Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.     

凝结芽胞杆菌TBC 169株对肠道致病菌的抑菌作用

目的 研究凝结芽胞杆菌TBC 169株对大肠埃希菌、痢疾志贺菌、伤寒沙门菌、普通变形杆菌、铜绿假单胞菌和金黄色葡萄球菌的抑制作用。方法 先将大肠埃希菌、痢疾杆菌等6种菌分别进行单独培养,测定不同培养时间内的pH和活菌数,然后将凝结芽胞杆菌TBC 169株分别和致病菌进行混合培养,再测pH和活菌数,并与单独培养时的测定情况进行比较。结果 凝结芽胞杆菌TBC 169株对大肠埃希菌、痢疾志贺菌、伤寒沙门菌等6种菌均有明显的抑制作用,尤其是对伤寒沙门菌和铜绿假单胞菌的抑制作用更强。结论 凝结芽胞杆菌TBC 169株对肠道致病菌有显著的抑制作用。     

Establishment of a poliovirus oral infection system in human poliovirus receptor-expressing transgenic mice that are deficient in alpha/beta interferon receptor

Poliovirus (PV) is easily transferred to humans orally; however, no rodent model for oral infections has been developed because of the alimentary tract's low sensitivity to the virus. Here we showed that PV is inactivated by the low pH of the gastric contents in mice. The addition of 3% NaHCO3 to the viral inoculum increased the titer of virus reaching the small intestine through the stomach after intragastric inoculation of PV. Transgenic mice (Tg) carrying the human PV receptor (hPVR/CD155) gene and lacking the alpha/beta interferon receptor (IFNAR) gene (hPVR-Tg/IfnarKO) were sensitive to the oral administration of PV with 3% NaHCO3, whereas hPVR-Tg expressing IFNAR were much less sensitive. The virus was detected in the epithelia of the small intestine and proliferated in the alimentary tract of hPVR-Tg/IfnarKO. By the ninth day after the administration of a virulent PV, the mice had died. These results suggest that IFNAR plays an important role in determining permissivity in the alimentary tract as well as the generation of virus-specific immune responses to PV via the oral route. Thus, hPVR-Tg/IfnarKO are considered to be the first oral infection model for PV, although levels of anti-PV antibodies were not elevated dramatically in serum and intestinal secretions of surviving mice when hPVR-Tg/IfnarKO were administered an attenuated PV.     

Interference of in vitro and in vivo growth of several intestinal bacteria by Lactococcus strains

The ability of Lactococcus strains to inhibit the growth of intestinal bacteria was examined. In in vitro cocultures, we observed that among eighteen Lactococcus strains tested, the ability to inhibit growth of Escherichia coli varied, with the L. lactis N7 showing the greatest growth inhibition. Strain N7 (8.94x1010 CFU/day for 7 days) was orally administered to mice, and the viable count of strain N7 in feces appeared at a level of 104-5 CFU/g. After administration, the proportion of Bacteroidaceae to total intestinal bacteria decreased. Lactococci may act as probiotic bacteria by inhibiting the growth of harmful bacteria.     

The Effects of Antilymphocytic Serum (ALS) on Trypanosoma lewisi Infection

SYNOPSIS. ALS was produced in rabbits immunized with cells collected from spleens and mesenteric lymph nodes of normal female Holtzman rats. The ALS was pooled and leucoagglutination tests were performed to determine the ability of the antiserum to agglutinate rat white blood cells in vitro. ALS employed in the study had a titer of 1 :512. Treatment of rats with ALS prior to and during Trypanosoma lewisi infection resulted in suppression of the immune response. Ablastin production was inhibited. The numbers of dividing forms of the parasites were higher in ALS-treated rats during the course of infection than in controls injected with saline or normal rabbit serum. ALS-treated rats continued to have a higher parasitemia than the controls and died 5–12 days post-infection. Hematocrit readings were lower in the ALS-treated rats infected with T. lewisi than in rats infected with T. lewisi or normal animals treated with ALS.     

Trypsin-dependent production of an antibacterial substance by a human Peptostreptococcus strain in gnotobiotic rats and in vitro.

