Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice.

One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization.

BACKGROUND: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. METHODS: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. RESULTS: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. CONCLUSION: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Monocolonization with Bacteroides ovatus protects immunodeficient SCID mice from mortality in chronic intestinal inflammation caused by long-lasting dextran sodium sulfate treatment.

This study was aimed to evaluate the role of commensal Gram-negative bacterium Bacteroides ovatus in murine model of chronic intestinal inflammation. The attempt to induce chronic colitis was done in Bacteroides ovatus-monoassociated, germ-free and conventional mice either in immunocompetent (BALB/c) mice or in mice with severe combined immunodeficiency (SCID), using 2.5 % dextran-sodium sulfate (DSS) in drinking water (7 days DSS, 7 days water, 7 days DSS). Conventional mice developed chronic colitis. Some of germ-free BALB/c and the majority of germ-free SCID mice did not survive the long-term treatment with DSS due to massive bleeding into the intestinal lumen. However, monocolonization of germ-free mice of both strains with Bacteroides ovatus prior to long-term treatment with DSS protected mice from bleeding, development of intestinal inflammation and precocious death. We observed that though DSS-treated Bacteroides ovatus-colonized SCID mice showed minor morphological changes in colon tissue, jejunal brush-border enzyme activities such as gamma-glutamyltranspeptidase, lactase and alkaline phosphatase were significantly reduced in comparison with DSS-untreated Bacteroides ovatus-colonized mice. This modulation of the enterocyte gamma-glutamyltranspeptidase localized to the brush border membrane has been described for the first time. This enzyme is known to reflect an imbalance between pro-oxidant and anti-oxidant mechanisms, which could be involved in protective effects of colonization of germ-free mice with Bacteroides ovatus against DSS injury.

Commensal intestinal bacterial strains trigger ankylosing enthesopathy of the ankle in inbred B10.BR (H-2(k)) male mice.

Joint disease ankylosing enthesopathy (ANKENT) naturally occurs in inbred mice with C57Bl/10 genetic background. ANKENT has many parallels to human ankylosing spondylitis (AS) and represents an animal model for AS. Environmental conditions (i.e., microbial load of the organism) are among the risk factors for ANKENT, similar to AS. The role of microflora in the development of ANKENT was investigated. ANKENT was tested in four experimental groups of germ-free mice associated with different numbers of various intestinal microbes and three control groups: germ-free, specific pathogen-free, and conventional (CV) mice. Mice were colonized either with anaerobic bacteria isolated from the intestine of a CV mouse or with bacterial strains obtained from the collection of microorganisms. Microbes were characterized and checked by microbiological cultivation methods and with the use of polymerase chain reaction amplification and rDNA sequence analysis. Joint disease developed in GF mice colonized with a mixture containing Bacteroides spp. and Enterococcus sp., and/or Veillonella sp. and Staphylococcus sp. No ANKENT appeared in males colonized with probiotic bacterium Lactobacillus sp. In control groups ANKENT occurred in SPF and CV animals; the GF animals remained healthy. The results confirmed that the germ-free conditions protect from joint inflammation, and thus microbes are necessary for ANKENT development. In colonized mice the ANKENT was triggered by luminal anaerobic bacteria, which are common components of intestinal microflora.

Effects of monocolonization with Escherichia coli strains O6K13 and Nissle 1917 on the development of experimentally induced acute and chronic intestinal inflammation in germ-free immunocompetent and immunodeficient mice.

Germ-free immunocompetent (BALB/c) and immunodeficient (SCID) mice were colonized either by E. coli O6K13 or by E. coli strain Nissle 1917 and intestinal inflammation was induced by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Controls were germ-free mice which demonstrated only mild inflammatory changes after induction of an acute intestinal inflammation with DSS as compared with conventional mice in which acute colitis of the colon mucosa similar to human ulcerative colitis is elicited. In mice monocolonized with the nonpathogenic E. coli Nissle 1917 the inflammatory disease did not develop (damage grade 0) while animals monocolonized with uropathogenic E. coli O6K13 exhibited inflammatory changes similar to those elicited in conventionally reared mice (damage grade 3). In the chronic inflammation model, immunocompetent BALB/c mice monocolonized with E. coli Nissle 1917 showed no conspicuous inflammatory changes of the colon mucosa whereas those monocolonized with E. coli O6K13 developed colon inflammation associated with marked infiltration of inflammatory cells. In contrast to germ-free immunodeficient SCID mice that died after application of DSS, the colon mucosa of SCID mice monoassociated with E. coli Nissle 1917 exhibited only moderate inflammatory changes which were less pronounced than changes of colon mucosa of SCID mice monoassociated with E. coli O6K13.

Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease.

Ankylosing enthesopathy (ANKENT) is a naturally occurring joint disease in mice with numerous parallels to human ankylosing spondylitis (AS). Similarities between AS and ANKENT include not only affected tissue (joint entheses) but also association of the disease with genetic background, including MHC genes, gender, and age. Young males with the C57Bl/10 background have been described to suffer from ANKENT and, among H-2 congenic strains, high frequency of afflicted joints has been recorded in B10.BR (H-2(k)) males. Interestingly, the incidence of ANKENT is higher in conventional (CV) males that in their specific-pathogen-free (SPF) counterparts. The latter finding suggests that microbes could play a role as an ANKENT-triggering agent. To further examine this hypothesis we have established a germ-free (GF) colony of B10.BR mice and observed ANKENT incidence in both GF males and their conventionalized (ex-GF) male littermates; 20% of ex-GF males developed ANKENT before 1 year of age. In contrast, no joint disease was observed under GF conditions (p < 0.0001). Our results show that live microflora is required in ANKENT pathogenesis.

Effects of dietary essential fatty acid deficiency on the development of the rat thymus and immune system.

The paper describes an effect of essential fatty acid (EFA) deficiency on the development of the rat thymus and pups' immune system. From birth until being weaned (22nd day), the pups were hand-fed artificial milk diets containing a low (EFA-D) or high (EFA-R) proportion of EFA in the lipid fraction. The weight parameters of the body, thymus and spleen, the fatty acid composition of the individual thymus phospholipid subclasses, and mitogen-induced proliferation of thymus and spleen lymphocytes were studied. The results show that the total body weight of the EFA-deficient (EFA-D) fed animals was significantly decreased in comparison with the EFA-rich (EFA-R) and rat milk hand-fed animals. For the EFA-D group of young rats a high level of the (n-9) and (n-7) series fatty acids [mainly oleic 18:1(n-9) and eicosatrienoic 20:3(n-9) acids] was characteristic of the fatty acids in phosphatidylcholine and phosphatidylethanolamine in the thymus, compensating for the reduction of the content of arachidonic acid 20:4(n-6). The biochemical index of the EFA nutritional deficit in the thymus tissue was observable as early as on day 7. The mitogen-induced (Con A) proliferation of the thymus and spleen lymphocytes was decreased both on the 30th and 40th day of life. The results show that the EFA nutritional deficit in the early postnatal period caused damage to the structure of thymus in the young rats, most probably as a result of the change in the composition of the lipid fraction. These changes also affected the development of the immune system of the whole organism.

Differences in enterocyte brush border enzyme activities in ageing rats reared in germ-free and conventional conditions.

The aim of this study was to evaluate the levels of disaccharidase and dipeptidyl peptidase i.v. activities in rat jejunal enterocytes under the influence of long-term germ-free conditions. We found that the brush-border lactase and dipeptidyl peptidase i.v. activities were two to three times higher in 2-month-old germ-free rats in comparison with their conventional counterparts. The highest effect of germ-free condition was observed on lactase activity in 6-month-old and dipeptidyl peptidase i.v. in 2-month-old rats. No difference between germ-free and conventional rats in sucrase and glucoamylase activities was found in 2-month-old rats. The difference develops with increasing age, sucrase activity becoming significantly higher in 6- and 12-month-old rats and glucoamylase in 12-month-old germ-free rats.

Milk-borne hormones: possible tools of communication between mother and suckling.

Early studies suggested endocrine type mother-pup interaction: 131I administered to suckling rats appeared via the urine of the suckling and mother's milk in the circulation of litter mates who were not injected with iodine; levels of thyroxin in rat milk were influenced by the status of the thyroid gland of the lactating rat. Administration of TRH (thyrotropin releasing hormone) to lactating mothers led to an appearance of unaltered hormones in the milk and stomach content of sucklings. TSH (thyroid stimulating hormone) or ACTH (adrenocorticotropic hormone) when given orogastrically to suckling rats increased thyroid hormones and corticosterone serum levels in suckling rats. Functional effects of gastrointestinal administration of insulin, bombesin (mammalian analog of gastrin-releasing peptide) and epidermal growth factor (EGF) are reviewed in detail (32 references).

Humoral and cellular immune responses in gluten-treated suckling or hand-fed rats.

