Inhibition of bone resorptionin vitro by compound 48/80

Summary The mechanisms that control cycles of bone formation and bone resorption are not well understood. In this report we provide evidence that compound 48/80 is a potent inhibitor of bone resorptionin vitro. Resorption was assessed by the release of calcium-45 from pre-labelled new-born mouse calvaria that were treated with compound 48/80 and/or parathyroid hormone (PTH) in organ culture. Our results demonstrate that co-incubation of calvaria with PTH plus compound 48/80 (concentrations 1–10 meg/ml) produces a marked reduction of calcium-45 release compared to PTH alone. Furthermore, pre-indubation of calvaria with compound 48/80, for as little as three hours, inhibits resorption by subsequent treatment with PTH alone. Measurement of lactate dehydrogenase (LDH) released into the culture medium indicated that treatment with compound 48/80, at the doses and time periods studied, was not cytotoxic. This novel effect of compound 48/80 may provide a useful tool for studying the cellular mechanisms involved in the bone resorption process.     

Protective role of metallothionein in acute lung injury induced by bacterial endotoxin

BACKGROUND: Metallothionein (MT) is a protein that can be induced by inflammatory mediators and participate in cytoprotection. However, its role in inflammation remains to be established. A study was undertaken to determine whether intrinsic MT protects against acute inflammatory lung injury induced by bacterial endotoxin in MT-I/II knock out (-/-) and wild type (WT) mice. METHODS: MT (-/-) and WT mice were given vehicle or lipopolysaccharide (LPS, 125 microg/kg) intratracheally and the cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung histology, expression of proinflammatory molecules, and nuclear localisation of nuclear factor-kappaB (NF-kappaB) in the lung were evaluated. RESULTS: MT (-/-) mice were more susceptible than WT mice to lung inflammation, especially to lung oedema induced by intratracheal challenge with LPS. After LPS challenge, MT deficiency enhanced vacuolar degeneration of pulmonary endothelial cells and type I alveolar epithelial cells and caused focal loss of the basement membrane. LPS treatment caused no significant differences in the enhanced expression of proinflammatory cytokines and chemokines nor in the activation of the NF-kappaB pathway in the lung between the two genotypes. Lipid peroxide levels in the lungs were significantly higher in LPS treated MT (-/-) mice than in LPS treated WT mice. CONCLUSIONS: Endogenous MT protects against acute lung injury related to LPS. The effects are possibly mediated by the enhancement of pulmonary endothelial and epithelial integrity, not by the inhibition of the NF-kappaB pathway.     

Inflammatory response after inhalation of bacterial endotoxin assessed by the induced sputum technique

BACKGROUND: Organic dusts may cause inflammation in the airways. This study was performed to assess the usefulness of the induced sputum technique for evaluating the presence of airways inflammation using inhaled endotoxin (lipopolysaccharide) as the inducer of inflammation. METHODS: To characterise the inflammatory response after inhalation of endotoxin, 21 healthy subjects inhaled 40 micrograms lipopolysaccharide and were examined before and 24 hours after exposure. Examinations consisted of a questionnaire for symptoms, spirometric testing, blood sampling, and collection of induced sputum using hypertonic saline. Eleven of the subjects inhaled hypertonic saline without endotoxin exposure as controls. Cell counts, eosinophilic cationic protein (ECP), and myeloperoxidase (MPO) were determined in blood and sputum. RESULTS: A significantly higher proportion of subjects reported respiratory and general symptoms after endotoxin inhalation. MPO and the number of neutrophils in the blood were higher and spirometric values were decreased after the lipopolysaccharide challenge. In the sputum MPO, ECP, and the numbers of neutrophils and lymphocytes were higher after the lipopolysaccharide challenge. No significant differences were found after the inhalation of hypertonic saline compared with before, except for a significantly lower number of lymphocytes in the sputum. CONCLUSIONS: The results support previous studies that inhaled endotoxin causes an inflammation at the exposure site itself, as well as general effects. Sampling of sputum seems to be a useful tool for assessing the presence of airways inflammation, and the inhalation of hypertonic saline used to induce sputum did not significantly interfere with the results found after inhalation of lipopolysaccharide.     

