Translocation ofLactobacillus murinus from the gastrointestinal tract

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other extraintestinal sites. The translocation rate of a newly described species of indigenous bacteria,Lactobacillus murinus, was compared with the translocation rates of indigenousLactobacillus acidophilus and nonindigenousSalmonella enteritidis. Groups of germfree or antibiotic-decontaminated, specific pathogen-free mice were monoassociated with each of these bacterial strains and tested at various intervals for translocation to the mesenteric lymph nodes. The translocation rates of the various bacteria expressed in decreasing order as the numbers of translocating bacteria per gram mesenteric lymph node wereS. enteritidis, L. murinus, andL. acidophilus. The degree of histologic damage to the gastrointestinal mucosa after monoassociation with these strains followed the same pattern. Thus,L. murinus translocates from the GI tract at a surprisingly high rate for an indigenous bacterial strain, and its translocation appears to be associated with mucosal alterations.     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans.

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette-Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell-mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium-specific IFN-gamma-secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid-encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyer's patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium-specific IFN-gamma production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid-encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.     

Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation

Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.     

In vitro tapeworm extract-induced proliferative responses of gut-associated lymphoid cells from Hymenolepis diminuta infected mice

The development of lymphoid cells reactive to tapeworm-associated antigens during the course of Hymenolepis diminuta rejection from mice was studied using an in vitro tapeworm extract (TWE)-induced cell proliferation culture system. Mice infected with three cysticercoids on day 0 developed three adult worms by day 7 but worms were rejected by day 21 post-infection. Concomitant with worm rejection was the development of TWE-sensitized lymphoid cells which responded by proliferation when stimulated in vitro with TWE. Sensitized cells were detected in gut-associated mesenteric lymph nodes but were not detected in spleen, axillary lymph nodes, or Peyer's patches of infected mice, or in lymphoid organs of non-infected mice. These studies suggest that rejection of H. diminuta from mice is associated with the activities of gut-associated, tapeworm antigen-sensitized immune cells localized in the mesenteric lymph nodes.     

Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes

Three hybridoma clones were isolated after hybridization of a mouse myeloma line with splenocytes from rats immunized with Forssman glycosphingolipid (Fo). Two of these clones produced Fo-specific monoclonal antibodies (MAB) of the IgM class, one MAB of the IgG2c class. In complement-dependent depletion experiments and immunofluorescence studies on the nature of Fo-positive leukocytes in CBA/J mice the following results were obtained: whereas blood monocytes, polymorphonuclear leukocytes, and lymphocytes were Fo negative, 5 to 10% of suspended spleen cells were positive. The majority of these were macrophage-like, glass- and nylon-adherent, nonspecific esterase-positive phagocytizing cells carrying Ia and globoside markers. These cells participated as accessory cells in the mixed lymphocyte culture reaction. In cell suspensions from axillary and inguinal lymph nodes, 2% were Fo positive. They were enriched up to 70% in the glass-adherent, esterase-positive population from this source. In contrast, no Fo-positive cells were detected in mesenteric lymph nodes, and less than 0.1% of the resident peritoneal macrophages bore this marker. The percentage of Fo-positive cells increased to 1% in thioglycollate-elicited peritoneal cells. Immunostaining of cryosections of lung and liver tissue showed alveolar macrophages and Kupffer cells, respectively, to be Fo negative.     

Modulation of the rats' immune status by monoassociation with anaerobic bacteria

The capacity of a pure culture of anaerobic intestinal bacteria to influence the host's cellular and humoral immune systems was investigated with germfree, monoassociated, and conventionally reared rats. Monoassociation of germfree rats with Bacteroides fragilis stimulated the production of serum gamma globulin, agglutinating antibodies, and an apparent IgG (immunoelectrophoresis) band. A comparison of the in vitro blastogenic potential of lymphocytes (spleen cells and mesenteric lymph node cells) from germfree, monoassociated, and conventionally reared rats indicated the following: (1) the microbial flora had no obvious effect on the capacity of nonstimulated lymphocytes to incorporate [3H]thymidine; (2) spleen cells from conventionally reared rats responded to phytohemagglutinin, concanavalin A, or pokeweed mitogen better than splenocytes from germfree rats; (3) colonization of germfree rats with Fusobacterium necrophorum increased the responsiveness of splenocytes to photohemagglutinin and concanavalin A; and (4) monoassociation of germfree rats with B. fragilis, but not with F. necrophorum or propionibacterium acnes, increased splenocyte blastogenesis to homologous (i.e., colonizing) bacterial antigens. This study indicated that some intestinal bacteria can modulate the immune status of the host; the extent and nature of this modulation depended on the particular species of colonizing bacteria.     

Apudocytes in experimental cholera vaccination

The morphofunctional state of apudocytes in the gastrointestinal tract and immunocompetent organs (spleen, mesenteric lymph nodes) of mice immunized with chemical bivalent cholera vaccine was studied. The study revealed that the APUD system of the intestine and the argyrophil elements of the immunocompetent organs of white mice gave a response to the oral administration of commercial cholera vaccine. The reaction of the APUD system of the gastrointestinal tract was manifested by a significant increase in the number of apudocytes and their greater synthesizing activity in the immunized animals during the period of maximum immunological transformation of the macroorganism. The immunization of mice with Vibrio cholerae facilitated the maintenance of homeostasis in the macroorganism and prevented appearance of morphological disturbances in its organs and system after subsequent challenge with V. cholerae.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.     

Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection.

The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P < 0.01]). Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L. casei GG population level in the gut dramatically decreased in these animals.     

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.     

