Studies on pyrogenic tolerance to bacterial lipopolysaccharide in rabbits.

The development of pyrogenic tolerance was studied in rabbits treated with varying doses of E. coli lipopolysaccharide (LPS). The following results were obtained. 1) Development of pyrogenic tolerance seemed to proceed in two steps: that is, the first in which tolerance appeared rapidly and the second in which tolerance proceeded more gradually or steadily in response to repeated injections of a constant dose. 2) Tolerance induced by the latter method was not absolute; the rabbits were still sensitive to increased doses of LPS. 3) Rabbits immunized with E. coli vaccine lost their pyrogenic sensitivity of parent LPS to some extent. 4) Following intracisternal injection of LPS into tolerant rabbit, pyrogenic response was not decreased but rather enhanced in comparison with control. 5) The contents of nor-epinephrine and serotonin in the brain did not differ between normal and tolerant rabbits. 6) The mechanisms of endotoxin tolerance remain to be further studied.     

Detoxified bacterial endotoxns. II. Preparation and biological properties of chemically modified crude endotoxins from Salmonella typhimurium

Martin, William J. (University of Utah, Salt Lake City), and Stanley Marcus. Detoxified bacterial endotoxins. II. Preparation and biological properties of chemically modified crude endotoxins from Salmonella typhimurium. J. Bacteriol. 91:1750-1758. 1966.-Chemical modification of a crude endotoxin prepared by the Roschka-Edwards (RE) procedure from a strain of Salmonella typhimurium yielded products which were nontoxic for mice and had reduced fever effects in rabbits. A reduction in rabbit pyrogenicity of approximately 100 times was noted with a potassium periodate-treated RE preparation when compared with the parent RE preparation. Measured in a similar fashion, pyrogenicity of a potassium methylate-treated RE preparation was reduced by a factor of 10 while pyrogenicity of a boron trifluoride RE preparation was unchanged. All of these endotoxoids, including the parent RE preparation, showed little toxicity for mice. Immunogenicity was determined in mice by comparing Boivin, RE, and endotoxoid preparations with a heat-killed, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. Employing a 10 ld(50) challenge, the protective immunogenicity of the respective vaccines was determined by active immunized mouse protection tests. Although two 100 mug immunizing doses of the Boivin, RE, and the respective endotoxoid preparations varied in mouse protection (potassium methylate RE > Boivin > RE > boron trifluoride RE > potassium periodate RE), it was evident that, with the exception of the potassium methylate preparation, the HP vaccine yielded greatest protection against the 10 ld(50) challenge with S. typhimurium. Further mouse protection experiments suggested that the minimal immunogenic dose of the potassium methylate RE vaccine preparation was approximately 50 mug. These data indicated an approximate fivefold difference between the minimal pyrogenic dose (10 mug) and the minimal immunogenic dose (50 mug). These findings further suggest that potassium methylate RE vaccine preparations should be considered in the search for less toxic enteric fever vaccines.     

Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage

Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.     

Feasibility of implementing cell-based pathway reporter assays in early high-throughput screening assay cascades for antibody drug discovery

Implementing functional cell-based screens in early antibody discovery has become increasingly important to select antibodies with the desired profile. However, this is limited by assay tolerance to crude antibody preparations and assay sensitivity. The current study aims to address this challenge and identify routes forward. Two common types of high-throughput screening (HTS) antibody sample, derived from either phage display or hybridoma techniques, have been screened across a wide range of CellSensor beta-lactamase reporter assays in a variety of cell backgrounds to more extensively characterize assay tolerance. Pathway-, sample-, and cell background-specific effects were observed. Reporter assays for agonism were less affected by crude antibody preparations, with 8 of 21 sample tolerant, and the potential to implement an additional 8 assays by choosing the best-tolerated sample type. Antagonist mode assays exhibited more complexity, with potentiating as well as inhibitory effects. However, 5 of 24 antagonist assays were fully tolerant, with the potential to implement an additional 11 assays. Different subsets of assays were affected in agonist versus antagonist mode, and hybridoma sample sets were better tolerated overall. The study clearly demonstrates the potential to use cell-based reporter assays in biologics HTS, particularly if the method of antibody production is considered in the context of the required assay mode (agonist/antagonist).     