静脉注射内毒素法制作大鼠肠道细菌易位模型

目的探讨静脉注射内毒素制作大鼠肠道细菌易位模型的方法。方法60只雄性SD大鼠(体重200～250g)随机分为4组(15只/组)。其中3组为实验组,按组别不同分别给予尾静脉注射内毒素2、4、6mg/kg;对照组给予尾静脉注射生理盐水0.5ml。观察各组大鼠的体温、呼吸频率、外周血白细胞计数及死亡率,7d后将各组大鼠分别开腹取门静脉血、下腔静脉血、肠系膜淋巴结、肝、脾和胰腺组织作细菌培养。结果注射内毒素后各组大鼠的体温、呼吸频率及外周血白细胞均有不同程度的升高。2mg/kg组大鼠无死亡,组织细菌培养阳性率为24.4%。4mg/kg组的死亡率为20.0%,细菌培养阳性率为47.2%,其中肠系膜淋巴结阳性率为83.3%。6mg/kg组的死亡率为46.7%,细菌培养阳性率为64.6%,其中肠系膜淋巴结阳性率为100.0%。结论4mg/kg剂量的内毒素静脉注射是制作大鼠肠道细菌易位模型的较为理想可行的方法。     

Preconditioning Donor Livers With Cromolyn or Compound 48/80 Prolongs Recipient Survival in a Rat Orthotopic Liver Transplantation Model

Background

Acute rejection (AR) remains a challenge in organ transplantation. Preconditioning donor organs can reduce AR and prolong survival. Whether preconditioning with cromolyn (CRM), a mast cell (MC) stabilizer, or compound 48/80 (CMP 48/80), a MC degranulator, can alleviate AR and prolong survival has not been studied.

Methods

We used the male-DA-to-female-Lewis-rat orthotopic liver transplantation (OLT) model. Donors were preconditioned with CRM in a MC stabilizing way (CRM group) or CMP 48/80 in a MC depleting way (CMP 48/80 group). Rats preconditioned with phosphate-buffered saline were used as controls (PBS group). After preconditioning, OLT surgeries were carried out. OLT male-Lewis-to-female-Lewis-rats were used as the syngeneic group (syngeneic group).

Results

Rats in the PBS group developed AR rapidly and died at 7.40 ± 1.14 days. Rats in the CRM and CMP 48/80 groups had significantly slower rejections and died at day 17.40 ± 1.67 or 14.20 ± 2.28, respectively (P < .05). Rats in the syngeneic group survived more than 60 days. Rejection activity indexes (RAIs) and liver functions were all alleviated through CRM or CMP 48/80 preconditioning. Interferon-γ messenger RNA (mRNA) expressions were reduced and interleukin-10 mRNA levels were higher in allografts in the CRM and CMP 48/80 groups, compared with the PBS group. These were confirmed by testing serum interferon-γ and interlerkin-10.

Conclusion

Preconditioning donor livers with CRM or CMP 48/80 can reduce AR and prolong survival of recipients after OLT.     

beta(2)-adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin.

