Experimental Study on Inactivation of Bacterial Endotoxin by Using Dielectric Barrier Discharge

The low-temperature plasma （LTP） generated by dielectric barrier discharge （DBD） was used to sterilize the E.coli endotoxin, which is usually difficult to kill by traditional methods. Three different concentrations of bacterial endotoxin （1 EU/mL, 0.5 EU/mL and 0.25 EU/mL） were treated by LTP for different time （20 s, 40 s and 60 s）. Tachypleus amebocyte lysate （TAL） method was employed to detect the concentration variation of bacterial endotoxin before and af- ter the plasma treatment, and endotoxic shock mice model was used to evaluate the inactivation effects of LTP on endotoxin for further study. Experimental results demonstrated that, DBD plasma can inactivate the bacterial endotoxin quickly and effectively, and when the LTP treatment time was increased, the concentrations of bacterial endotoxin decreased gradually （after 60 s plasma treatment, its inactivation effect was beyond the Chinese pharmacopoeia standard）, and the average survival time of mice gradually extended. The possible inactivation mechanisms are proposed to be related to reactive oxygen species （ROSs）.

Comparison of result judgment algorithm of test for interfering factors in the bacterial endotoxins test among Chinese, Japanese, European, American, and Indian pharmacopeias

Background The bacterial endotoxins test （BET） is a method used to detect or quantify endotoxins （lipo-polysaccharide,LPS） and is widely used in the quality control of parenteral medicines/vaccines and clinical dialysis fluid.It is also used in the diagnosis of endotoxemia and in detection of environment air quality control.Although BET has been adopted by most pharmacopoeias,result judgment algorithms （RJAs） of the test for interfering factors in the BET still differ between certain pharmacopoeias.We have evaluated RJAs of the test for interfering factors for the revision of BET described in the Chinese Pharmacopoeia 2010 （CHP2010）.Methods Original data from 1 748 samples were judged by RJAs of the Chinese Pharmacopoeia 2010,the Japanese Pharmacopoeia 2011 （JP2011）,the European Pharmacopoeia 7.0 （EP7.0）,the United States Pharmacopoeia 36 （USP36）,and the Indian Pharmacopoeia 2010 （IP2010）,respectively.A SAS software package was used in the statistical analysis.Results The results using CHP2010 and USP36,JP2011,EP7.0,and IP2010 had no significant difference （P=-0.7740）.The results using CHP2010 of 1 748 samples showed that 132 samples （7.6%） required an additional step; nevertheless there was no such requirement when using the other pharmacopeias.The kappa value of two RJAs （CHP2010 and EP7.0） was 0.6900 （0.6297-0.7504） indicating that the CHP2010 and other pharmacopoeias have good consistency.Conclusions The results using CHP2010 and USP36,JP2011,EP7.0,and IP2010 have different characteristics.CHP2010 method shows a good performance in Specificity,mistake diagnostic rate,agreement rate,predictive value for suspicious rate,and predictive value for passed rate.The CHP2010 method only had disadvantages in sensitivity compared with other pharmacopeias.We suggest that the Chinese pharmacopoeia interference test be revised in accordance with the USP36,JP2011,EP7.0,and IP2010 judgment model.

Detection of bacterial endotoxin in paclitaxel liposome

Based on current research, there are three technologies during the test of bacterial endotoxin of liposomes：（1） extraction of bacterial endotoxin from liposomes;（2） addition of bacterial endotoxin in the process of recovery test; and（3） elimination of the interference factors from drugs and excipients. In the present study, we pointed out that the key technologies to test bacterial endotoxin from paclitaxel liposome included following steps： extraction of bacterial endotoxins from ethanol-dissolved liposomes; preparation of positive control of recovery solution by adding 0.01 m L standard endotoxins in 1 m L liposome ethanol solution; and the use of 0.5% human albumin to eliminate the interference from detection, and accurate detection of the bacterial endotoxin of liposomes.

Five simple models for interfering factors test of bacterial endotoxins test

Endotoxins have been credited for over 50% of sepsis cases, with significantly greater mortality.1 A literature indicates wide-spread agreement that early detection of endotoxemia, endotoxin in the bloodstream, is the major key for patient survival from sepsis.2 Today the most popular endotoxin detection system is bacterial endotoxins test （BET）, adopted by most pharmacopoeias. Interference test is a part of the bacterial endotoxin inspection method, used to judge whether the sample can be applied in BET. However, Limuloid resources are exhausted in China, which is an important source for LAL. Here, we reported 5 simple models for interfering factors test in the BET, and compared new models with the United States Pharmacopoeia 36 （USP36）.