The pathophysiological effect of scarlet fever toxin

The intravenous injection of scarlet fever toxin leads to acute changes in the white blood picture. The changes are correlated to the dose; a small dose causes lymphopenia and granulocytosis, while higher doses are followed by initial granulocytopenia. Tolerant rabbits do not have granulocytopenia, but lymphopenia persists. Tolerance to the leucopenic effect of the toxin probably develops somewhat sooner and lasts longer than tolerance to its pyrogenic effect. The blood picture of a cortisone-treated rabbit after the intravenous injection of scarlet fever toxin resembles the blood picture of a tolerant rabbit. The significance and associations of these observations are briefly discussed.     

Site-specific interaction of ATPase-pumped protons with photosystem II in chloroplast thylakoid membranes

The chloroplast thylakoid ATPase proton pump-driven H+ accumulation in the dark was compared to the light-dependent proton pump driven by either photosystem II or I, in regard to the effects of the resultant acidity on chemical modification reactions. The assays used to detect the acidity effects were: (a)the incorporation of [3H]-acetic anhydride into membrane protein -NH2 groups, and (b) the effect of a certain level of that chemical modification on inhibition of photosystem II water oxidation activity. Based on labeling data with [3H]-acetic anhydride, 20-30 nmol.(mg chl)-1 of -NH3+ groups appear to be metastable in the dark in untreated membranes. The term metastable is used because proton leak-inducing treatments in the dark lead to about 20-30 nmol . (mg chl)-1 increase in acetic anhydride labeling probably due to reaction with the -NH2 form of amine groups. Addition of low levels of uncoupler or a brief thermal treatment caused a loss of protons from the membrane equivalent to the increase in acetic anhydride derivatization. The increase in acetic anhydride derivatization caused inhibition of water oxidation activity. Using thermally sensitized membranes, photosystem II but not photosystem I electron transport (each giving a steady-state proton accumulation of about 50 nmol H+ . (mg chl)-1 restored the lower level of acetic anhydride reactivity as in previous results (Baker et al., 1981). In dark-maintained, thermally treated membranes, ATPase activity, i.e., the proton pump associated with it, also restored the lower level of acetic anhydride labeling, and again acetic anhydride no longer inhibited water oxidation. Because photosystem I activity did not elicit this type of response to acetic anhydride, there appears to be a pathway for ATPase pumped protons which allows them to reach a restricted domain, perhaps intramembrane, common with the photosystem II water oxidation mechanism and unavailable to protons pumped by photosystem I. The membrane structure(s) which determines this site specificity is not yet understood.     

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. I. Tolerance and effect of antibody in fever response, and heart damaging effect in rabbits.

Induced tolerance to the pyrogenic action of group A streptococcus peptidoglycan decreased after one week and was no longer detectable after the second week. However, one or two further doses of peptidoglycan rapidly restored the tolerance. The passive transfer of plasma from rabbits tolerant to streptococcus peptidoglycan to nontolerant animals failed to transfer tolerance. Antiserum to streptococcus peptidoglycan neutralized the pyrogenic effect of not only streptococcus but also staphylococcus and pneumococcus peptidoglycan; it did not influence the febrile response to endotoxin. Histopathologic changes in the rabbit heart produced by the intravenous injection of staphylococcus or pneumococcus peptidoglycans were similar and were characterized by various stages of degeneration and necrosis. The changes were less pronounced than after streptococcus peptidoglycan. Antiserum to streptococcus peptidoglycan had modest or no counteracting effect on the development of heart alterations after staphylococcus or pneumococcus peptidoglycan.     