BACKGROUND: beta(2)-Adrenoceptor activation regulates tumour necrosis factor (TNF)-alpha and interleukin-6 (IL-6) production in cultured renal cells. However, it remains uncertain whether, in vivo, the administration of beta(2)-adrenoceptor agonists regulate renal TNF-alpha and IL-6 mRNA following lipopolysaccharide (LPS) stimulation to cause endotoxaemia. This study was performed in order to evaluate the effect of beta(2)-adrenoceptor agonist on renal TNF-alpha and IL-6 production. METHODS: Four-week-old Wistar rats pre-treated with the beta(2)-adrenoceptor agonist terbutaline or formoterol, and/or the beta- and beta(2)-adrenoceptor antagonists (propanolol, ICI118,551), were injected with LPS (1 mg i.p.), and then 2, 4 or 6 h later, kidneys (cortex, medulla), spleen, thymus and plasma were collected to assay TNF-alpha and IL-6 mRNA levels and their respective protein release. RESULTS: Administration of beta(2)-adrenoceptor agonists suppressed TNF-alpha mRNA expression in the whole kidney, by 61% (P<0.05), as well as plasma, spleen and thymus TNF-alpha protein and mRNA expression 2 hours after injection of LPS. On the other hand, although IL-6 levels in plasma, spleen and thymus mRNA expression were suppressed significantly by administration of beta(2)-adrenoceptor agonists, the basal- and LPS-induced IL-6 mRNA levels in the whole kidney were increased 1.6- and 1.2-fold (P<0.05), respectively, by treatment with beta(2)-adrenoceptor agonists. beta(2)-Adrenoceptor agonist suppressed LPS-induced TNF-alpha mRNA expression by 35% (P<0.05) and stimulated LPS-induced IL-6 mRNA expression by 1.5-fold (P<0.05) in the medullary region of kidney. CONCLUSIONS: beta(2)-Adrenoceptor agonists down-regulate renal TNF-alpha mRNA expression following LPS-induced endotoxaemia. This effect was particularly apparent in the renal medulla. IL-6 mRNA expression in the renal medulla was up-regulated by the agonists whereas plasma, spleen and thymus IL-6 levels were completely inhibited by the agonist, which suggests the existence of tissue specific regulation of IL-6 production in the kidney by beta(2)-adrenoceptor activation.     

益气活血药对阿霉素肾病大鼠凝血机制的影响

目的 探讨益气活血方药（简称肾舒）对阿霉素诱导的肾病综合征大鼠的治疗作用和对凝血机制的影响。方法 将Wistar雄性大鼠随机分为正常组、模型组（A组）、3个治疗组[肾舒组（B组）、肾舒加激素组（C组）、激素加双嘧达莫组（D组）]。按Bertani法制作肾病综合征模型。以24h尿蛋白定量、血清白蛋白、总蛋白、甘油三酯、胆固醇、尿素氮、血肌酐作为观察指标,观察肾舒对肾病综合征模型的治疗作用;以D-二聚体（D-Dimer）及血小板a-颗粒膜蛋白（GMP-140）为观察指标,观察肾舒对凝血机制的影响;取肾组织作光、电镜检查,观察肾脏组织结构的变化。结果B、C、D组与A组比较,24h尿蛋白定量、甘油三酯、胆固醇、D-Dimer及GMP-140含量下降,差异有统计学意义（P〈0．05,P〈0．01）;血浆白蛋白、总蛋白含量均升高（P〈0．01）。C组与B、D组比较,在升高血浆总蛋白、白蛋白,降低24h尿蛋白定量、血甘油三脂、胆固醇方面,差异有统计学意义（P〈0．05）。B组在降低D-Dimer及GMP-140方面,与C、D组比较差异有统计学意义（P〈0．01）。病理结果显示,B、C、D组见足突仅少部分融合,与A组比较,差异有统计学意义。结论益气活血治疗,能改善阿霉素诱导的肾病综合征大鼠全身和肾脏局部凝血机制,并与激素协同作用,提高对肾病综合征的疗效。     

Differential effects of aprotinin and tranexamic acid on endotoxin desensitization of blood cells induced by circulation through an isolated extracorporeal circuit

OBJECTIVE: To compare the effects of aprotinin and tranexamic acid on blood cytokine secretion induced by the circulation of blood through an isolated extracorporeal circuit. DESIGN: Prospective, placebo-controlled study. SETTING: University hospital. PARTICIPANTS: Healthy volunteers (n = 18). INTERVENTIONS: Blood (400 mL) first was drawn from volunteers, then circulated through an isolated extracorporeal circuit. Three groups were compared depending on the addition or not of an antifibrinolytic agent in the circuit (control group [n = 8], tranexamic group [n = 5], aprotinin group [n = 5]). Samples for measurement were taken before and at different time points after the start of circulation through the extracorporeal circuit. Cytokine (tumor necrosis factor-alpha, interleukin [IL]-6, IL-8, and IL-10) concentrations in the plasma and in the supernatant of lipopolysaccharide-stimulated whole blood cell cultures were analyzed. MEASUREMENTS AND MAIN RESULTS: In the control and tranexamic acid groups, tumor necrosis factor-alpha, IL-6, and IL-10 secretion by whole blood cell cultures were rapidly decreased, whereas IL-8 secretion was unaffected. In the aprotinin group, IL-8 secretion was also decreased (p < 0.05). CONCLUSION: These results show that aprotinin, but not tranexamic acid, modulates the inflammatory response by reducing the IL-8 secretion of blood cells activated by contact with foreign surfaces.     

