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After ligation of the mesenteric arteries an intestinal ischaemia was obtained. Endotoxin was measured from the peritoneal cavity, the portal und the peripheral blood. The limulus test for Endotoxin was used after phenol-water-extraction of the plasma. Microbiological investigations were done simultaneously. The endotoxin concentrations correlated to the clinical state of gram negative sepsis.
Fecal peritonitis was produced by a sphincter-system leading to intestinal perforation. In 10 beagle dogs endotoxin was measured with the Limulus test. There was a correlation between endotoxin appearance and microbiological findings in the peritoneal cavity and in the blood. Endotoxinaemia correlated with the clinical severity of peritonitis.
After the study of a group of severely burned patients with septicemia the authors reached the conclusion that the diagnosis of endotoxemia should be based on the following criteria: clinical condition, laboratory results and autopsy findings. Clinical findings can be subdivided into anamnestic data (such as age, liver function etc.) and direct clinical observation (taking into account states of stress and some specific surgical procedures). The most significant laboratory findings in the investigated group of patients were hypoxy, metabolic acidosis, lowered platelet count and positive Limulus test. Autopsy findings corresponded to findings in septicemia and shock. The authors also discuss the significance of activated lymphocyte accumulation in the liver following endotoxin administration in animals.
The interaction of bacterial endotoxins, deep rough mutant lipopolysaccharide LPS Re and the 'endotoxic principle' lipid A, with recombinant human serum albumin (rHSA) was investigated with a variety of physical techniques and biological assays. With Fourier-transform infrared spectroscopy and differential scanning calorimetry, the influence of albumin on the acyl chain melting behavior of the endotoxins was measured. Also, the effect on the functional groups of the endotoxins, in particular with respect to their orientation, was studied, including competition experiments with polymyxin B. Furthermore, the influence of endotoxin binding to rHSA on the protein's secondary structure was investigated. The results indicate a non-electrostatic binding with no change of the backbone orientation of LPS and only a slight change of the secondary structure of rHSA. Correspondingly, the amount of charge neutralization of the endotoxins due to rHSA measured by the electrophoretic mobility exhibited only a slight reduction of the surface potential. From these measurements and isothermal titration calorimetry, the lipid:protein binding stoichiometry was estimated to [LPS]:[rHSA], 10:1 molar. The determination of the aggregate structure of the endotoxins by X-ray small-angle scattering exhibited a complex change of a cubic into a non-lamellar structure. No influence of rHSA on endotoxin intercalation into phospholipid liposomes induced by lipopolysaccharide-binding protein could be detected by fluorescence resonance energy transfer. Finally, the LPS-induced cytokine production of human mononuclear cells was only slightly increased at high molar rHSA excess, while the coagulation of amebocyte lysate in the Limulus test yielded a complex change due to rHSA binding of LPS.
The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.
BACKGROUND/AIMS: The purpose of this study was to evaluate the occurrence of false-positive endotoxemia after hepatectomy for hepatocellular carcinoma in patients with cirrhosis. The chromogenic conventional limulus test (Toxicolor test) revealed transiently increased blood endotoxin levels after hepatic resection for hepatocellular carcinoma in patients with liver cirrhosis. However, clinical signs of endotoxemia were not observed in all of the cases. METHODOLOGY: Pre- and postoperative changes of blood endotoxin levels and beta-glucan were measured in 20 patients with liver cirrhosis who underwent hepatic resection for hepatocellular carcinoma, using the Toxicolor test, the Endospecy test (which specifically reacts with endotoxin) and the Gluspecy test (which specifically reacts with beta-glucan). The changes in endotoxin levels and beta-glucan in ascitic fluid during surgery were also studied. RESULTS: The Toxicolor test revealed transiently increased blood endotoxin levels, but Endospecy test results were not elevated. The changes in the Gluspecy test were almost the same as in the Toxicolor test. During surgery, beta-glucan levels in the ascitic fluid increased remarkably due to their release from the surgical gauze. Digestion studies using 1-3-beta-D-glucanase on specimens which showed high Toxicolor and Gluspecy test values resulted in a lack of response for both tests. CONCLUSION: The cause of false-positive endotoxemia was determined to be caused by beta-glucan, which was released from the surgical gauze.
We evaluated the clinical utility of a new endotoxin-specific chromogenic limulus test in febrile patients with haematological malignancies. The specificity is assured by the removal of factor G, which is sensitive to (1-->3)-beta-D-glucan, from horseshoe crab amoebocyte lysate. The sensitivity and specificity of the test to systemic gram-negative bacterial infections were 69.7 and 96.3%, respectively. Meanwhile, gram-negative bacteria grew in only 39.7% of endotoxaemic samples. Thus, it seems appropriate to consider gram-negative bacteraemia and endotoxaemia as different entities. Endotoxaemia was significantly associated with septic shock and infectious death, especially in patients with neutropenia. The new test, the results of which are available within 3 h, should help physicians to recognise this ominous sign early and to initiate a prompt countermeasure to endotoxaemia.
