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PubMed1994

Antitumor activity of bacterial endotoxins and their subunits in in vitro test.

The tumoricidal effect of endotoxins and their subunits of Shigella dysenteriae serovar 1 of both growth forms and certain other representatives of the Enterobacteriaceae family was tested against Németh-Kellner mouse lymphoma cells using an in vitro assay based on the use of sodium chromate solution yielding labelled hexavalent 51Cr ions. The most effective in vitro activity was evidenced in both growth forms by S. dysenteriae 1 lipopolysaccharide-protein complex (LPSP) (76-92%), lipid A and lipoid B isolated from LPS (77-82%) and lipid A and lipoid B from LPSP (53-70%). A direct dependence of the level of the Limulus test and pyrogenicity on the tumoricidal activity of a preparation was not demonstrated. The influence of selected cations (Cu, Fe, Ca, Mg, Zn) bound to selected substances on antitumor activity was monitored. The method of probit analysis is recommended as it enables estimation, based on a number of concentrations, of the regression line of probable effectiveness of a given preparation.

Animals↗
PubMed1985

Elimination of false-positive limulus amebocyte lysate tests in patients with hyperlipidemia.

Varying concentrations of lipopolysaccharide (LPS) and mannan suspensions were mixed with either saline or plasma from normal volunteers, heated to 100 degrees C for 10 min, and then subjected to the limulus amebocyte lysate test (LAL). A positive LAL in saline required minimum LPS and mannan concentrations of 10(-11) and 10(-5) g/ml, respectively, while the minimum concentrations premixed with plasma were 10(-13) and 10(-9) g/ml, respectively. Thus, use of plasma instead of saline increased assay sensitivity 100-fold for LPS and 10,000-fold for mannan. In the second part of the experiment, normal plasma was separated into lipid and nonlipid phases by Folch's method. LAL analysis of each phase revealed equal sensitivity for LPS and mannan in the nonlipid phase, but no sensitivity in the lipid extract. Subsequently, 200 ml of a fat emulsion (Intralipos) was administered to the normal volunteers, and LAL and lipid analyses were performed. The LAL turned positive in all volunteers. When LAL was positive, triglycerides (TG), chylomicron (Chyl), and very low-density lipoprotein (VLDL) increased significantly compared with when LAL was negative. It is concluded that plasma lipids increase the sensitivity of LAL and directly activate LAL when TG, Chyl, and VLDL concentrations are high. This effect of plasma lipids on LAL can be eliminated by extracting LPS and mannan in the nonlipid phase.

Escherichia coli↗
PubMed1993

An evaluation of serodiagnostic tests in patients with candidemia: beta-glucan, mannan, candida antigen by Cand-Tec and D-arabinitol.

The serodiagnostic tests, beta-glucan, mannan, candida antigen by Cand-Tec, and D-arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta-glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test-D and Endospecy), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand-Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D-Arabinitol was the least sensitive, because a D-arabinitol/creatinine ratio greater than 2.0 mumol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti-fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.

Adolescent↗
PubMed1997

A novel component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans.

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermedia ATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate and protein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) on deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. We designated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity on splenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction were resistant to heat inactivation (100 degrees C for 1 h) and protease treatments and were scarcely inhibited by polymyxin B. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with a phenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages from C3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeN mice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hot phenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. These findings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reported to date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not from the LPS itself.

Adjuvants, Immunologic↗
PubMed1979

Effect of polymyxin B sulfate on endotoxin activity in a gram-negative septicemia model.

The antiendotoxin effect of polymyxin B was investigated in experimentally induced septicemia in rabbits. The Pasteurella multocida organisms were sensitive to the antibacterial action of penicillin but not to polymyxin B. Animals pre-treated with polymyxin showed positive blood cultures and significantly reduced plasma endotoxin levels (Limulus test) with normal white blood cell and platelet counts when analyzed 6 hr after the injection of live organisms. Polymyxin therapy given after the animals had established septicemia-endotoxemia reduced the plasma endotoxin levels and improved the survival, but had no effect on the leukopenia and thrombocytopenia. The best survival data were obtained in rabbits who were treated with both penicillin and polymyxin. The data suggest that polymyxin is effective in neutralizing the endotoxic effects from live organisms and that the timing and perhaps duration of the polymyxin treatment is of critical importance. In addition, a modified Limulus lysate method was developed which showed that quantitative plasma endotoxin determination could be made more sensitive by prior heating of the plasma to remove the natural inhibitors.