An antibacterial substance appeared within 1 day in feces of gnotobiotic rats harboring a human intestinal Peptostreptococcus strain. It disappeared when the rat bile-pancreatic duct was ligatured or when the rats ingested a trypsin inhibitor. Anaerobic cultures of the Peptostreptococcus strain in a medium supplemented with trypsin also exhibited an antibacterial activity, which was also inhibited by the trypsin inhibitor. In vitro the antibacterial substance from both feces and culture medium was active against several gram-positive bacteria, including other Peptostreptococcus spp., potentially pathogenic Clostridium spp. such as C. perfringens, C. difficile, C. butyricum, C. septicum, and C. sordellii, Eubacterium spp., Bifidobacterium spp., and Bacillus spp. Whatever the order of inoculation of the strains, a sensitive strain of C. perfringens was eliminated within 1 day from the intestine of rats monoassociated with the Peptostreptococcus strain. These findings demonstrate for the first time that very potent antibacterial substances can be produced through a mechanism involving intestinal bacteria and exocrine pancreatic secretions.     

Effect of diosgenin on lipid metabolism in rats.

The purpose of this study was to determine whether diosgenin suppresses cholesterol absorption in rats, and to examine relevant changes in cholesterol and bile acid metabolism. Diosgenin fed with the diet for 1 week inhibited cholesterol absorption as determined by the serum isotope ratio technique, as well as by measuring in the feces the amount of unabsorbed radioactivity from orally administered [3H]cholesterol. In addition, diosgenin suppressed the serum and liver uptake of radioactivity from co-administered [3H]cholesterol as well as the accumulation of liver cholesterol in the cholesterol-fed rat; diosgenin was substantially more active than cholestyramine or beta-sitosterol. In vitro, diosgenin had no effect on the activity of rat pancreatic esterase. Diosgenin decreased the elevated cholesterol in serum LDL and elevated cholesterol in the HDL fraction of cholesterol-fed rats; diosgenin had no effect on serum cholesterol in normocholesterolemic rats. In contrast to cholestyramine, diosgenin markedly increased neutral sterol excretion without altering bile acid excretion; in vitro, diosgenin had no effect on bile acid binding. Diosgenin treatment increased hepatic and intestinal cholesterol synthesis as well as the activity of hepatic HMG CoA reductase. This was accompanied by increased biliary concentration of cholesterol, but not of bile acids. Diosgenin had no effect on cholesterol synthesis when added to normal rat liver homogenates. It was concluded that diosgenin interferes with the absorption of cholesterol of both exogenous and endogenous origin; such interference is accompanied by derepressed, i.e., increased, rates of hepatic and intestinal cholesterol synthesis. The increased unabsorbed cholesterol together with enhanced secretion of cholesterol into bile resulted in increased excretion of neutral sterols without affecting the biliary and fecal excretion of bile acids.     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Antileptospiral activity of serum. I. Normal and immune serum

Johnson, Russell C. (University of Minnesota, Minneapolis), and Louis H. Muschel. Antileptospiral activity of serum. I. Normal and immune serum. J. Bacteriol. 91:1403-1409. 1966.-Normal serum was found to exert a leptospiricidal effect, mediated by the complement system, against the nonpathogenic leptospires. Although resistant to normal serum, the pathogenic serotypes were susceptible to antiserum plus complement. Several variables in these immune leptospiricidal reactions were investigated. A reaction period of 3 hr at 37 C between serum substances and 1-day-old cells provided a maximal leptospiricidal effect. The normal serum of the rabbit, guinea pig, bovine, and human were leptospiricidal against the nonpathogenic serotypes, and, in conjunction with rabbit antiserum, rabbit and bovine complement were leptospiricidal against the pathogenic serotypes. Studies with C(14)-labeled leptospires indicated that the immune leptospiricidal reaction was associated with a loss of permeability control. Thus, like the gram-negative bacteria, the treponemes, erythrocytes, and nucleated mammalian cells, the leptospires may be included as cell types susceptible to the antibody-complement system.     

