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.     

Routes of E. coli 055 bacterial penetration through the intestinal wall in gnotobiotic and ordinary animals]

Peculiarities of permeability of intestinal barrier in germfree animals for enteropathogenic E. coli 055 were studied. Both germfree and conventional guinea pigs and rats were used. An increase of bacteriemia was revealed in gnotobiotes during the first day of oral E. coli 055 monocontamination; bacteriemia was transient in conventional animals. Under electron microscopy alterations of intercellular contacts and formation of spaces between enterocytes containing numerous microorganisms were found in the intestinal mucous membrane in gnotobiotes. Also more pronounced changes of microvessels of the intestinal mucous membrane were discovered in gnotobiotes. The processes of ingestionan and digestion of Escherichia coli by enterocytes and leukocytes were noted in conventional animals. The revealed derangements of the intestinal barrier in gnotobiotes explain the cause of higher bacteriemia in germfree animals. An important role of the microbial factor in the formation of intestinal barrier is indicated by the data obtained.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

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.     

Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]

The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.     

Resistance of germfree rats to indomethacin-induced intestinal lesions.

Indomethacin given orally to conventional rats produced in three days a syndrome, often fatal, of intestinal lesions characterized by multiple ulcers and peritonitis. Male germfree rats were found to be resistant to this effect of indomethacin, while female germfree rats developed very mild lesions. Germfree rats became sensitive again to such lesions when monocontaminated with E. coli. In such animals, however, the lesions were less severe than in conventional animals, presumably because more than one microorganism is necessary for the full syndrome to develop. These results suggest that microorganisms are necessary for the development of indomethacin-induced intestinal lesions. Secondary bile acids, absent in germfree animals, may also be necessary. The prostaglandin deficiency caused by indomethacin appears to weaken the resistance of the intestinal mucosa to microorganisms and/or their toxins. The latter may then penetrate the mucosa, damage the cells and produce ulcers and perforations. Since several prostaglandins also protect against indomethacin-induced lesions, the hypothesis is advanced that certain prostaglandins may protect the mucosa ("cytoprotection") by preventing the spread of microorganisms and/or their toxin through the intestinal wall.     

Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.     

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).     

Lysosomal enzyme activities in polymorphonuclear leukocytes,macrophages, serum,and spleen of conventional,germ-free,and antigen-free minnesota miniature swine

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of gnotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.     

The dynamics of the basic factors of nonspecific resistance in North Asian tick-borne typhus]

At the acute period of tick-borne typhus an increase in the activities of the main nonspecific resistance factors of the body, and particularly lysozyme, betalysins, complement and properdin, is observed. During convalescence the concentration of lysozyme normalizes and the content of betalysins remains sufficiently high, this indicating that, even in the absence of clinical sings of the disease, the disturbances in homeostasis persist. A decrease in the levels of complement and properdin during convalescence is seemingly linked with their consumption for the formation of immune complexes, eliminated from the body.     

Properdin plays a protective role in polymicrobial septic peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.     

Studies on the phagocytic activity of the reticuloendothelial system (RES) to rough and smoothEscherichia coli in young conventional and germfree guinea pigs

The functional (phagocytic) capacity of the reticuloendothelial system (RES) of young conventional and germfree guinea pigs was studied using thein vivo blood clearance test of living bacteria (rough and smoothEscherichia coli). It was found that as previously shown in newborn germfree piglets, the smooth strain was taken up from the blood stream of germfree guinea pigs very slowly whereas roughEscherichia coli was phagocytosed effectively. The inability of the RES of germfree guinea pigs to phagocytose the smooth strain is not due to a functional incapability of phagocytic cells, but it reflects rather the lack of serum opsonins to this strain. This was demonstrated in experiments in which smooth bacteria, sensitized prior to injection into the blood circulation with specific antiserum, were phagocytosed as effectively as the rough strain. It is assumed that effective phagocytosis of rough strain is due to the presence of non-specific opsonins (e.g. components of the complement system). In young conventional guinea pigs both strains,i.e. smooth and rough, were taken up from the blood stream very effectively thus indicating that sufficient levels of serum opsonins for both strains were present. This fact could be correlated with the finding that in sera of conventional guinea pigs haemagglutinating antibodies to both strains ofEscherichia coli could be detected, whereas in sera of young germfree guinea pigs, no antibodies to usedEscherichia coli strain were found. The importance of serum opsonins for effective phagocytosis of bacteria by RE cellsin vivo is discussed.     

