浙江省热原检查用兔敏感性状况调查

目的调查浙江省热原检查用兔对细菌内毒素的敏感性情况,为提高热原检查结果的准确性和可靠性提供参考。方法对全省所有取得生产许可证单位的家兔,用国家颁发的细菌内毒素标准品,＂热原检查法＂进行检查,剂量分别为5EU/Kg和10EU/Kg,记录并比较各单位家兔的平均升温值和升温率。结果对细菌内毒素,各兔场家兔的敏感性有一定的差异。静脉注射5EU/kg,平均升温值为0.40℃～0.87℃,升温率为35%～83%;静脉注射10EU/kg,平均升温值为0.74℃～1.16℃,升温率为70%～94%。结论不同生产单位的家兔对细菌内毒素的敏感性不同,有必要对热原检查用家兔进行细菌内毒素敏感性检查。     

Age-related differences in thermoregulatory responses to endotoxin in rabbits.

E. coli endotoxin evokes fever in rabbits immediately after birth. In 0--3 day-old rabbits the fever is monophasic and brown fat thermogenesis is mainly responsible for the reaction. In 6--10 day-old animals the fever is usually biphasic and increased heat conservation also contributes to the response. An inverse relationship exists between the endotoxin dose and the latent period before the onset of fever, while the height of the fever is independent of the endotoxin dose. The response is similar as that of adult rabbits, except that after all endotoxin doses the latent period is longer and the magnitude of the response slightly smaller in the newborn.     

Maternal fever at parturition and its effects on the newborn rabbit.

The febrile response to administration of endotoxin has been reported to be suppressed in both pregnant animals at term and in their newborn. In a previous study we found that newborn rabbits under appropriate conditions to develop a febrile reaction to injected endotoxin. In this investigation we sought to discover whether pregnant rabbits at term had a febrile response to endotoxin, and if so, its effect on thermoregulation in their newborn. Endotoxin (E. Coli LPS) was injected into 19 pregnant rabbits at term. Six delivered spontaneously within an hour. At one hour, 13 were given oxytocin, and a further 8 delivered within five minutes. The colonic temperature (Tc) of the mothers before endotoxin administration and at delivery, and of their young, was measured. The results were compared with those of 10 pregnant rabbits not given endotoxin, and their young. Within 15 min of delivery newborn rabbits from each litter were placed on a thermal gradient to assess their thermoregulatory responses. Pregnant rabbits at term developed an impressive febrile response to injected endotoxin and their young were born with high colonic temperatures. Newborn rabbits from febrile mothers selected higher thermal environments and maintained a higher colonic temperature than the newborn of non-febrile mothers. We conclude that fever is sustained in the first hours of life in the newborn of mothers injected with endotoxin. The possible mechanisms are of considerable interest. None of the pregnant rabbits died after endotoxin administration, but the stillbirth rate was 50% compared with 10% in non-febrile does.     

Intravenous alpha-MSH reduces fever in the squirrel monkey

alpha-MSH reduces fever in rabbits when administered IV, ICV, or by gavage; however, the applicability of this finding to higher species, specifically to primates, has not been determined. In this study, we chose the squirrel monkey as an appropriate primate model since it responds reliably to peripheral administration of bacterial endotoxins that cause fever in man. From pilot studies, doses of S. typhosa endotoxin necessary to produce maximum fever and doses of alpha-MSH which did not cause hypothermia were determined for each animal. In the main experiments endotoxin was given via an indwelling catheter in the saphenous vein, followed by alpha-MSH injections when the rectal temperature increased 0.3 degrees C. alpha-MSH (100-400 micrograms) reduced the area under the fever curve an average of 50.0%, but had no effect on afebrile temperature. Molar equivalent amounts of the antipyretic drug acetaminophen had little effect on fever. These findings support the idea, based on research on rabbits, that alpha-MSH has a role in central modulation of fever.     

The protective effect of endotoxin in X-irradiated mice

In mice X-irradiated with lethal dose (750 r) and mid-lethal dose (550 r) the protective effect of bacterial endotoxin isolated from strains ofSalmonella typhi has been found to be limited to a short period before irradiation and administration of endotoxin in repeated doses did not enhance protection. Application of endotoxin 4 days after X-rays resulted in increase of lethality of irradiation and earlier deaths of experimental animals were observed. Active intravenous and intraperitoneal immunization with endogenous strains ofEscherichia coli isolated from the intestinal flora of mice had a demonstrable protective effect when compared with passive immunization.     