Effects of aqueous extract of Notobasis syriaca on lipopolysaccharide-induced inflammation in rats

Objective: To investigate the effects of a dry aqueous extract of Notobasis syriaca(N. syriaca)on lipopolysaccharide(LPS)-induced inflammation in rats. Methods: Rats were fed the dried extract [500 mg/(kg·d)] for three consecutive days and then were intraperitoneally injected with LPS(1 mg/kg). Two hours after LPS injection, rats were sacrificed and blood and brain regions were collected. Inflammatory mediators' levels in plasma and homogenates of brain regions were determined by ELISA. Results: Pretreatment with the N. syriaca extract resulted in significant anti-inflammatory effects(P<0.05), including: i) attenuated LPS-induced hypothermia; ii) decreased hypothalamus and hippocampus prostaglandin E_2 levels in the LPStreated rats; and, iii) reduced hypothalamus and hippocampus interleukin-6 and tumor necrosis factor-a levels in the LPS-treated rats. Conclusions: These results suggest that N. syriaca possesses anti-inflammatory properties. Thus, it is possible that long-term consumption of this plant may result in beneficial pharmacological effects.

A new method for concentration analysis of bacterial endotoxins in perfluorocarbon

This communication demonstrates the feasibility of the gel-clot method for the analysis of bacterial endotoxins in water extracts of perfluorocarbon which is a water insoluble liquid medical device. Perfluorocarbon （10 mL） was shaken with 10 mL water for 15 min at 2000 r/min and the endotoxin present was extracted to the aqueous phase without interference inhibition/enhancement of the product and the recovery of endotoxin added to perfluorocarbon was determined, A validation study confirmed that endotoxins presented in perfluorocarbon pass over into the aqueous phase at concentrations of 20, 10 and 5EU/mL with recoveries from 86.8% to 96.8%. Therefore, the gel-clot test is suitable for detecting bacterial endotoxins in perfluorocarbon which is a water insoluble medical device.

Prophylactic uses of integrin CD18-βA peptide in a murine polymicrobial peritonitis model

AIM:To evaluate the prophylactic properties of integrin CD18-βA peptide in a murine model of abdominal polymicrobial peritonitis and sepsis.METHODS:Bacterial sepsis was induced in Institute of Cancer Research(ICR) mice by cecal ligation and puncture(CLP) surgery.Inflicted mice were then injected with either sterile saline or CD18-βA peptide intraperitoneally at 2 h after surgery,and were sacrificed at 12 and 24 h after surgery.Blood samples were immediately collected,and analyzed for endotoxin activity and tumor necrosis factor(TNF)-α and interleukin(IL)-6.Lungs and liver were studied for CD45+ leukocyte and CD3 mRNA content.Pulmonary expression of intercellular adhesion molecule(ICAM)-1,vascular cell adhesion molecule(VCAM) and E-selectin was also determined.RESULTS:Intraperitoneal injection of CD18-βA peptide significantly suppressed circulating endotoxin activity(P<0.01) at 24 h,as well as serum levels of TNF-α(P<0.05 at 12 and 24 h) and IL-6(P<0.01 at 12 h,P<0.05 at 24 h) in CLP-inflicted mice.CD18-βA peptide also abrogated leukocyte infiltration into liver and lungs as unveiled by reduced CD45+ leukocyte and CD3 mRNA contents.Furthermore,the peptide significantly reduced pulmonary expression of VCAM(P<0.01 at 12 h,P<0.001 at 24 h),E-selectin(P<0.01 at 12 and 24 h),and ICAM-1(P<0.01 at 12 h,P<0.001 at 24 h).These actions of CD18-βA peptide collectively protected septic mice against lethality(P<0.01).CONCLUSION:CD18-βA peptide is a potent endotoxin antagonist that can protect surgical patients against sepsis-associated lethality.