Stimulating effect of prodigiozan on nonspecific reactivity in typhoid fever, dysentery and staphylococcal infection]

A number of factors of nonspecific reactivity, i.e. complement, lysozyme, properdin, blood serum bactericidal activity, leucocyte phagocytal activity, phagocytal index and completed phagocytosis were studied on 160 guinea pigs with experimental typhoid fever, dysentery and staphylococcal infections. The data of the study showed that with the use of prodigiozan the non-specific reactivity of the host increased even within a short period of time. At the same time the pathological changes in the internal organs in their histochemical investigation decreased.     

Susceptibility of Gnotobiotic Swine to Escherichia coli Isolated from Nonenteric Human Infections

Newborn, germfree piglets were susceptible to Escherichia coli associated with human, nonenteric infections and should provide a useful model in the study of generalized E. coli infections.     

The sensitivity of smooth and rough mutants ofSalmonella typhimurium to bactericidal and bacteriolytic action of serum,lysozyme and to phagocytosis

The sensitivity of smooth and rough mutants ofSalmonella typhimurium belonging to different chemotypes to bactericidal and bacteriolytic action of antibodies, complement and lysozyme was investigated. By the action of specific antibodies and complement the majority of strains was killed and in the presence of sufficient amount of lysozyme even lysed. Sera of newborn piglet which do not contain antibodies however, were able to kill regularly only strains of lower chemotypes (Rc and Rb). Smooth strains were usually resistant to the action of piglet serum whereas strains of Ra chemotype were killed only by sera of some piglets. Using the liver clearance method a significant correlation between the lipopolysaccharide chemotype of the particular strain and the degree of phagocytosis was found.     

Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

State of the nonspecific humoral factors of the body's natural resistance in patients with suppurative and septic infections having various ABO system blood groups]

The results of surveying 140 patients with severe purulent and septic infections of staphylococcal etiology, when compared with the distribution of the blood groups (as classified according to the ABO system) in 180 healthy donors, revealed that generalized purulent infections occurred most frequently in patients with blood groups A (II) and AB (IV), and more seldom in patients with blood groups O (I) and B (III). The average content of lysozyme, complement and normal antibodies to E. coli, as well as the average level of general bactericidal activity in the blood sera of the patients were considerably lower than in the blood sera of healthy donors; at the same time content of lysozyme, complement and normal antibodies in the blood sera of patients having different groups of blood did not reflect the degree of their predisposition or resistance to staphylococcal infections. The general bactericidal activity of the blood serum was found to correlate with the degree of predisposition or resistance to purulent septic infections of staphylococcal etiology to a greater extent than other characteristics.     

A technique for rearing germfree piglets obtained without surgery

A relatively simple procedure is described for obtaining germfree piglets which does not involve hysterectomy or hysterotomy. Newborn pigs were delivered into an isolator and their freedom from microbial contamination was ensured by applying bactericidal solutions externally and a combination of antibiotics in solution per os. 40 piglets so derived have been maintained free from detectable micro-organisms, some for up to 140 days. Equipment is described which allowed the long-term holding of these animals so that nutritional balance studies could be completed.     

Body temperature and the reaction to pyrogenal in germ-free and ordinary animals

Body temperature, as well as pyrexia in response to pyrogenal in germfree and conventional mice and miniature piglets were studied. A decrease of the mean body temperature in the intact germ-free mice and miniature piglets in comparison with conventional animals of the corresponding species was revealed. The absence of marked pyretic response to pyrogenal after intraperitoneal injections of 10 minimal pyrogenic doses to mice and after intramuscular injections of 500 minimal pyrogen doses of pyrogenal to miniature piglets was observed in germfree animals. The data obtained indicated an important role of autoflora in the development of the organism capacity to temperature reaction and pyrexia.     

Serum vibriolytic activity as an index of antibacterial immunity in cholera

As a result of study of the vibriolytic activity of the serum during the immunity formation in the vaccinated animals in comparison with the specific antibodies titres and nonspecific immunity factors (complement and lysozyme) there was revealed a dependence of the reaction on the vaccine dose and the immunization method; there was also found a relationship between the vibriolytic activity and the serological indices in the sera of volunteers. On the basis of study a conclusion was drawn that the vibriolytic activity of the serum could serve as an index of antibacterial immunity in cholera.