Animals↗
PubMed1996

Enolase antigen, mannan antigen, Cand-Tec antigen, and beta-glucan in patients with candidemia.

We compared the specificities and sensitivities of four tests used for the serodiagnosis of candidemia in 39 patients with candidemia, including 10 patients with superficial Candida colonization, 10 patients with deep mycosis, and 20 healthy subjects. The results obtained by the dot immunoblotting assay for detecting the enolase antigen (48 kDa) were compared with those of assays for detecting mannan antigen, heat-labile antigen (a threshold titer of four times), and beta-glucan (> or = 60 pg/ml). Enolase antigen was detected in 28 (71.8%) patients with candidemia, while 30 (76.9%), 10 (25.6%), and 27 (84.4%) patients were positive for the heat-labile antigen by the Cand-Tec assay, the mannan antigen by the Pastorex Candida assay, and beta-glucan by the limulus test, respectively. Ten patients with superficial Candida colonization, 5 patients with invasive pulmonary aspergillosis, 5 patients with cryptococcosis, and 20 healthy subjects were negative for both enolase antigen and mannan antigen. Two patients with superficial Candida colonization, one patient with invasive pulmonary aspergillosis, and two patients with cryptococcosis were positive by the Cand-Tec assay. The beta-glucan concentration was more than 60 pg/ml in all patients with invasive pulmonary aspergillosis; however, it was less than 10 pg/ml in all patients with cryptococcosis. The specificity of enolase antigen in the serodiagnosis of candidemia was 100%, but the sensitivity was 71.8%. The specificity and sensitivity of Cand-Tec, the assay for mannan antigen, and the assay for beta-glucan were 76.9 and 87.5%, 25.6 and 100%, and 84.4 and 87.5%, respectively. Our results demonstrated that antigen detection tests are useful for the diagnosis of candidemia; however, none is satisfactory for the serodiagnosis of candidemia. We suggest that a combination of two assays may increase the accuracy of diagnosis of candidiasis.

Adult↗
PubMed1986

Production of a neutrophil chemotactic factor by endotoxin stimulated alveolar macrophages in vitro.

Alveolar macrophages (AM) isolated from normal guinea-pigs and from those chronically exposed to endotoxin (LPS) were cultured in the presence of various concentrations of LPS (from 0.5 ng to 5 micrograms/ml). The presence of a neutrophil chemotactic factor (NCF) in culture supernatants was tested in migrations chambers. Contamination of all reagents has been tested using LAL test (Limulus Amoebocyte Lysate). The results indicate a production of NCF with low LPS concentrations (0.5 and 5 ng/ml) within the first 6 h of incubation: when larger doses are used the response decreases and a significant inhibition is observed with 5 micrograms LPS/ml (P less than or equal to 0.05). When contaminated medium was used, all responses observed were three times higher than with LPS-free medium (P less than or equal to 0.01). However, the response pattern was the same. AM from chronically exposed animals exhibit the same response patterns: the magnitude of NCF production was higher than with normal AM but not significantly. The data suggests that initial conditions of AM in vitro or in vivo with reference to LPS contamination have to be determined as they are of importance when AM NCF production has to be tested.

Animals↗
PubMed1979

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.

Aerobiosis↗
PubMed1986

Suppressive effect of Escherichia coli on adjuvant-induced arthritis in germ-free rats.

Our previous finding, that germ-free F344 rats develop severe adjuvant-induced arthritis, whereas specific pathogen-free rats develop mild disease, prompted us to investigate the role of bacterial flora in promoting the development of this disease. Germ-free rats given Escherichia coli experienced disease suppression. Germ-free rats treated with 3 strains of Lactobacillus experienced an enhancement of the disease, although it was not significant. Germ-free rats treated with a combination of E coli and lactobacilli had disease suppression similar to that of E coli monoassociated rats. Thus, these findings suggest that E coli may play a dominant role in modulating the development of the disease in this particular strain of F344 rats, possibly through its lipopolysaccharide (as evidenced by positive results on limulus tests). These findings also suggest that microflora play an important role in modifying the development of joint disease.