Macrophage hyperreactivity to endotoxin induced by streptococcal pyrogenic exotoxin in rabbits

Pretreatment of rabbits with streptococcal pyrogenic exotoxin (SPE) resulted in an enhancement of their febrile response to subsequent endotoxin challenge. This suggested that SPE may enhance the macrophage capacity to respond to endotoxin in vivo to produce an endogenous pyrogen. It was also demonstrated that peritoneal macrophages derived from SPE-treated rabbits exhibited hyperreactivity to endotoxin in vitro as assessed by endotoxin-induced increase in glucose consumption. These data indicate that SPE has the ability to enhance macrophage reactivity to endotoxin.     

Human interferon for clinical trials: removal of pyrogen by a simple two-step procedure

Commonly used preparations of human interferon are pyrogenic when used in humans for clinical trials. We describe a simple two-step procedure for the purification of human fibroblast interferon, employing Blue Sepharose chromatography and high-speed centrifugation. Such preparations are devoid of pyrogenic activity in a rabbit test system and are much more suitable for use in human clinical trials than previously used preparations.     

A novel bivalent Pasteurellosis-RHD vaccine candidate adjuvanted with Montanide ISA70 protects rabbits from lethal challenge

In the present study, a bivalent vaccine against Pasteurella multocida and rabbit hemorrhagic disease virus (RHDV) was formulated with Montanide™ ISA70 oil adjuvant (Seppic, Paris, France). Its efficacy was evaluated and compared to similar monovalent preparations and commercially available monovalent vaccines. White new Zeeland rabbit groups (n = 10) received 2 successive doses of the tested vaccines and were challenged 2 weeks after 2nd dose with Pasteurella multocida and RHDV or either pathogens according to their vaccination schedule. Challenged not-vaccinated group of rabbits (n = 10) was included as a control. The bivalent and monovalent ISA70 preparations were found stable, safe, sterile, pure and of low viscosity. Group 3 (GP3) which received bivalent vaccine showed the highest antibody geometric mean titers against Pasteurella multocida and RHDV evaluated by ELISA and hemagglutination inhibition (HI) respectively. Following virulent challenge; Gp3 rabbits were 90% protected from challenge over other groups that showed 80% protection. Detection of either pathogen in the livers of dead and euthanized rabbits had failed except for non-vaccinated controls. The bivalent vaccine candidate was fully protective. Immunization against both pathogens can be achieved by single vaccination.     

The pyrogenic effect of scarlet fever toxin

Scarlet fever toxin was found to liberate leukocytic pyrogen from granulocytesin vitro. In comparative experiments withSalmonella paratyphi B endotoxin and scarlet fever toxin it was tested whether leukocytes from rabbits tolerant to one of these toxins are able to synthetize and liberate endogenous pyrogen. Leukocytes from rabbits tolerant to endotoxin liberated leukoeytic pyrogen following challenge with endotoxin or with scarlet fever toxin. Leukocytes from animals tolerant to scarlet fever toxin liberated leukocytic pyrogen in the presence of endotoxin, but were insensitive to homologous, i.e. scarlet fever toxin. Similarly, leukocytes from cortisone-treated animals did not liberate leukocytic pyrogen if they were incubated with scarlet fever toxin, but liberation of leukocytic pyrogen did take place under challenge with endotoxin. Leukocytes from normal animals incubated in Hanks solution without toxin did not synthetize endogenous pyrogen.     

Ascaris suum: protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.     

Detoxified bacterial endotoxins. I. Preparation and biological properties of an acetylated crude endotoxin from Salmonella typhimurium

Martin, William J. (University of Utah, Salt Lake City), and Stanley Marcus. Detoxified bacterial endotoxins. I. Preparation and biological properties of an acetylated crude endotoxin from Salmonella typhimurium. J. Bacteriol. 91:1453-1459. 1966.-Acetylation of a crude endotoxin prepared by the Roschka-Edwards (RE) procedure from a strain of Salmonella typhimurium yields a product with reduced pyrogenicity in rabbits as well as one which is nontoxic in mice. A reduction in pyrogenicity of approximately 100 times was noted with this acetylated crude endotoxin when compared with the parent RE preparation. A comparison was made of immunogenicity with mice of Boivin, RE, and acetylated Roschka-Edwards (Acet-RE) preparations with a heat-killed, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. Less than pyrogenic doses of all vaccines were not protective. The least pyrogenic preparation (Acet-RE) was immunogenically effective in about five times the minimal pyrogenic dose. The data suggest that the Acet-RE preparation should be considered further in the search for enteric fever vaccines with lowered potential for undesirable systemic responses.     

