Nonspecific resistance in germ-free and E. coli monocontaminated miniature piglets]

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Difference in antibody production to hererologus erythrocytes in conventional, specific-pathogen-free (SPF), germfree and antigen-free mice.

The expression of antibody-producing capacities against hamster erythrocytes (HRBC), known to be weakly immunogenic in mice, was compared among conventional, SPF, germfree and antigen-free mice. ICR strain germfree and antigen-free mice showed antibody production to HRBC comparable to that in conventional or specific-pathogen-free (SPF) mice. In the NC strain, some of the conventional mice produced low titers of antibody after a single injection of HRBC, but none of the germfree mice showed such a transient antibody production. In the ICR-KIG strain, which was selected from the colony-bred ICR strain, antibodies with high titers were produced after a single injection of HRBC under both conventional and germfree conditions. The onset of conversion from 2-mercaptoethanol (2-ME) sensitive to 2-ME resistant antibody after a single injection of HRBC was not delayed in the germfree mice when compared with the conventional or SPF mice. Antibody production to sheep erythrocytes (SRBC), known to be highly immunogenic in mice, was not influenced by exogeneous stimulation from microorganisms or diet. No differences in antibody production to SRBC were detected irrespective of the maintenance conditions of the mice. Stimulation with microorganisms or diet may not be required as essential elements for the maturation of antibody-producing capacities, but such a stimulation appears to modify the antibody response. The modifying effect was more prominent in the antibody response against weakly immunogenic antigens than against highly immunogenic ones.     

Experimental protective action of kanamycin, ampicillin and their combination with methyluracil and pyrogenal]

Efficacy of kanamycin, ampicillin and their combinations with methyluracyl and pyrogenal in experimental Coli infections was studied. The antibiotics were administered an hour after the infection. Methyluracyl and pyrogenal were used according to 2 schemes. Scheme No. I: the drug is used daily for 7 days in increasing doses, the last dose is administered 24 hours before the infection. Scheme No. 2: the drug is used once at the moment of the infection. The methyluracyl doses were: 0.5, 1.0, 2.5 mg and 5 mg and 5 mg per a mouse during the following 4 days. The pyrrogenal doses were: 5, 10, 15, 25, 30 and 35 minimum pyrogenic doses. 5 mg of methyluracyl and 35 minimum pyrogenic doses of pyrogenal were used according to scheme No. 2. The most pronounced increase in the efficacy of kanamycin, ampicillin and their combination was observed in the animals treated simultaneously with methyluracyl and pyrogenal according to scheme No. 1. The efficacy of kanamycin and ampicillin increased 3 and 2.68 times respectively. ED50 of kanamycin and ampicillin used in combination in the animals treated with methyluracyl and pyrogenal was lowered 4 and 2.9 times respectively as compared to that in the animal groups treated only with the antibiotic combination and 21 and 15.2 times respectively when the antibiotics were used alone. Sanation of the animal organs was also rather successful. A single administration of methyluracyl and pyrogenal simultaneously with the infection (scheme No. 2) had a lower effect on the efficacy.     

Stimulating effect ofStreptococcus faecalis on phagocytosis in gnotobiotic piglets

The concentration of active phagocytes in peripheral blood remained in germfree pigs up to the age of one year approximately at the same level as found at the age of 7 d and did not exceed 0.3×10⁹/L of blood, whereas a steady increase was established in conventional pigs. Monoassociation of gnotobiotic piglets withStreptococcus faecalis increased during 24 h the concentration of circulating granulocytes and the concentration of active phagocytes. An even more pronounced effect was obtained when formolizedS. faecalis cells were applied intraperitoneally to germfree piglets. This treatment elevated the phagocytosis index also in conventional piglets, as well as in germfree piglets previously given cyclophosphamide.Escherichia coli O 83 or a mixture of anaerobic bacteria did not cause any serious changes in the activity of phagocytosis in gnotobiotic piglets.S. faecalis seems to be a natural factor stimulating both the release of granulocytes from their depots as well as their phagocytic activity.     

The size pH, and redox potential of the cecum in mice associated with various microbial floras.

Cecal size and in situ redox potential and pH of cecal contents were determined in conventionally reared mice and mice reared under a variety of gnotobiotic conditions: germfree, monoassociated with a cecal Clostridium sp., hexaflora-associated and thermoduric polyflora-associated. The mean Eh was approximately +200 mV in germfree and -200 mV in conventional mice. The Eh was close to zero in the monoassociated mice, thus occupying a position intermediate between the germfree and conventional mice. The potentials observed in the hexaflora and the thermoduric flora groups were indistinguishable from those of conventional animals. The degree of normalization was more advanced with respect to the redox potential than to the cecal size in the various gnotobiotic groups. In the thermoduric polyflora-associated group, normalization was observed in both cecal size and redox potential. This demonstrates that normalization can be accomplished with a relatively simplified microflora, at least with regard to the parameters studied.     