Ablation of the blood-testis barrier in rats and guinea pigs by 48/80, a histamine releaser, and cadmium chloride

When adult male guinea pigs were injected unilaterally intratesticularly with compound 48/80 (1 mg in 0.05 ml saline), their testicular peritubular capillaries were engorged with blood, intratubular edema was present on the ipsilateral side, and the blood-testicular (BT) barrier (measured by the entrance of acriflavin into the seminiferous tubule) was ablated. The contralateral testis, when injected with saline, showed no pathological changes nor a breakdown of the BT barrier. Subcutaneous injection of guinea pigs with cadmium chloride resulted in a more intense intratubular fluorescence than was observed for 48/80-treated animals. The H1 and H2 receptor blockers (diphenhydramine and cimetidine, respectively) reduced the intensity of capillary engorgement and edema. Intraperitoneal injections of 48/80 (0.5 mg) was lethal to rats, and both diphenhydramine and cimetidine induced survival even with as much as 1 mg of 48/80. Intratubular fluorescence was less severe than that observed for similarly treated guinea pigs or from cadmium chloride-treated rat testes. Neither treatment (48/80 or cadmium chloride) altered the blood-epididymal barrier of either guinea pigs or rats. Species differences were observed in the lethal effects of exogenously administered histamine.     

大鼠急性心肌缺血后肺组织细胞的变化

目的观察大鼠急性心肌缺血早期肺组织细胞凋亡情况,进一步证实此过程中肺损伤的程度。方法健康成年雄性SD大鼠24只,随机均分为两组,假手术组(S组),急性心肌缺血模型(C组)。取肺组织切片行TUNEL染色,计算CAO3、6h细胞凋亡率(RA);肺组织蛋白中caspase-3活性变化。结果 C组肺组织RA明显高于S组(P<0.05)。两组CAO6h时RA值和caspase-3活性明显高于CAO3h时(P<0.05)。肺组织蛋白中caspase-3活性C组高于S组,但差异无统计学意义。结论急性心肌缺血后大鼠肺组织细胞可能通过caspase-3激活途径凋亡数增加,并随时间延长而增加。     

内毒素预处理诱导大鼠供肝缺血再灌注交叉耐受机制的研究

目的探讨以内毒素耐受为基础诱导的移植肝脏对缺血再灌注损害（I／RI）交叉耐受的相关机制。方法雄性SD大鼠，随机分为正常对照组、缺血再灌注组（I／R组）和内毒素耐受组（ET组）。以未经任何处理的大鼠肝脏为正常对照；ET组供体第1天经尾静脉给予脂多糖0.1mg／kg体重，第2、3、4、5天给予0.5mg／kg体重；I／R组供体给予等体积0．5ml无菌PBS液。第8天，切取供体，以未经任何处理的大鼠为受体，两袖套法建立大鼠原位肝移植模型。按门静脉血流恢复后第0、60及180min分为3个亚组。逆转录-聚合酶链式反应及蛋白免疫印记法测定肝组织的白细胞介素-1受体相关激酶-4（IRAK-4）mRNA和蛋白表达水平；酶连免疫吸附法检测肝组织核因子-κB出（NF-κB）活性及血清TNF-α含量。结果再灌注后0、60及180rain．I／R组与ET组的IRAK-4蛋白与mRNA表达水平，NF-κB活性以及TNF-α含量均高于正常对照组（P〈0．01）；再灌注后0min，I／R组与ET组的NF-κB活性以及TNF-α含量差异无统计学意义（P〉0．05），但IRAK-4蛋白与mRNA表达水平高于ET组（P〈0．01）；再灌注60min及180min，I／R组的上述各指标均明显高于后者（P〈0．01）。结论抑制IRAK-4表达是以内毒素耐受为基础诱导的移植肝脏对I／RI交叉耐受的相关原因，但能否以IRAK-4为减轻I／RI基因治疗的理想靶点尚有待进一步的研究。     