A new inhibitor of coupled oxidative phosphorylation, 5-hydroxynaphthalenedicarboxylic anhydride, a derivative of a carcinogenic polynuclear hydrocarbon.

5-Hydroxy-1,2-naphthalenedicarboxylic anhydride is closely related to its precursor dibasic acid which is a metabolite of the carcinogenic polynuclear hydrocarbon dibenz[a,h]anthracene. The anhydride inhibited respiration of coupled mitochondria. This inhibition was relieved by 2,4-dinitrophenol. Several mitochondrial volume change processes energized by ATP were also inhibited by the anhydride. Both the mitochondrial ATPase activity induced by 2,4-dinitrophenol and the ATPase activity of submitochondrial particles induced by magnesium ion were inhibited by the anhydride. The spectrum of inhibitory activity was not associated with acetic anhydride, succinic anhydride, or phthalic anhydride. The data indicate that 5-hydroxy-1,2-naphthalenedicarboxylic anhydride inhibits the machinery of oxidative phosphorylation in a manner similar to rutamycin. 5-Hydroxy-1,2-naphthalenedicarboxylic anhydride is the first molecule derived from a carcinogen with such inhibitory properties.     

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

毒素源性大肠杆菌CFA/I、CS_3重组菌株的粘附力、免疫原性和保护性的研究

本研究采用可逆性肠结扎成兔腹泻(RITARD)动物模型检测了毒素源性大肠杆菌(ETEC)定居因子抗原Ⅰ(CFA/Ⅰ)和表面抗原3(CS_3)重组菌株的肠道粘附力、免疫原性和保护性。经肠道及口服接种时重组菌株对肠道均有较强的粘附力,与CFAs阴性宿主菌相比P<0.01。口服免疫家兔时,第四周血清抗体滴度达到高峰,分别为1∶9 000和1∶80 000;肠道免疫家兔后血清效价在第二周达到高峰,滴度分别为1∶8 000和1∶60 000。在抗体滴度高峰时进行ETEC野生株攻毒,结果显示口服组抗腹泻保护率为66.7％～75％,肠道组为50.1％～71.4％。攻毒后免疫家兔的排菌天数较对照组明显减少。     

Immunization of female rabbits with heat-solubilized bovine zonae: Production of anti-zona antibody and inhibition of fertility

Bovine zonae were solubilized by heating to 80°C in buffered saline. On injection of female rabbits with this solution, zona-specific antisera were produced that could be assayed on bovine eggs in vitro by immunofluorescence, coating, and inhibition of sperm-binding. When the latter was complete, the rabbits were found to be infertile. Immunofluorescence assays showed that the rabbit anti-bovine zona serum reacted as strongly with rabbit and rhesus monkey zonae as with homologous zonae. Strong cross-reaction was also observed with marmoset and dog zonae, but with hamster zonae the reaction was relatively weak.     

Biological activity of synthetic subunits of Streptococcus peptidoglycan. II. Relation of peptidoglycan subunits and analogues to fever effect and induction of tolerance

A series of synthetic subunits and analogues of streptococcal peptidoglycan was prepared and used in fever and tolerance experiments on rabbits. The lengthening of the chain of the peptide moiety of peptidoglycan did not result in pyrogenic activity, except for hexapeptide. Attachment of the muramyl residue rendered the peptides pyrogenic. The activity of such materials varied in degree and was rather in an indirect relation to peptide chain length. A change in the configuration of C4-OH or C3-OR in the muramyl residue resulted in a profound decrease in pyrogenicity. No inhibitory effect of N-acetylmuramyl-D-alanyl-D-isoglutamine on muramyldipeptide (MDP) pyrogenicity could be demonstrated. Repeated administration of MDP resulted in the induction of tolerance to the pyrogenicity of this substance in rabbits. These animals were not tolerant to the pyrogenicity of peptidoglycan. Nontolerance was also observed in reciprocal experiments with these materials as well as in trials with hexapeptide and peptidoglycan given in either order. The data are consistent with the assumption that peptidoglycan contains more than one biologically active subunit. There is a structure-to-function relationship. The knowledge of the biological effects of the synthetic analogues is essential for the prospect of their use under model or human conditions.     