Obtaining and rearing germ-free miniature piglets for biomedical research

The method of obtaining and rearing germfree miniature piglets for medical-biological research is described. The gnotobiotes were reared in rigid organic glass isolation cells up to 3 months. Milk diets supplemented with minerals and vitamins were used for nutrition. The gnotobiotes gained weight well during the whole observation period.     

Colony stimulating activity of serum from germfree normal and leukemic mice

Serum from germfree Swiss/HaM mice exhibited a reduced capacity to stimulate granulocytic and mononuclear cell colony formation by DBA/1 bone marrow cells in vitro when compared with serum from conventional Swiss/HaM mice. Sera from germfree preleukemic and leukemic AKR mice exhibited strong colony stimulating activity, indicating that the increased colony stimulating activity previously observed in the serum of conventional leukemic mice is not the consequence of bacterial or fungal infections supervening in leukemic animals with deficient immune responses.     

Gnotobiotic techniques

A brief review is given of the development of equipment used for rearing germfree animals in various laboratories and of our exparience with designing germfree isolators and with the technology of rearing germfree piglets is presented. The philosophy of isolation as applied to the animal as a biological unit, the elimination of contamination and the theoretical prerequisites of isolation were considered by Reyniers (1943, 1959) and Trexler (1960).     

Effect of cytostatic drugs on fever development following administration of bacterial pyrogen]

A study was made of the development of pyretic reaction to the administration of a bacterial lipopolysaccharide (pyrogenal) after preliminary treatment of rabbits with actinomycin D and cortisone. Such treatment failed to change the reactivity of thermoregulating centres to the endogenous pyrogen. Intravenous injection of bacterial pyrogen was followed by marked shortening of pyretic reaction; the reaction was markedly inhibited in response to its intracysternal administration. An important role played by polymorphonuclear leukocytes in the formation of endogenous pyrogens in the mechanism of pyrexia induced by bacterial pyrogens was shown in this work.     

Course of the post-irradiation syndrome after irradiation with lethal doses in newborn conventional,germ-free andEscherichia coli-monoassociated piglets

The influence of whole-body irradiation with lethal doses of ionizing radiation (⁶⁰Co) was studied in conventional, germ-free andEscherichia coli-monoassociated newborn piglets. The dose 1,200 R produced an acute intestinal death (i.e. within 3–4 days) in conventional animals, whereas survival was three times as long in their germ-free counterparts. Artificial colonization of the intestinal tract of germ-free piglets with non-pathogenic strain ofEscherichia coli, prior to irradiation with the same dose, produced the conventionalization of these animals and reduction in the survival time almost to the level of conventional animals. In conventional animals, profound focal regressive changes of the epithelium accompanying the denudation of intestinal villi were found already on the 2nd–3rd day after irradiation with 1,200 R. On the other hand, the intestinal epithelium of germ-free piglets, irradiated with 1,200 R, was found to be intact on the 7th–9th day of post-irradiation, and the first signs of damage started to occur around the 9–10th days. The morphological characteristics of the intestinal mucous membrane ofEscherichia coli-monoassociated piglets were comparable to those of conventional, irradiated piglets. The role of the presence of the microbial factor for the turnover and radiosensitivity-resistance of enterocytes, and for the survival-death rate of animals irradiated with doses producing the post-irradiation gastro-intestinal syndrome, is discussed.     

Piglets as a model for studying the activity of staphylococcal shock syndrome toxin (TSST-1)

In this study the influence of toxic shock syndrome toxin produced by Staphylococcus aureus on the organism of piglets of Minnesota race is presented. Animals were tested in two groups: conventional and gnotobiotic and they were given the toxin intradermally in two doses 10 and 100 micrograms/kg of body weight. It was shown that piglets are sensitive to the toxin which induced rise of body temperature, various clinical symptoms and in functional changes of several organs as shown by analytical studies. Gnotobiotic animals showed lower susceptibility to the toxin as compared to conventional piglets what suggest a participation of endotoxin in pathologic process.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

Influence of Indigenous Microbiota on Amount of Protein and Activities of Alkaline Phosphatase and Disaccharidases in Extracts of Intestinal Mucosa in Mice