Local Schwartzman phenomenon in axenic rabbits]

Local Schwartzman phenomenon was produced by coli-endotoxin in all the germfree rabbits tested (11 in all) at the age of 102 to 135 days. Any kinds of natural antibodies were not detected in sera of the rabbits, which in fact were found to be agammaglobulinaemic in most cases, as revealed by immunoelectrophoresis. These facts suggested that the germfree rabbits utilized here had not been sensitized to bacterial endotoxins. From the results obtained here, it may be concluded that the existence of hypersensitivity to endotoxin is not necessary to the production of local Schwartzman phenomenon by bacterial endotoxin.     

Ventricular myosin from young and adult animals with respect to the thyroid state

Studies were conducted to analyze the effect of the thyroid hormone on ventricular myosin during ontogenesis of mice, rats and rabbits. Hypothyroidism was induced in mice and rats by administering propylthiouracyl in drinking water. Rabbits were made hyperthyroid by chronic administration of thyroxine. The change in the thyroid state of rats and rabbits influenced young and adult animals differently depending on whether V1 or V3 was the major ventricular isomyosin form present. Measurements of Ca2+-ATPase activity of myosins from young and old control animals and from animals with changed thyroid state showed that hypothyroidism in rats is associated with a greater decrease of myosin ATPase in young rats which contain V1 isomyosin only, when compared with old rats which contain a preponderance of V3 isomyosin and less of the V1 form. In rabbits, ATPase activity of ventricular myosin was more elevated after thyroxine administration in adult rabbits, which contain V3 isomyosin only, than in young rabbits in which myosin consists of V1 and V3 isomyosins. Ventricular myosins of young and adult mice did not differ in their ATPase activity and the treatment of mice with propylthiouracyl had only slight effect on myosin ATPase. It can be concluded based on these results that the hypothesis concerning hypothyroidism inducing transformation of V1 into V3 isomyosin does not hold generally.     

Studies on the Dutch rabbit for pyrogen test

A study was conducted on Dutch rabbits to examine their applicability for the pyrogen test in comparison with the Japanese white rabbit which, conventionally, has been mainly used for the test in Japan. The following results were obtained. Adult Dutch rabbits, so small as to weight only about 60% of average adult weight of the Japanese white, showed a food consumption as low as 40% of that in the latter. None in the Dutch rabbit group suffered dislocation of the hip-joint during restraint on a neck stock while it was frequently encountered among Japanese white rabbits. The body temperature during restraint was unstable in infantile Dutch rabbits (7-8 weeks old)(, but stable in the young (14 weeks old) and in the adult (20-47 weeks old). Adult Dutch rabbits exhibited greater febrile responses to intravenously injected bacterial pyrogen (LPS) than infantile and young Dutch rabbits and adult Japanese white rabbits. A good linear regression was observed between the dose of LPS (0.01-0.1 microgram/kg, iv) and the febrile response in adult Dutch rabbits as well as in adult Japanese white rabbits. From these data, it is concluded that the Dutch rabbit has an advantage over the Japanese white for the pyrogen test in respect of febrile responsiveness and cost of rearing.     

Demonstration of a macrophage migration enhancement factor in the sera of young rabbits