The production of migration inhibitory factor (MIF) in rabbit lymphocytes by other than immunological mechanism

The ability of certain substances to activate lymphocytes in terms of the production of biologically active substances was studied. These substances were tested by following their migration inhibitory activity. The capacity of concanavalin A and commercial preparations of phytohaemagglutinin (PHA-M and PHA-P) to induce formation of MIF was confirmed. In addition, similar activities were found even in antirabbit antilymphocyte serum (ALS) and erythrogenic toxin (ET). The production of MIF was usually found in material obtained from rabbits treated with complete Freund adjuvant (CFA). On the other hand, material obtained from non-treated rabbits, was found to be inactive with the exception of thymus from young rabbits. The results support the significance of an increasing pharmacological potency of cells in the course of sensitization with CFA. The evidence concerning the release of MIF by ET was extended by similar finding in rabbits made tolerant to the pyrogenic activity of this particular toxin. Lymphocytes obtained from rabbits that were unable to respond to ET by fever, could not liberate MIF (at the same time) upon incubation with ET, even though these lymphocytes produced MIF normally after incubation with PPD tuberculin or concanavalin A. The importance of these results is discussed in terms of the immunologically nonspecific activation of lymphocytes that can mimic immunologically specific events.     

Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes

Treatment of rabbits with 1% (v/v) acetone for 1 week resulted in the appearance in blood serum of 88 +/- 14 14 nmol/ml 1-hydroxyacetone (acetol) and 70 +/- 9 nmol/ml 1,2-propanediol. Untreated rabbits had no detectable 1,2-propanediol or acetol. Hepatic microsomes from control, ethanol-, and acetone-treated rabbits catalyzed the hydroxylation of acetone at rates of 0.32 +/- 0.01, 2.01 +/- 0.43, and 3.64 +/- 0.23 nmol/min/mg of protein, respectively. The same microsomal preparations catalyzed the hydroxylation of acetol at rates of 0.33 +/- 0.04, 0.94 +/- 0.20, and 1.08 +/- 0.12 nmol/min/ mg of microsomal protein, respectively. Isozyme 3a purified from acetone- or ethanol-treated rabbits was identical as judged by comparison of the high performance liquid chromatographic profiles of tryptic digests of the two proteins. Antibody to isozyme 3a inhibited greater than 90% of the acetone monooxygenase activity from untreated, acetone-, or ethanol-treated rabbits. In contrast, the antibody only inhibited 30% of the acetol monooxygenase activity of microsomes from untreated rabbits. The inhibition was increased to about 70% after acetone or ethanol treatment. Although the activities were inhibited to different extents, a comparison of the rates attributable to isozyme 3a from antibody inhibition experiments indicated that both activities were induced to a similar extent by ethanol. Similarly, acetone also increased both activities to the same extent but was more effective than ethanol. In a reconstituted system, isozyme 3a was the only isozyme of six forms from rabbit liver to exhibit acetone monooxygenase activity. Isozyme 3a was the most active enzyme in the hydroxylation of acetol, but isozymes 2, 3b, and 4 also were able to catalyze the reaction. Antibody to isozyme 3a also inhibited greater than 90% of the acetone hydroxylase activity and 70% of the acetol hydroxylase activity of microsomes from acetone-treated rats. Two proteins were immunochemically stained on Western blots of microsomes from untreated and acetone-treated rats, one of which was increased by acetone treatment. These results suggest that isozyme 3a in rabbit and an immunochemically homologous enzyme in rat are responsible for acetone and acetol hydroxylation, the initial steps in the proposed gluconeogenic pathways for acetone.