The protein content and the activities of alkaline phosphatase, maltase, and sucrase were measured at 0800, 1000, 1200, 1400, and 1600 in saline extracts of the proximal small bowels of germfree and of ex-germfree mice colonized with an indigenous microbiota. In extracts prepared from germfree mice, the total activities of all of the enzymes were relatively constant throughout the sampling period. Likewise, the total activity of alkaline phosphatase in extracts prepared from associated mice varied little as a function of time. By contrast, the total activities of maltase and sucrase in the extracts from these latter animals varied significantly from sample to sample. The total activity levels in extracts from germfree mice were approximately twofold greater than the levels in extracts from associated mice. The specific activities of alkaline phosphatase and sucrase did not vary from sample to sample in extracts prepared from either type of mouse. In contrast, the specific activity of maltase in extracts prepared from both germfree and associated mice differed significantly from sample to sample. The specific activities of all three enzymes were greater in extracts from germfree animals than in those from associated animals. The protein content of extracts prepared from germfree mice also was greater than that of extracts prepared from associated animals at every sampling time. The amount of protein extractable from the mucosa of the small bowels of the former animals varied significantly at different sampling times during the day, whereas the amount of protein extractable from the tracts of associated animals remained relatively constant throughout the day. The indigenous microbiota apparently stabilizes in some way the amount of protein extractable from the mucosa of the mouse small bowel.     

The required role of endogenously produced lipoxin A4 and annexin-1 for the production of IL-10 and inflammatory hyporesponsiveness in mice

The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-alpha production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-alpha production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.     

Selective decontamination,induced colonization resistance and connected immunological changes in piglets

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.     

Modifications of the local immune response to Vibrio cholerate attributed to the intestinal microbial flora of the mouse.

Oral immunisation studies in germfree, specific pathogen-free (SPF) and conventionalised mice illustrated that the autochthonous gut flora can have a suppressive effect on the induction of a local intestinal immune response to Vibrio cholerae. Temporary colonisation of the small bowel by viable vibrios occurred only in the germfree animal. The lack of colonisation in SPF and conventionalised mice was presumably a cause of their lower coproantibody responses. Prevention of colonisation was probably due to bacterial antagonism rather than to cross-reaction antibodies. This conclusion was reinforced by studies involving oral immunisation of SPF mice maintained on streptomycin, and of conventionalised ex germfree mice. In addition to the increased protective coporantibody response of animals with reduced gut flora, there were increased levels of non-complement-fixing protective antibodies in their serum, which were probably derived from the guy lamina propria.     

Supressor activity of spleen cells during medically induced immunologic tolerance]

SRBC tolerance was induced in mice (CBA X C57BL/6) F1 by single intraperitoneal injection of 6 X 10(9) SRBC and of cyclophosphamide (100-200 mg/kg) in 44-46 hours. Spleen cells of tolerant mice obtained at various periods after the tolerance induction (in 12-26 days) failed to decrease their immune response to SRBC after administration to intact syngeneic recipients. Contrary to intact mice, tolerant animals were incapable of producing suppressor cells after a single SRBC immunization. Only when 3 additional injections of high SRBC doses (6 X 10(9)) were given to tolerant mice the spleen cells in them acquired the capacity to inhibit the immune response after administration to normal mice. It is supposed that the absence of suppressor cells in induction of the immunological tolerance by means of cyclophosphane was caused by the processes of clone elimination. Suppressor cells can originate in tolerant animals under the effect of intensive antigenic stimulation, this leading to enhancement of the tolerance state as a result of additional SRBC injections.     

Studies on the mitogen responses of germfree allogeneic chimeras. II. Maturation of two cell types and partial restoration of responsiveness of the short-term chimeras.

Germfree allogeneic bone marrow chimeras (ABMC) were produced by the i.v. injection of approximately 10(7) bone marrow cells from germfree DBA/2 mice into lethally irradiated germfree C3H mice. In the germfree state, the short-term ABMC showed no histologic signs of graft-vs-host reactions (GVHR), yet splenic lymphocytes were unable to respond to PHA, Con A, or SRBC. Attempts to remove responsiveness by the implantation of a DBA/2 thymus under the host kidney capsule also resulted in failure. However, when the donor thymus was enclosed in a cell-impermeable chamber to eliminate a GVH reaction, responsiveness to Con A was restored. The PHA and SRBC responses were unaffected by this treatment. Daily injections of thymosin caused both an increased Con A response and increased numbers of PFC, although the PHA response was again unaffected. Thus, soluble substances from thymic tissue can be used to overcome partially the histocompatibility barrier present in the ABMC that affects at least two different functional cell populations.     

Barrier-fixing function in germ-free animals]

The state of barrier-fixative function was studied in germfree and conventional guinea pigs and rats. Under conditions of conventional animals contamination with E. coli 055 (in doses of 500 million and 10 milliard microbial bodies for subcutaneous and oral inoculation, respectively) only an early transitory bacteremia developed at the early postinfection periods. As to bacteriemia in gnotobiotes, it increased progressively leading to the animal death in the course of 2 to 3 days. A decreased fixative and bactericidal capacity of the regional lymphatic apparatus and deep structures of the mononuclear-phagocytic system was revealed in germfree animals. An experimental confirmation of the participance of antibodies in the manifestation of the barrier-fixative function to E. coli was obtained. These studies demonstrated an important role of the microbial factor in the formation of the macroorganism barrier-fixative function.