Alveolar macrophages (AM), harvested from the lungs of untreated normal young rabbits (New Zealand White) 14 days to 8 weeks of age, exhibited a state of migration stimulation compared to AM from normal adult rabbits (5 to 6 months of age). Migration of AM from normal adult rabbits (New Zealand White) was stimulated 2.0- to 2.5-fold when incubated with sera from 39- to 46-day-old rabbits compared with sera from normal adult rabbits. Furthermore, 4-day spleen cultures obtained from animals 28 to 59 days of age yielded supernatants that also stimulated the migration of adult AM. The spleen cell culture supernatants from 42- to 49-day-old animals had the greatest activity and stimulated the migration of adult AM 2.5- to 3.2-fold compared to the supernatants from adult normal rabbits. The peak production of migration enhancement factor (MEF) by splenic lymphoid cells coincided with the peak activities found in the sera. It was observed that nonadherent peanut agglutinable lymphoid cells produced MEF. When sera or culture supernatants containing MEF were mixed with MIF-containing adult sera or spleen cell culture supernatants, the respective activities were neutralized. The large migrations of normal neonatal AM were diminished by the addition of MIF-containing sera obtained from BCG-sensitized/challenged rabbits. In contrast, AM from BCG-sensitized rabbits, which exhibited a state of reduced migration, were enhanced by MEF-containing sera from untreated young rabbits. Three peaks of MEF activity were detected in Sephadex G-100 column fractionated sera from 42-day-old rabbits having MWs of approximately (Peak I) 80,000, (Peak II) 43,000, and (Peak III) 8000 to 18,000; most of the activity was found in peaks II and III. Two peaks of MEF activity were detected in Sephadex G-100 column-fractionated spleen cell culture supernatants from 42-day-old rabbits having MWs of approximately (Peak I) 35,000 to 43,000 and (Peak II) 10,000 to 14,000; most of the activity was in peak I which corresponds to peak II of the serum fractionation experiment. Collectively, these data indicate that MEF is a lymphokine that could be important in the modulation of cell-mediated immune effector responses.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

Potentiation and suppression of mouse liver cytochrome P-450 isozymes during the acute-phase response induced by bacterial endotoxin

Infection and inflammation are known to affect the metabolism and disposition of drugs and carcinogens. We report a detailed study of the effects of bacterial endotoxin on the constitutive and inducible expression and activities of cytochrome P-450 isozymes from families P-450I, P-450IIB, P-450IIC and P-450III. In general high doses of high endotoxin caused very marked suppression of P-450 isozymes and associated activities. However, this effect was differential, the expression of certain isozymes being only slightly reduced whereas others were suppressed to almost undetectable levels. Low doses of endotoxin also gave differential effects on cytochrome P-450 expression. Of particular interest was the very marked potentiation of the inductive effect of both 3-methylcholanthrene and phenobarbital. In the case of 3-methylcholanthrene the 10-fold induction of activity was increased to 24-fold by concomitant endotoxin administration. In this regard it was interesting that 3-methylcholanthrene was an effective inducer of a wide variety of acute-phase proteins including metallothionein, serum amyloid A, fibrinogen and hemopexin. These data show that endotoxin, and therefore bacterial infection and inflammation, can have profound and differential effects on components of the cytochrome-P-450 monooxygenase system which could result in significant changes in susceptibility to the effects of drugs, chemical toxins and carcinogens.     

Effect of temperature on the induction of interferons by endotoxin and virus

Postic, Bosko (University of Pittsburgh, Pittsburgh, Pa.), Catherine DeAngelis, Mary K. Breinig, and Monto Ho. Effect of temperature on the induction of interferons by endotoxin and virus. J. Bacteriol. 91:1277-1281. 1966.-The effect of ambient and body temperature on interferon formation in rabbits injected intravenously with virus differed from that seen after injection of endotoxin. Newcastle disease virus-induced interferon (VII) was elevated by increasing ambient temperature to 35 C, whereas cooling of the rabbit at 4 C resulted in low VII levels. Neither of these conditions affected the titers of endotoxin-induced interferon (EII). However, a significant enhancement of EII levels was found in sera of shorn rabbits, in which the body temperature was lower than in unshorn animals by 1.0 to 1.5 F and the pyrogenic response to endotoxin was less by about 2 F. This enhancement of EII by relatively low body temperatures was also in striking contrast to the reported enhancing effect of high body temperature of the rabbit on the lethal action of endotoxin. It is suggested that the temperature optimum for formation of EII is lower than for formation of VII.     

Endotoxin activity of Moraxella osloensis against the grey garden slug,Deroceras reticulatum

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.     

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.     