GUT BARRIER FUNCTION DAMAGE FOLLOWING MULTIPLE FIREARM INJURIES IN A PORCINE MODEL

Objective. To study the characteristics and pathogenesis of gut barrier damage following multiple firearm injuries in a porcine model. Methods. Twenty four small pigs were divided into 4 groups: control group (n=6, group C), group H (n=6, gunshot induced tangential fracture of parietal bone), group L (n=6, gunshot induced comminuted fracture of bilateral femora) and group M (n=6, combined group H+L). Gastric intramucosal pH (pHi), plasma endotoxin levels in portal vein, and plasma D lactate levels were measured and blood samples were cultured at different intervals after trauma. The animals were sacrificed at 72 h following trauma and intestinal tissues were harvested for pathological examination and diamine oxidase (DAO) activity measurement. Results. In group M at 72 h, pHi was significantly lower than that of group H and L (P< 0.01), and plasma endotoxin level was significantly higher than that of group H (P< 0.01) and group L (P< 0.05). Simultaneously, in groupM, D lactate level was markedly higher than that of group H (P< 0.01), and incidence of positive blood culture was much higher than that of group H and L (P<0.05). Necrosis and exfoliation were revealed at ileum villus top in all traumagroups, especially in group M, in which ileum DAO activity declined most significantly as well. Conclusion. Multiple trauma is prone to cause gastrointestinal ischemia even without hemorrhagic shock. The damage of gut barrier in multiple trauma appears to be more severe than that in one site trauma, thereby promoting gut derived endotoxemia and bacterial translocation and contributing to the development of endogenous infection.SURGICAL TREATMENT OF MALIGNANTESOPHAGEAL TUMORS IN PUMC HOSPITAL Guo Huiqin,Li Zejian ,Zhang Fan1 ,Zhang Zhiyong,Xu Letian ,Li Weidong2,Wang Xiuqin2and Wu Min2Department of Thoracic Surgery, PUMC Hospital, CAMS &PUMC, Beijing 100730Key words malignant esophageal tumors; early diagnosis; FHIT geneTo study how to prolong the postoperative survival time of the patientswith malignant esophageal tumors. The clinical data of 1098 patients with malignant esophageal tumors from 1961 to 1992 were retrospectively analyzed. The deletion of fragile histamine triplet (FHIT) gene (a tumor suppressor gene) in 30 fresh esophageal samples obtained in 1996 was detected with PCR and RT PCR method. The resectability was raised gradually and the operative morbidity and mortality decreased year by year, but there was no significant improvement on the postoperative 5 year survival rate. Delayed diagnosis and irradical resection influenced the long term survival. The deletion of cDNA of FHIT gene was 64.2%in esophageal cancer and 20%in the resected margin of the cancer. We believe that high grade atypical hyperplasia in esophageal epithelium and deletion of FHIT gene in esophageal cancer and its resected margin are pathological and molecular markers for early diagnosis of esophageal cancer respectively, and the latter may be one of the molecular markers for the resection. Early diagnosis and treatment, radical resection, and postoperative nutritional support are very important for the improvement of the postoperative survival time of the patients.

Effects of Salmonella infection on hepatic damage following acute liver injury in rats

BACKGROUND： Acute liver injury is a common clinical disorder associated with intestinal barrier injury and disturbance of intestinal microbiota. Probiotic supplementation has been reported to reduce liver injury; however, it is unclear whether enteropathogen infection exacerbates liver injury. The purpose of this study was to address this unanswered question using a rat model. METHODS： Oral supplementation with Salmonella enterica serovar enteritidis（S. enteritidis） was given to rats for 7 days. Different degrees of acute liver injury were then induced by intraperitoneal injection of D-galactosamine. The presence and extent of liver injury was assayed by measuring the concentrations of serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin. Histology was used to observe liver tissue damage. Additionally, we measured the changes in plasma endotoxin, serum cytokines and bacterial translocation to clarify the mechanisms underlying intestinal microbiota associated liver injury.RESULTS： The levels of liver damage and endotoxin were significantly increased in the Salmonella infected rats with severe liver injury compared with the no infection rats with severe liver injury（P〈0.01）; The peyer＇s patch CD3＋ T cell counts were increased significantly when the Salmonella infection with severe injury group was compared with the normal group（P〈0.05）. S. enteritidis pretreatment enhanced intestinal barrier impairment and bacterial translocation.CONCLUSIONS： Oral S. enteritidis administration exacerbates acute liver injury, especially when injury was severe.Major factors of the exacerbation include inflammatory and oxidative stress injuries induced by the translocated bacteria and associated endotoxins, as well as over-activation of the immune system in the intestine and liver.