内毒素介导的急性肺损伤大鼠白细胞介素13表达及肺组织激活剂蛋白1活性的变化

目的　观察内毒素 /脂多糖 (LPS)致伤大鼠的血浆白细胞介素 (IL)13含量及肺组织激活剂蛋白 1(AP 1)活性的变化 ,探讨AP 1与IL 13表达的关系。　 方法　将 12 0只Wistar大鼠根据注射LPS剂量不同随机分为A(2mg/kg)、B(4mg/kg)、C(6mg/kg)、D(8mg/kg)组 ,并设立相应的等渗盐水对照组 (NS组 ) ,取伤后 1、2、4、6h为观察时相点 ,每组每时相点 6只大鼠。致伤Wistar大鼠 ,复制全身炎症反应综合征 急性肺损伤 (SIRS ALI)模型。在致伤后 1、2、4、6h,采用酶联免疫吸附测定法 (ELISA)检测血浆IL 13含量 ,采用凝胶迁移率分析法 (EMSA)检测肺组织中AP 1活性。　 结果　LPS可使IL 13含量及AP 1活性同步升高。A、B、C、D组与NS组比较 ,差异均有显著性意义及非常显著性意义 (P <0.0 5～ 0.0 1)。C、D组的血浆IL 13含量及肺组织中的AP 1活性均显著增加 ,D组两指标的峰值分别为 (45 .6 2± 5 .78) pg/ml、(177.6 8± 6.81)NII%,均出现在伤后 2h。　 结论　IL 13表达增强时AP 1活性亦升高 ,并与SIRS ALI发生有关。     

Characterization of certain hepatocyte-proliferating and/or protective factors induced by the sensitization of freezing-thawing hepatic tissue

Although tumor cryosurgery would be expected to produce beneficial immunological effects from the enhancement of anti-tumor activity, under certain conditions the tumor may become enlarged and metastases promoted due to increased immunosuppressive activity and a high zone tolerance. In the present study, we examined whether hepatocyteproliferating factors were produced by the inoculation of freezing-thawing hepatic tissue (FTHT). Serum obtained from rats inoculated with FTHT increased DNA synthesis, according to measurement by [³H]thymidine incorporation in primary cultured rat hepatocytes. This increase was dependent on the serum concentration, with serum obtained on day 14 after the inoculation being the most potent for hepatocyte proliferation. The sensitized serum promoted DNA synthesis nearly as much as serum obtained from a 70% hepatectomized rat, but slightly less than 10ng/ml hepatocyte growth factor. The sensitized serum also protected hepatocytes from carbon tetrachloride (CCI₄)-induced hepatotoxicity. Optical density measured by the 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrozolium bromide (MTT) cytotoxicity assay was increased, and the release of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in medium was decreased by treating hepatocytes damaged by CCI₄ with the sensitized serum. These results suggest that certain hepatocyte-proliferating and protective factors are induced in serum by the inoculation of freezing-thawing hepatic tissue, and that the sensitized serum may be useful in the treatment of liver failure.     

Comparative study of acute tissue damage induced by the CO2 laser versus microelectrodes in cordectomies

OBJECTIVE: This study compared the acute tissue damage produced by a CO2 laser and microelectrodes in samples of vocal cords from patients undergoing laryngeal endoscopic surgery for stage T1 squamous cell carcinoma. STUDY DESIGN AND SETTING: Based on prior surgical experience with the CO2 laser and microelectrodes, the study protocol used hematoxylin-eosin staining of vocal cords treated with a CO2 laser (n=20) or microelectrode (n=20). RESULTS AND CONCLUSION: The acute tissue damage produced by the CO2 laser was similar to that induced by microelectrodes in cutting mode. The tissue damage resulting from the use of the microelectrode in coagulation mode was comparatively greater.