Animals↗
PubMed1998

Elution of lipopolysaccharides from polyacrylamide gels.

A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.

Acrylic Resins↗
PubMed1997

In vitro phagocytosis of Escherichia coli and release of lipopolysaccharide by adhering hemocytes of the silkworm, Bombyx mori.

A primary culture containing adhering hemocytes mainly granular cells from the silkworm, Bombyx mori, was used to investigate in vitro phagocytosis of Escherichia coli. Phagocytosis was confirmed to occur in this system by microscopic observation. Lipopolysaccharide (LPS) concentration in the culture medium was measured by a Limulus test and a higher LPS concentration was detected in phagocytosis-occurred samples than in control samples, which omitted either E. coli cells or adhering hemocytes. Moreover, it was found that LPS containing sample but not control samples strongly induces gene expression of cecropin B, an antibacterial protein. These results suggest that bacterial cell wall components like LPS released by phagocytosis play an important role in the induction of insect antibacterial proteins.

Animals↗
PubMed1983

Toxicity studies with human fibroblast interferon.

Acute toxicity studies of interferon derived from human fibroblasts (FHIF) were performed in mice and rats with doses between 6 X 10(4) and 480 X 10(4) IU/kg i.v. and an observation period of 14 days. No severe toxic effects, such as loss of weight or other pathological clinical signs were observed. In local innocuity studies (i.v., i.a., paravenous) in rabbits FHIF proved to have no adverse effects. 180,000 IU/kg were pyrogen free in rabbits, in the limulus test the content of lipopolysaccharides (LPS) was 208 pg/ml as compared to the FDA standard EC-2. Studies were performed to assess the pharmacological safety of FHIF in 10 monkeys (Macaca fascicularis). The i.v. injection of 60,000 IU/kg had no pathological effects on circulation, respiration, number of blood cells, or body temperature. In six monkeys a short decrease of arterial blood pressure was noted a few minutes after injection of 120,000 IU/kg i.v. Both doses caused an increase of thrombelastographic time (TEG). The proposed single human dose of approximately 60,000 IU/kg has been proved safe in the acute toxicity studies and the pharmacological safety assessment in mice, rats, rabbits, and monkeys. The LPS content is low.

Animals↗
PubMed1983

Protective activities of ribosomal ribonucleic acid and lipopolysaccharide of Pseudomonas aeruginosa: a comparative study.

Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C3H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS.

Animals↗
PubMed1986

Superoxide anion generating capacity and lysosomal enzyme activities of Kupffer cells in galactosamine induced hepatitis.

To elucidate the function of the reticuloendothelial system of liver in hepatic injury, we investigated the effect of endotoxins on superoxide anion (O-2) generating capacity and lysosomal enzyme activities of Kupffer cells isolated from rats treated with galactosamine (Gal N), with Gal N supplemented with polymyxin B (Polymyxin B-Gal N), with lipopolysaccharide (LPS) and from control rats. After collagenase digestion of the liver and centrifugation over metrizamide gradient, Kupffer cells were prepared by the dish adherence procedure. O-2 production by the cells was examined as chemiluminescence during phagocytosis of latex particles and beta-glucuronidase activities were analyzed. High titers of endotoxemia were detected in LPS and Gal N rats by limulus test, while a low endotoxemia titer was found in Polymyxin B-Gal N rats. Hepatocyte damage was found in Gal N rats, but little was recognized in LPS and Polymyxin B-Gal N rats. In the latter groups, Kupffer cells, activated by endotoxins, showed the enhancement of chemiluminescence and a release of lysosomal enzyme. Though lysosomal enzyme was released from Kupffer cells in Gal N rats, chemiluminescence was slightly suppressed in spite of the high titer of endotoxemia. These results appear to be related to the consumption of O-2 during liver injury. The functional state of Kupffer cells was thus changed by the grade of endotoxemia and hepatic injury.

Animals↗