We analyzed the immune response to gliadin in suckling rats and rats hand-fed with an artificial milk formula, an animal model of gluten enteropathy. Animals of both groups were intragastrically given either gliadin or albumin (control animals) or gliadin from birth till day 55. When compared to the controls, spleen lymphocytes from both groups of gliadin-treated rats cultivated in vitro exhibited a significant increase of spontaneous 3H-thymidine incorporation. Moreover, the proliferation of spleen and mesenteric lymph node (MLN) lymhocytes from both groups of gliadin-treated suckling and hand-fed rats was specifically increased by the in vitro gliadin challenge. Spleen B cells from gliadin-treated rats spontaneously produced higher amounts of gliadin-specific antibodies than those from the controls, however, in vitro stimulation by gliadin caused no further increase in antibody production. Apoptotic DNA fragmentation in MLN cells was higher in gliadin-treated rats than in albumin-treated ones, independently of the milk diet during the suckling period.

Development of antibody formation in germ-free and conventionally reared rabbits: the role of intestinal lymphoid tissue in antibody formation to E. coli antigens.

The occurrence of cells which produce 'natural" bactericidal and haemolytic antibodies (the so-called background plaques) was studied by the plaque method in lymphatic tissues of germ-free and conventionally reared rabbits of various ages. In conventional rabbits the cells which produce "natural" bactericidal antibodies against E. coli 086 appear early after birth in organized intestinal lymphatic tissue, their number increases during development, then they appear in mesenteric lymph nodes and spleen and their number decreases due to aging. Natural haemolytic antibodies are produced throughout the life predominantly in the spleen of conventional rabbits. In germ-free rabbits the appearance of "natural" bactericidal or haemolytic antibodies was not demonstrated during the tested period of 16 weeks of life. Also when comparing the immunological capacity after parenteral immunization or after stimulation of cells from various organs cultivated in diffusion chambers, the response in germ-free rabbits was very low or lacking. The differences between the reactivity of germ-free and conventional rabbits were especially marked when studying the specific and non-specific (polyclonal) response after administration of E. coli suspension. The nature and possible causes underlying the differences in immunological reactivity in various species of germ-free animals are discussed.

Essential fatty acid deficiency and bone fragility in rats.

This study demonstrates the effect of essential fatty acid deficiency on the postnatal skeletal development in the rat. Four groups (n = 10) of newborn Wistar rats were fed diets containing high and low proportions of essential fatty acids in the lipid fraction until day 16 after birth. Suckled littermates were used as controls. X-ray and histological studies showed the occurrence of multiple pathological fractures of the long bones in 1-month-old rats fed a diet deprived of essential fatty acids. No effect of high (51,000 IU/100 g diet) and low (5,100 IU/100 g diet) concentrations of vitamin D2 was observed in our experiment. Thus, these data suggest the importance of essential fatty acids for bone pathology in the rat.

Effects of microflora antigens on lymphocyte migration patterns in germfree and conventional rats.

The aim of this work was to help to explain the role of antigen in the migration pattern of lymphoid cells by using the possibility to work with GF and CONV inbred rats. Our findings indicate that: The homing of 51Cr-labelled cells from PP into different organs of GF and CONV rats was comparable. The homing of MLN cells into the gut and salivary glands was three times higher than homing of PLN cells. The homing of MLN cells of CONV rats into the gut of CONV rats is higher in comparison with the homing into the gut of GF rats. The changes in blood flow do not seem to have any influence on the homing activity because MLN cells from GF rats migrate comparably to the gut of GF and CONV animals. Higher localization of MLN cells from CONV rats in the gut of CONV rats (as compared with these cells from GF donors) seems to be a result of antigen activation of these cells. Our results imply that antigens of microflora present in the intestine enhance the homing of MLN cells into the gut provided that these cells were activated by microflora antigens.

Enteropathy induced in young rats by feeding with gliadin--similarity with coeliac disease.

Young rats (10- and 21-day-old) were given gliadin by intragastric application, in the form of a 5% and 0.5% solution. A 5% gliadin (1 ml) was intolerable for young rats, destroying the structure of their jejunal mucosa. A 0.5% gliadin (1 ml) was well tolerated by 21-day-old animals and caused no changes in enterocytes. Repeated administration of 0.5% gliadin on days 0 and 17 caused an increased lymphocyte infiltration in the lamina propria and an increase in the number of cells producing IgA immunoglobulins. Repeated intragastric administration of 0.5% gliadin until 1 month of age, followed by a single administration of 0.5% gliadin at 2 months, caused shortening of the jejunal villi and changes in apical enterocytes. The most pronounced immunological changes were observed in these repeatedly treated animals. Increased numbers of lymphocytes producing IgG in the lamina propria were found and the intestinal contents of these animals exhibited an increased level of IgG and IgA anti-gliadin antibodies when compared to control rats. The results indicate that some morphological and immunological changes observed in young rats fed with gliadin resemble those observed in patients with coeliac disease. The described experimental model is supposed to be suitable for studying the pathogenetic aspects of human coeliac disease.