Host response to Helicobacter pylori infection before initiation of the adaptive immune response

Helicobacter pylori persistently colonizes the human stomach. In this study, immune responses to H. pylori that occur in the early stages of infection were investigated. Within the first 2 days after orogastric infection of mice with H. pylori, there was a transient infiltration of macrophages and neutrophils into the glandular stomach. By day 10 postinfection, the numbers of macrophages and neutrophils decreased to baseline levels. By 3 weeks postinfection, an adaptive immune response was detected, marked by gastric infiltration of T lymphocytes, macrophages, and neutrophils, as well as increased numbers of H. pylori-specific T cells, macrophages, and dendritic cells in paragastric lymph nodes. Neutrophil-attracting and macrophage-attracting chemokines were expressed at higher levels in the stomachs of H. pylori-infected mice than in the stomachs of uninfected mice. Increased expression of TNFalpha and IFNgamma (Th1-type inflammatory cytokines) and IL-17 (a Th17-type cytokine) was detected in the stomachs of H. pylori-infected mice, but increased expression of IL-4 (a Th2-type cytokine) was not detected. These data indicate that a transient gastric inflammatory response to H. pylori occurs within the first few days after infection, before the priming of T cells and initiation of an adaptive immune response. It is speculated that inappropriate waning of the innate immune response during early stages of infection may be a factor that contributes to H. pylori persistence.     

Quantitative interrelationships between the T- and B-cell populations of mouse lymphoid organs]

The number of thymocytes and splenic T-lymphocytes correlate negatively. After the treatment of mice with cortisol, this negative correlation changes for a positive one. It is proposed that the negative correlation between cortisone-sensitive thymocytes and splenic T cells is mediated by cortisone-resistant thymocytes. The negative correlations were also found between amounts of T-cells in mesenteric lymph nodes and in Peyer's patches, the positive correlations being seen between numbers of splenic and lymph nodes T-lymphocytes. No correlations between B-cell populations in different organs were revealed. Apparently, correlations between lymphocyte populations reflect some aspect of hierachic structure of the lymphoid system.     

Inhibition of Bacterial Translocation from the Gastrointestinal Tract of Mice Injected with Cyclophosphamide

Effects of intraperitoneal injection of cyclophosphamide, an immunosuppressant, on the degree of bacterial translocation and morphological changes of Peyer's patches (PP) in the intestine were investigated with antibiotic-decontaminated SPF mice and germfree mice monoassociated with Escherichia coli C25. It has been reported that treatment with cyclophosphamide induces bacterial translocation. Cyclophosphamide treatment in this study, however, significantly decreased E. coli C25 translocation from the gastrointestinal tract to the mesenteric lymph nodes (MLN), although the numbers of lymphoid cells, especially B cells, in the PP, MLN, and spleen were remarkably reduced. Four injections of cyclophosphamide at a dose of 100 mg/kg inhibited bacterial translocation more than one injection at a dose of 200 mg/kg in SPF mice. Germfree mice, however, treated with one dose of 200 mg/kg showed the same inhibition of bacterial translocation as those given 100 mg/kg four times. In cyclophosphamide-treated mice, lymph follicles in the PP were obviously smaller than those in control mice, M-cells were similar in appearance to absorption epithelial cells except for short microvilli, and immune cells among the M-cells had disappeared. These data suggested that inhibition of bacterial translocation in mice treated with cyclophosphamide may be the result of morphological and physiological changes of epithelial cells in the gastrointestinal tract, especially M-cells, as a point of entry of invading bacteria, independent of the changes in immunological function. Received: 16 November 1995 / Accepted: 12 December 1995     

Development of Neospora caninum cultured with human serum in vitro and in vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti-N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti-N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.     

Metabolic changes induced by w-3 polyunsaturated fatty acid rich-diet (w-3 PUFA) on the thymus, spleen and mesenteric lymph nodes of adult rats

The maximal activity of key enzymes of glycolysis, pentose phosphate pathway, TCA cycle and glutaminolysis were measured in the immune tissues of rats fed w-3 PUFA during 6 weeks. Total lipid peroxidation and glutathione peroxidase activity were also measured. The hexokinase activity was enhanced 4-fold in the spleen and thymus, doubled in the liver and was diminished in mesenteric lymph nodes (35%). Citrate synthase activity was decreased in the spleen and lymph nodes and increased in the thymus. G-6-PDH activity was increased 2-fold in the spleen and mesenteric lymph nodes and by 20% in the thymus whereas it was reduced (66%) in the liver. Glutathione peroxidase activity and total lipid peroxides increased in all tissues of rats fed w-3 PUFA. The results presented here suggest that w-3 PUFA, by causing important metabolic changes in the immune tissues and lipid peroxidation may lead to changes of immune function.     

乳酸杆菌制剂对应用抗生素大鼠体内TLR2 mRNA转录水平及相关因素影响的研究

目的动态观察乳酸杆菌制剂对应用抗生素大鼠肠道菌群结构和TLR2 mRNA转录水平的影响。方法采用细菌培养法定量检测肠道双歧杆菌、乳酸杆菌、肠杆菌和肠球菌;利用反转录聚合酶链反应技术测定大鼠肠黏膜组织、肠系膜淋巴结、肝脏和脾脏细胞TLR2 mRNA转录水平。结果应用抗生素可致肠道菌群失调和TLR2 mRNA转录水平的早期受抑制。乳酸杆菌制剂干预可迅速提高肠道乳酸杆菌数量,及早扶正肠道菌群结构,减轻由于应用抗生素引起的Toll样受体mRNA转录受抑程度。结论乳酸杆菌制剂早期干预可及早扶正肠道菌群结构,减轻TLR2 mRNA转录水平受抑制程度,为临床合理应用抗生素,早期益生菌干预提供理论依据。     

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.