Establishment of specific pathogen-free (SPF) rat colonies using gnotobiotic techniques.

Gnotobiotic Wistar rats were produced using gnotobiotic techniques, which were established in the production of a SPF mouse colony, in order to establish a barrier-sustained colony. One strain of Escherichia coli, 28 strains of Bacteriodaceae (B-strains), three strains of Lactobacillus (L-strains) and a chloroform-treated fecal suspension (CHF, Clostridium mixture) were prepared from conventional Wistar rats as the microflora source. Two groups of limited-flora rats, E. coli plus B-strains and E. coli plus CHF, were produced. After confirmation that Clostridium difficile was not detected in the CHF-inoculated rats, two groups of limited-flora rats were transferred to an isolator and housed together in a cage. These rats were then orally inoculated with L-strains. The gnotobiotic rats showed colonization resistance to Pseudomonas aeruginosa, and the number of E. coli in the feces was 10(5) to 10(6)/g. The gnotobiotic rats were transferred to a barrier room as a source of intestinal flora for SPF colonies. In the SPF rats, basic cecal flora was mainly composed of Bacteroidaceae, clostridia, fusiform-shaped bacteria and lactobacilli, and did not change over a long period. Their flora became similar to that of conventional rats.     

沼泽红假单胞菌2c菌株的安全性评价

本实验旨在对1株沼泽红假单胞菌Rhodopseudomonaspalustris2c菌株(2c)作为益生菌株的安全性进行系统研究。结果表明,2c菌株体外对多数抗生素敏感;在体内不具有急性毒性,不会在血液中产生内毒素,在宿主体内自身不会易位,也不会造成其它肠道细菌易位,对肝肾功能无不良影响,对回肠黏膜及其细胞形态和结构无不良影响。2c菌株具备益生菌安全特性,值得进一步研究。     

Smouldering epidemic of Yersinia pseudotuberculosis in barn rats.

Yersinia pseudotuberculosis was isolated from 8 (8 Rattus norvegicus) of 270 (259 R. norvegicus and 11 R. rattus) rats examined. Seasonal variation was not found in the incidence of isolations. The isolation occurred almost equally in both young and old rats. The isolated strains were determined as serovar IB in one rat, and serovar IVA in seven rats. The strains were isolated from the contents of the intestinal tract (the duodenum, jejunum, ileum, cecum, colon, and rectum), the spleen, liver and mesenteric lymph nodes; they were not detected in the kidneys. Agglutinin titer in the eight rats was no more than 32.     

THE STABILITY OF BACTERIAL SUSPENSIONS : III. AGGLUTINATION IN THE PRESENCE OF PROTEINS,NORMAL SERUM,AND IMMUNE SERUM.

1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates.     

Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus.

Rotaviruses are the single most important cause of severe diarrhea in young children worldwide, and vaccination is probably the most effective way to control the disease. Most current live virus vaccine candidates are based on the host range-restricted attenuation of heterologous animal rotaviruses in humans. The protective efficacy of these vaccine candidates has been variable. To better understand the nature of the heterologous rotavirus-induced active immune response, we compared the differences in the mucosal and systemic immune responses generated by heterologous (nonmurine) and homologous (murine) rotaviruses as well as the ability of these infections to produce subsequent protective immunity in a mouse model. Sucking mice were orally inoculated with a heterologous simian or bovine rotavirus (strain RRV or NCDV) or a homologous murine rotavirus (wild-type or tissue culture-adapted) strain EHP at various doses. Six weeks later, mice were challenged with a virulent murine rotavirus (wild-type strain ECW) and the shedding of viral antigen in feces was quantitated. Levels of rotavirus-specific serum immunoglobulin G (IgG) and fecal IgA prior to challenge were measured and correlated with subsequent viral shedding or protection. Heterologous rotavirus-induced active protection was highly dependent on the strain and dose of the virus tested. Mice inoculated with a high dose (10(7) PFU per mouse) of RRV were completely protected, while the protection was diminished in animals inoculated with NCDV or lower doses of RRV. The ability of a heterologous rotavirus to stimulate a detectable intestinal IgA response correlated with the ability of the virus to generate protective immunity. Serum IgG titer did not correlate with protection. Homologous rotavirus infection, on the other hand, was much more efficient at inducing both mucosal and systemic immune responses as well as protection regardless of the virulence of the virus strain or the size of the immunizing dose.     