Intravenous admixtures containing potassium collected from three hospital pharmacies were analyzed for compounding accuracy, sterility and pyrogenicity. The study was performed in two stages. During stage I, pharmacists and technicians were not informed of the study, but during stage II they were informed. In each stage 10 samples were collected from each person in the two personnel groups, analyzed and the results compared between the two personnel groups and the two stages. Results of the study showed that without monitoring (stage I) pharmacists had a higher mean percent error and contamination level than technicians. With monitoring, however, pharmacists showed a lower mean percent error and contamination level than technicians. Both personnel groups showed a decline in their mean percent error in the second stage, but there were still 83 (39.5%) errors in compounding accuracy greater than +/- 6%. No positive results with the Limulus test for pyrogens were obtained. It is recommended that a planned program of quality control be instituted for the preparation of i.v. admixtures by both pharmacists and technicians.
Lipopolysaccharide (LPS) extracted with phenol and water from Yersinia pestis was compared with LPS of Escherichia coli for stimulation of deoxyribonucleic acid synthesis in mouse spleen cells (lymphocyte mitogenesis), gelation of limulus lysate, pyrogenicity in the rabbit, and susceptibility to inhibition of these activities by polymyxin B sulfate (PBS). LPS of Y. pestis stimulated deoxyribonucleic acid synthesis in mouse spleen cell cultures over the same quantitative range as LPS of E. coli. In the limulus tests and rabbit pyrogenicity studies, the LPS of Y. pestis was active but about 10 times less potent than E. coli LPS on a weight basis. PBS in concentrations from 1 to 10 microgram/ml diminished the rate of deoxyribonucleic acid synthesis in spleen cell cultures stimulated by LPS of both Y. pestis and E. coli. Addition of PBS to LPS of both Y. pestis and E. coli in a ratio of 100 parts of PBS to 1 part of LPS by weight increased by 10-fold the concentration of LPS required to produce gelation of limulus lysate and inhibited significantly pyrogenic responses in rabbits. These results demonstrating similarities of LPS of Y. pestis and E. coli may suggest that the pathogenesis of plague is similar to that of other gram-negative bacterial infections.
We developed a microplate method for determining endotoxin in whole blood with Endospecy, an endotoxin-specific chromogenic limulus test reagent. The factors in blood that would interfere with the test were successfully removed by exposing samples to 0.66 mol/l HNO3 containing 0.25% Triton X-100. Recoveries of various endotoxins spiked into whole blood of humans and experimental animals were almost complete, and were not enhanced by sample dilution. Normal endotoxin concentration in human whole blood was less than 10 pg/ml in reference to Escherichia coli 0111:B4 endotoxin. Measurements on paired whole blood and platelet-rich plasma (PRP) samples from 50 normal and 132 diseased subjects showed a good correlation (r = 0.901, P less than 0.01). This microplate whole blood method has advantages over the conventional PRP method in that it requires less time, less amount of sample and limulus reagent, and less risk of contamination during the procedure.
The anti-endotoxic effect of syringic acid (SA) isolated from Radix Isatidis (Banlangen, BLG) was studied. SA was extracted and isolated from BLG and diluted into 1% solution. The content of SA-pretreated endotoxin (ET) was quantitatively determined using Limulus test. The ability of fever induction of ET pretreated with SA was measured using endotoxin-induced fever test in rabbits. The LPS-induced death in mice pretreated with and without SA was compared. Results showed that after pretreatment with SA, 83.16% of ET was destroyed, the ET-induced fever in rabbits relieved markedly and the LPS-induced death rate in mice dropped from 68% to 20%. It was concluded that SA isolated from BLG had anti-endotoxic effects.
Incidence and severity of systemic endotoxemia in patients with chronic viral hepatitis B and C were studied using the limulus test. Healthy subjects and patients with acute viral hepatitis B and C comprised reference groups. The incidence of "physiological" systemic endotoxemia in healthy subjects was 31.3%, while in chronic hepatitis systemic endotoxemia was found in 69-84% patients and it was more severe in this case. A significant correlation was revealed between systemic endotoxemia with virus replication and the degree of basic clinical and laboratory signs in patients with chronic viral hepatitis B.
As a procedure for the diagnosis of fungemia, Toxicolor and Endospecy (limulus tests using a synthetic chromogenic substrate) were used to measure (1----3)-beta-D-glucan (beta-glucan), which is a membranous component of fungus. The plasma samples were pretreated with a new perchloric acid method. These methods permit measurement of the beta-glucan within about two hours. Patients in whom fungemia was diagnosed were effectively treated with miconazole.