Autoimmunity: from physiology to pathology. Natural antibodies, mucosal immunity and development of B cell repertoire.

Presence of spontaneously produced immunoglobulins bearing a broad spectrum of "natural" antibody specificities (including autoantibodies) in sera and other body fluids results mainly from inapparent immunization and polyclonal B cell activation by microflora and food antigens occurring mostly on mucosal surfaces. Early postnatal ontogeny in external environment is characterized by rapid growth and functional maturation of secondary lymphatic tissues as a consequence of this "natural" mucosal immunization. Under normal circumstances a state of "oral" tolerance to intestinal antigens is actively established after this period. Studies performed in germ-free, antigen-free and maternal antibody-deprived animals showed that low amounts of natural antibodies (mainly of IgM isotype) are formed without any known cause of stimulation. These "nonstimulated" antibodies, similarly as hybridomas originating from nonimmunized newborns, correspond to the preimmune repertoire of antibodies characterized by poly-specificity, high connectivity and reactivity against self antigens. Together with other innate humoral and cellular factors, they probably represent the first line of anti-infectious resistance. Moreover, due to their connectivity they are supposed to play an important role in B cell repertoire shaping (forming an idiotypic network), through interaction with a broad spectrum of immunological components they act as regulatory molecules, and through their participation in catabolic events they can promote morphogenetic changes during fetal development. Beneficial therapeutic effects of nonspecific gammaglobulin (IVIG) application observed recently in patients with autoimmune diseases suggest that they can influence autoimmune reactivity by a not yet analyzed mechanism. Other functions of natural autoantibodies can be suggested and expected to be found in the near future.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin-producing cells in lymphatic tissues of germfree and conventional rabbits as detected by an immunofluorescence method.

Lymphatic tissues of GF and CV rabbits were observed. No cells producing IgA and IgM antibodies were detected in appendix, sacculus rotundus, ileum terminale and thymus of GF rabbits. IgA cells were found in lymph nodes of GF rabbits.

Prolonged survival of AVN Wistar rats with transplanted Yoshida sarcoma and increase of granular lymphocytes after administration of Bacillus firmus and their crude lipids.

Bacillus firmus is a Gram-positive, aerobic, sporulating, nonpathogenic air contaminant which, according to earlier findings, is a strong polyclonal activator of B lymphocytes. The crude lipids of this microbe induced significant resistance of mice against listerial infection. The administration of bacterin, like that of crude lipids obtained by the extraction of cell suspension with chloroform-methanol to rats, strain AVN Wistar, transplanted later with Yoshida sarcoma, significantly prolonged the survival of the animals in comparison with the control group. At the same time the number of granular lymphocytes was increased. The destruction of tumor cells in the peritoneal exudate of immunostimulated rats was also determined.

Passage of foreign proteins across the intestinal wall of newborn rabbits.

Foreign proteins penetrate across the wall of rabbit jejunum takes place within the first eight hours after birth. Sensitization by foreign protein (cow's milk protein) during early postnatal ontogeny is the cause of death of artificially fed germfree rabbits at the age of 21-23 d.

Lymphatic tissue of the intestinal tract of germfree and conventional rabbits.

The lymphatic tissue of ileum, sacculus rotundus and appendix was poorly developed without germinal centres in germfree rabbits; on the other hand, in conventional rabbits, the lymphatic tissue was abundant with numerous germinal centers. In germfree rabbits, the stroma of villi of the jejunum and ileum possessed low cellularity, in conventional rabbits, the villi were wide with rich cellularity. The basal position of lymphocytes was predominant in the villi of ileum of germfree rabbits, in conventional rabbits a high percentage of lymphocytes was in the apical position.

Experimental colitis does not increase the prevalence of ANKENT, a spontaneous joint disease in mice.

A possible relationship between intestinal inflammation and joint disease development was investigated. Clinical symptoms of colitis--diarrhea and rectal bleeding--were confirmed by findings of inflammatory processes in the colon in dextran sodium sulfate-treated mice and joint ankylosing enthesopathy (ANKENT) developed in 12.8 % mice with chronic colitis and 13.6 % mice in the control group. Consequently no significant difference in ANKENT frequency was found between mice with and without chronic colitis and the occurrence of ANKENT in both groups was typical for conventional conditions. ANKENT cannot be triggered solely a generalized inflammatory process in the gut.