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

Susceptibility of Germ-free Mice to Infectious Megaenteron

Germ-free (GF)-ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c: K(B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.     

Smouldering epidemic of Yersinia pseudotuberculosis in barn rats.

Yersinia pseudotuberculosis was isolated from 8 (8 Rattus norvegicus) of 270 (259 R. norvegicus and 11 R. rattus) rats examined. Seasonal variation was not found in the incidence of isolations. The isolation occurred almost equally in both young and old rats. The isolated strains were determined as serovar IB in one rat, and serovar IVA in seven rats. The strains were isolated from the contents of the intestinal tract (the duodenum, jejunum, ileum, cecum, colon, and rectum), the spleen, liver and mesenteric lymph nodes; they were not detected in the kidneys. Agglutinin titer in the eight rats was no more than 32.     

Transfer of tubulointerstitial nephritis in the Brown Norway rat with anti-tubular basement membrane antibody: quantitation and kinetics of binding and effect of decomplementation

Lewis (LEW) rats immunized with Brown Norway (BN) rat renal basement membrane (RBM) and adjuvants produce high titer circulating anti-BN tubular basement membrane (TBM) antibodies, in addition to developing an autoimmune cell-mediated form of nodular tubulointerstitial nephritis (TIN). This immune LEW serum, which reacted with BN TBM but not LEW TBM by immunofluorescence, was capable of passively transferring TIN as early as 24 hr after administration of volumes as low as 3 ml i.v. to normal BN recipients, producing focal lesions histologically and immunopathologically similar but less extensive than those studied previously in this strain after active immunization with heterologous RBM. In contrast, a total of 45 ml of serum (in multiple doses) from BN rats immunized with bovine RBM and adjuvants produced only one small lesion of TIN in a recipient BN rat. This difference in serum transferability of anti-TBM-associated TIN appears to relate to quantitative differences in anti-particulate and soluble (collagenase-extracted) BN RBM antigen reactivity measured by radioimmunoassay. Paired-label quantitative studies of passively transferred LEW anti-BN RBM IgG demonstrated a slow accumulation of renal-bound antibody over 6 days, and corresponded with kidney elution and immunofluorescence studies after transfer of immune LEW sera to normal BN rats. Approximately 167 micrograms of kidney-fixing antibody per gram of kidney were calculated to be required for the development of the earliest cellular infiltration. C3 depletion with cobra venom factor greatly diminished the development of destructive TIN lesions associated with multinucleate giant cells after passive transfer of LEW anti-BN RBM antibody to BN rats. This study, using immune LEW sera containing high levels of anti-BN RBM antibody, has defined and quantitated a role for anti-TBM antibody and complement in the initiation of TIN in BN rats.     

Precocious cessation of intestinal macromolecular transmission and sucrase development induced by insulin in adrenalectomized suckling rat.

1. The role of insulin in the regulatory mechanisms governing cessation of intestinal absorption of macromolecules and sucrase development was studied in the adrenalectomized suckling rat (ADX rat). 2. Intestinal absorption of bovine immunoglobulin (IgG) infused orally was dose-dependently suppressed to 35-75% in ADX rats repeatedly injected subcutaneously with insulin. 3. When insulin was administered orally, the IgG absorption was also suppressed. 4. Intestinal sucrase activity was also induced precociously by insulin administered subcutaneously and orally. 5. These results suggest that insulin plays a role in the maturation of suppression mechanisms for macromolecular transmission and sucrase development in the small intestine of the suckling rat.     

Intestinal absorbability of wheat allergens, subunits of a wheat alpha-amylase inhibitor, expressed by bacteria.

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric alpha-amylase inhibitor as well as major allergens to baker's asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying the penetration characteristics of the CM proteins through intestinal mucosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.