Effect of age on collagen fibril diameter in rabbit patellar tendon repair

The effect of aging on soft tissue repair is poorly understood. We examined collagen fibril diameter in repairing patellar tendons from young adult and aging rabbits. We hypothesized that repairing tendons from older (geriatric) rabbits would have similar diameter fibrils compared with the younger (young adult) rabbits. Full-length, full-thickness, central-third (2.5 to 3 mm) patellar tendon injuries were made by cutting out the center of the tendon in twelve 1-y-old and thirteen 4- to 5.5 (average, 4.25)-y-old female New Zealand White rabbits. The contralateral tendon served as an unoperated control. The rabbits were euthanized at 6, 12, and 26 wk after surgery. The collagen fibril diameter was examined by electron microscopy at the patellar end, middle, and tibial end of the patellar tendon. There was no significant decline in collagen fibril diameter at any location in the aging rabbit healing patellar tendons compared with those of the 1-y-old rabbits. This study found that collagen fibril diameter was not altered with increasing age in the healing rabbit patellar tendon.     

Recombinant tumor necrosis factor enhances macrophage destruction of Trypanosoma cruzi in the presence of bacterial endotoxin

We examined in this work whether rTNF inhibits the capacity of Trypanosoma cruzi to multiply within murine macrophages or enhances the ability of the phagocytic host cells to destroy internalized parasites. We found that rTNF would not alter the fate of the trypanosomes within macrophages over a 48-h incubation period unless the latter cells were also treated with 1 ng/ml bacterial endotoxin (LPS). Treatment of macrophages with rTNF plus LPS, but not separate treatment with either rTNF or LPS, resulted in a significant decrease in the number of organisms per 100 macrophages with respect to values obtained with mock-treated macrophages. In addition, there was a significant reduction in the proportion of infected macrophages over the 48-h incubation period, indicating parasite clearance by the host cells. The combined effects of rTNF and LPS were seen when macrophages from CBA/J were used but not with LPS-insensitive macrophages from C3H/HeJ mice. Increased trypanosome killing by CBA/J macrophages treated with rTNF plus LPS was not seen when catalase was present in the culture medium, indicating a role for hydrogen peroxide in the cytotoxic effect. These results show that rTNF can affect the fate of T. cruzi within macrophages if LPS is present and point to destruction of internalized organisms rather than inhibition of parasite multiplication as the most likely explanation.     

Effects of endotoxin on immunity in aging mice

In vivo administration of bacterial lipopolysaccharides (LPS) to young adult mice causes a dose-dependent depression of antibody formation. In contrast, LPS produced no effects on the responses of older mice and these animals appeared to be refractory to the depressive effects observed in the young adults. When young adults were pretreated with endotoxin their baseline control responses were also severely depressed and the LPS response profile resembled that of the normal 9-month-old animals. These results suggest that endotoxin effects may contribute to the genesis of immune abnormalities in the aged.     

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.     

Peculiarities of the development of chloramine induced pulmonary edema in rabbits in various age periods]

Rabbits under three weeks of age were found to be less tolerant to chloramine (CA) than adult rabbits. For them the lethal dose of CA was 40--50 mg/kg. They perished from intoxication with the symptoms of protracted collapse without any manifestations of edema. For older rabbits the lethal dose of CA was 60--70 mg/kg. Death occurred with phenomena of acute and sharp edema of the lungs. In young rabbits atypical edema of the lungs occurred only with the administration of very high doses of CA--200--250 mg/kg. Edematous fluid is present in small amount only; there is a prevalence of hemorrhage, this being possibly caused by a direct alterating action of the CA excreted through the lungs on the walls of the arterioles and capillaries of the vascular system of the lungs.     

Potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits: Possible relevance of hyperreactivity of macrophages to endotoxin

Abstract Streptococcal pyrogenic exotoxin (SPE) potentiates lethal shock induced by endotoxin. We have previously reported that macrophages derived from SPE-treated rabbits showed hyperreactivity to endotoxin, and that the effect of SPE on macrophages was mediated by a lymphokine(s). Here we show that culture supernatants of SPE-stimulated lymphocytes, when administered into rabbits three hours before or together with endotoxin, potentiate a variety of endotoxin-induced pathophysiological changes and even lethal shock. These results suggest that SPE-induced lymphokine(s) mediates the potentiating effect of SPE on the lethal endotoxin shock through enhancing endotoxin reactivity of macrophages which play the central role in mediating endotoxin toxicity.