22 h. SA pretreatment improved 7-day survival after CLP from 18.2% to 70% (P < 0.005). Glucocorticoid receptor inhibition did not affect the effect of HSP induction on iNOS expression. The similarity of the effect of HSP on vascular reactivity and iNOS expression in two distinct sepsis models suggests that this effect may be clinically important and that a causative relationship between HSP induction, iNOS inhibition, and reversal of vascular reactivity is likely.     

Cannabinoid antagonist AM 281 reduces mortality rate and neurologic dysfunction after cecal ligation and puncture in rats

OBJECTIVES: The purpose of this study was to examine whether anandamide, an endogenous cannabinoid receptor ligand, is involved in the pathogenesis of septic encephalopathy. DESIGN: Prospective, controlled study. SUBJECTS: Male Wistar rats (7 wks old) were randomly divided into four groups as follows: group 1, control (0.5 mL of saline injected subcutaneously); group 2, sham (surgical abdominal incision and suturing were performed, but ligation and puncture of the cecum were omitted); group 3, cecal ligation and puncture (CLP); group 4, CLP + AM 281 ([N-morpholin-4-yl]-5-[2,4-yl]-5-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide) as the cannabinoid receptor antagonist (1 mg/kg intraperitoneally). INTERVENTIONS: Sepsis was induced by CLP under pentobarbital anesthesia (10 mg/kg intraperitoneally) with 1% isoflurane. A 2-Fr high-fidelity micromanometer catheter was inserted into the left ventricle via the right carotid artery to assess hemodynamics. Each of the rats was neurologically assessed at 30 mins and 12, 24, and 48 hrs after the treatment. The cytoplasmic levels of caspase-3 in the hippocampi were assayed before surgery and at 30 mins and 24 and 48 hrs after surgery using Western blotting techniques. To examine the effects of AM 281 on neurologic function and mortality rate, we set another control group treated solely with AM 281. Selective inducible nitric oxide synthase inhibitor, L-N6-(1-iminoethyl)-lysine (4 mg/kg), was injected intraperitoneally immediately after CLP to produce the CLP + L-N6-(1-iminoethyl)-lysine group to exclude the influence of depressed hemodynamics on neurologic impairment. MEASUREMENTS AND MAIN RESULTS: It was found that administration of AM 281 could prevent the hemodynamic changes induced by sepsis. Reflex responses, including the pinna, corneal, paw or tail flexion, and righting reflexes, and the escape response significantly decreased in the CLP and CLP + L-N6-(1-iminoethyl)-lysine groups at 48 hrs after the surgery. In contrast, no changes in these reflex responses were found between the CLP + AM 281 and control and sham groups. In addition, no effects of the administration of AM 281 on neurologic function and mortality rate in the control group were found. Tissue caspase-3 levels were elevated at 48 hrs after CLP in the CLP alone group (means +/- sd: control, 3.9 +/- 0.4; sham, 4.2 +/- 0.4; CLP, 7.1 +/- 1.0 [p < .01]; CLP + AM 281, 4.0 +/- 0.5 densitometric units). In addition, administration of AM 281 also decreased the mortality rate (p < .05). CONCLUSIONS: Administration of AM 281 prevented the hemodynamic changes and development of neurologic dysfunction occurring in association with septic shock, and could decrease the mortality rate in experimentally induced septic shock in rats. Although further studies are necessary to determine whether endogenous cannabinoids cause septic encephalopathy in rats directly or via their effects on systemic hemodynamics, the beneficial effects of AM 281 on these rats might have significant therapeutic implications in cases of septic encephalopathy.     

Effects of sesame oil on oxidative stress and hepatic injury after cecal ligation and puncture in rats

Oxidative stress is known to be involved in the development of organ failure and death in sepsis. Sesame oil attenuates oxidative stress induced by endotoxin; however, whether sesame oil is still effective in rats with sepsis has never been investigated. The aim of the present study was to determine the effect of sesame oil on oxidative stress-associated hepatic injury in cecal ligation and puncture-induced rats with sepsis. We examined the effect of sesame oil (4 mL/kg daily for 1 week) on lipid peroxidation, hydroxyl radical, superoxide anion, and nitrite levels in rats with sepsis. In addition, hepatic injury was also assessed by blood biochemistry. Sesame oil significantly decreased lipid peroxidation and serum nitrite levels, but affected neither superoxide anion nor hydroxyl radical in cecal ligation and puncture-treated rats. Furthermore, sesame oil significantly attenuated cecal ligation and puncture-induced hepatic injury in rats. Nevertheless, oxidative stress and hepatic injury were not affected by corn oil or mineral oil in rats with sepsis. Thus, attenuation of oxidative stress and hepatic injury may be associated with inhibition of nitric oxide in sesame oil-associated protection in rats with sepsis.     

CD4+ T-cell depletion is not associated with alterations in survival, bacterial clearance, and inflammation after cecal ligation and puncture

Our recent studies indicate that mice depleted of T cells that bear the alphabeta T-cell receptor (alphabeta T cells) show less inflammation, less physiological dysfunction, and improved survival after cecal ligation and puncture (CLP) compared with control mice. Classic CD4(+) and CD8(+) T cells comprise most of the alphabeta T-cell population. We previously showed that CD8(+) T cells, in conjunction with natural killer (NK) cells, participate in CLP-induced inflammation. However, the contribution of CD4(+) T cells to the early inflammatory response caused by CLP is largely undefined. In the present study, we evaluated CLP-induced mortality, bacterial clearance, and inflammation in mice that were depleted of CD4(+) T cells. Compared with control mice, CD4 knockout mice and wild-type mice treated with anti-CD4 did not show significant differences in survival, cytokine production, and systemic bacterial counts. The combined depletion of CD4(+) T and NK cells resulted in improved survival and decreased cytokine production compared with mice possessing a full lymphocyte complement, especially when CD4(+) T and NK cell-deficient mice were treated with imipenem. These improvements were nearly identical to those observed in mice depleted only of NK cells. These studies show that CD4(+) T cells do not seem to play a critical role in facilitating the early inflammatory response caused by CLP.     

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.     

Changes in alpha 1-acid glycoprotein serum concentrations and glycoforms in the developing human fetus.

alpha 1-Acid glycoprotein concentrations and reactivity to concanavalin A were measured in maternal and fetal serum and amniotic fluid obtained from 24 women undergoing diagnostic cordocentesis at 20 to 33 wk gestation and in 30 additional fetal sera (19 to 34 weeks gestation). Maternal alpha 1-acid glycoprotein serum levels were five to ten times higher than fetal and amniotic levels. Fetal alpha 1-acid glycoprotein levels were found to increase with advancing gestational age. Using crossed immunoaffino electrophoresis with concanavalin A, alpha 1-acid glycoprotein patterns were identical in maternal serum and amniotic fluid but totally different in fetal serum. The fetal concanavalin A pattern changed progressively during fetal life towards that of the newborn. These data confirm earlier assumptions of fetal synthesis of alpha 1-acid glycoprotein and provide normal reference values for alpha 1-acid glycoprotein in fetal serum. In addition, the specific fetal concanavalin A pattern indicates that the alpha 1-acid glycoprotein glycosylation process during fetal life differs from that in post-natal life.