Methylcellulose-elicited peritoneal macrophages from BALB/c mice were cultivated in vitro and inoculated with dengue virus (DV). At intervals thereafter portions of the culture fluids were taken and titrated for viral infectivity. Extracts from Ascaris suum and Parascaris equorum, either crude or Sephadex G-100 fractionated, were examined for effects on the multiplication of DV. The macrophage cultures treated with the above substances produced larger amounts of DV compared with untreated control cultures. The enhancing effect of the substances depended on doses added and duration of treatment and was suppressed by co-treatment with carrageenan, a specific macrophage-inhibiting agent, but was not related to the viability of cultured cells. In fluorescent antibody (FA) as well as infectious center assay experiments, it was shown that the DV-infected cells were found more frequently in treated cultures than in untreated control cultures. In the treated cultures phagocytosis by cultured cells was also of a higher magnitude than that in untreated cultures. In cocultures of macrophages and splenocytes from the same line of mice, no additive effect of splenocytes was noted. The limulus amebocyte lysate clotting enzyme reaction (Limulus test) indicated that involvement of bacterial lipopolysaccharides in the enhancement phenomena was negligible. The data so far obtained suggest that the enhancing effect was due to direct action of the parasitic extracts on macrophages. Four Sephadex G-100 fractions from the crude extracts showed similar activities; however, the effects of fractions I and III appeared to be comparatively strong. Significance of the findings in relation to the pathogenesis of DV infection was discussed.
Mild hydrolysis of Haemophilus influenzae type a lipopolysaccharide by ion exchangers yielded a lipid A extracted by chloroform. It contained phosphorus, glucosamine, and fatty acids. Myristic, palmitic, 3-hydroxymyristic, and oleic acids and two other unidentified long-chain fatty acids were found. The free lipid A was not toxic for mice at doses of up to 50 mg/kg and did not provoke a Shwartzman reaction. The Limulus test activity was positive up to 10(-12) g/ml, but the pyrogenicity in rabbits was lower than with the original lipopolysaccharide. However, the lipid A did induced a mitogenic response and polyclonal B-cell activation in mouse spleen cell cultures. Complexing lipid A with bovine serum albumin gave a nontoxic preparation which lost these immunological activities. Immunochemical studies showed that the major reactive determinants of this lipid-protein complex were altered after such a linkage. Consequently, the nontoxic and mitogenic lipid A isolated from H. influenzae type a did not exhibit all of the classical activities of lipid A preparations.
The biological and endotoxic activities of protective antigens (PAgs) prepared by the chloroform-methanol-water method from Leptospira interrogans serovars lai, copenhageni and canicola were examined. The PAg preparations did not show a local Shwartzman reaction in the rabbits at doses of 100 micrograms and 50 micrograms/site and lethal toxicity to galactosamine-sensitized mice at the dose of 12.5 micrograms to 50 micrograms/mouse. PAgs exhibited a weak cytotoxic action on peritoneal exudate macrophages of C3H/HeJ and C3H/HeN mice at the dose of 500 micrograms/ml in vitro, but did not show cytotoxicity for BHK-21 cells kidney cells of the Syrian hamster, CHO-K1, ovary cells of the Chinese hamster, and CHL, lung cells of the Chinese hamster, at doses of 5 and 500 micrograms/ml. Gelation activity in the Limulus test was only observed at PAg concentrations over 100 ng/ml, which dose was 10,000 times that of lipopolysaccharide (LPS) of Escherichia coli O55:B5. Furthermore, an adjuvant activity of PAgs was not observed in the production of anti-sheep red blood cell antibody in mice. Mitotic conversion of spleen cells from C3H/HeJ and C3H/HeN mice was observed by the addition of PAgs in vitro. These results indicated that the biological properties of PAgs were different from those of LPS prepared from gram-negative enterobacteria, that PAgs had no endotoxic activity and that the biological safety of PAgs as vaccine was proved.
The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.
Porphyromonas gingivalis 381 lipid A, characterized by beta-(1-6)-linked glucosamine disaccharide 1-phosphate, with one hydroxyacyl group and one acyloxyacl group, i.e., 3-hydroxy-15-methyl-hexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2 and 2' positions, respectively, was less endotoxically active than the synthetic Escherichia-coli-type lipid A (compound 506), which possesses beta-(1-6)-linked glucosamine disaccharide 1,4'-bisphosphate, with two acyloxyacyl groups at the 2' and 3' positions and two 3-hydroxytetradecanoyl group at the 2 and 3 positions and the synthetic Salmonella-type lipid A (compound 516), which has three acyloxyacyl groups at the 2,2' and 3' positions and one hydroxyacyl group at the 3 position. P. gingivalis lipid A exhibited no or very low endotoxic activities, i.e., lethal toxicity in galactosamine-loaded mice, preparative ability for local Shwartzman reaction, pyrogenicity and Limulus test as compared with compounds 506 and 516. However, polyclonal B-cell activation of BALB/c mouse splenocytes was as strong as that of compound 506. Furthermore, P. gingivalis lipid A had stronger immunoadjuvant and hemagglutinating activities than compound 506. The absence of ester-linked phosphate at the 4' position and ester-linked fatty acids, and the presence and positions of fatty acids possessing considerable lengths of acyl chains are unique features of P. gingivalis lipid A, and they differentiate this lipid A from enterobacterial and other lipids A. The good balance between endotoxic properties and beneficial immunobiological activities of P. gingivalis lipid A may be attributable to these features.