Influence of microbial bile salt desulfation upon the fecal excretion of bile salts in gnotobiotic rats

The fecal excretion of intraperitoneally injected 24-14C-labeled taurocholate (TCA), taurolithocholate (TLCA) and the respective 3-sulfate esters (TCA-3-S; TLCA-3-S), were compared in germfree (GF) rats, conventional (CV) rats, and in gnotobiotic rats associated with Clostridium Cl-8 or this same strain Cl-8 plus the bile desulfating Clostridium S1, respectively. TCA and TLCA were about two times more rapidly excreted by CV animals than by GF animals; the time required for 50% excretion of total label injected (t 1/2) was 6.6 days vs 14.9 for TCA, and 4.4 vs 8.9 for TLCA. In GF and in CV animals, TCA-3-S and TLCA-3-S were excreted more rapidly than their nonsulfated analogues; the t 1/2 values of TCA-3-S and TCA were 2.7 days vs 14.9 in GF rats, and 3.1 vs 6.6 days in CV animals. The t 1/2 values of TLCA-3-S and TLCA were 2.7 days vs 8.9 in GF rats, and 1.5 vs 4.4 days in CV rats. In gnotobiotic rats associated with Clostridium strains S1 + Cl-8, fecal bile salts were nearly 100% deconjugated and desulfated and the 50% excretion times of TCA-3-S and TLCA-3-S approximated to those of TCA and TLCA in GF animals. T 1/2 of TCA-3-S in gnotobiotic S1 + Cl-8 animals was 12.2 days vs 14.9 for TCA in GF animals. In gnotobiotic S1 + Cl-8 animals the t 1/2 of TLCA and TLCA-3-S was 12.5 and 11.0 days, respectively. These results illustrate clearly the important effect the intestinal microflora has upon the metabolic half-life of bile salts. Moreover, they demonstrate that desulfation of bile salts by the intestinal microflora takes place in intestinal segments from where a certain degree of reabsorption is still possible, and thus point to the fact that microbial desulfation is an important variable in the overall elimination of bile salts.     

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.     

The clinical response of gnotobiotic calves, pigs and lambs to inoculation with human, calf, pig and foal rotavirus isolates

The infectivity, pathogenicity and immunogenicity of 5 human, 6 calf, 2 pig and 2 foal rotavirus isolates were studied in gnotobiotic calves, piglets and lambs. Three of the human isolated produced subclinical infection in newborn gnotobiotic piglets and the piglets developed neutralising antirotavirus antibody. When challenged with pig rotavirus 2 weeks later, the piglets did not develop diarrhoea, but rotavirus was detected in the faeces. In contrast, piglets inoculated with the other 2 human isolates failed to show evidence of infection and there was no specific antibody detected. These piglets developed diarrhoea when challenged 2 weeks later with pig rotavirus. The 4 human isolated failed to infect gnotobiotic calves an lambs.     

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.     

The opsonic activity of complement to rough strains ofEscherichia coli in vivo in newborn precolostral pigs

The participation of complement of newborn precolostral piglets (in the absence of antibody) in the opsonization of roughEscherichia coli was investigated using blood clearance test. It was found that the complement inhibitor-yeast mannan inhibits thein vivo blood clearance when injected i. venously into newborn precolostral piglets. This inhibition takes place at the serum level and not by saturation of RES cells by mannan: sera from mannan-treated piglets are not bactericidal in anin vitro bactericidal assay against roughEscherichia coli; secondly, the presence of antibody removes the blockade of phagocytosis by mannan indicating that the RES cells are functionally capable to perform phagocytosis. On the basis of our findings we postulate that in newborn precolostral piglets in which antibody is not present in their sera, complement alone (or with cooperation with other unknown serum component) acts as an opsonin on roughEscherichia coli and is responsible for the blood clearance rate of these bacteria.     

Critical role of prostaglandin E2 overproduction in impaired pulmonary host response following bone marrow transplantation

The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.     

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.     

Effect of controlled antigenic stimulation on lymphocyte subsets in pigs and pig fetuses

The effect of controlled antigenic stimulation in immunologically virgin organisms,i.e. pig fetuses treated with NDCM (Nocardia delipidated cell mitogen) and germ-free (GF) piglets associated with a non-pathogenicE. coli O86, on peripheral blood lymphocyte subsets defined by the expression of CD5 and CD8 was studied by double color flow cytometry. Stimulation of both fetuses and GF piglets increased the frequency of CD8^low+ lymphocytes. A prominent subset of CD5⁻ CD8^low+ NK cells was present in GF andE. coli associated piglets and their frequency was slightly higher inE. coli associated animals. The most pronounced difference between stimulated and non-stimulated animals was in a relative proportion of an ill-defined lymphocyte subset with an unusual CD5^low+ CD8^low+ expression. Both NDCM injection into fetal blood circulation and association of GF piglets withE. coli resulted in a marked increase of frequency of CD5^low+ CD8^low+ lymphocytes in peripheral blood.     

Some properties of piglet,calf, pig and bovine conglutinin

In sera of newborn piglets which were prevented from sucking maternal colostrum a low titre of conglutinin activity was demonstrated. For comparison of properties of this piglet conglutinin sera derived from adult pigs, cows and calves were used. Conglutinin from precolostral piglet serum behaves in different way as compared with immunoconglutinin from adult pig following reduction with 2-mercaptoethanol and in reaction with EDTA and thus resembled bovine natural conglutinin. In density gradient ultracentrifugation and inhibition reaction with zymosan andn-acetyl-d-glucosamine reacts piglet conglutinin as pig immunoconglutinin. Conglutinin present in precolostral calf serum behaves like a typical natural conglutinin of adult cattle.     

Impaired alveolar macrophage function in smoke inhalation injury

The high incidence of both bacterial pneumonia and the adult respiratory distress syndrome (ARDS) associated with smoke inhalation injury (SII) may result, at least in part, from smoke-induced injury to the alveolar macrophage (AM). Specifically, we hypothesized that AM antimicrobial function, ability to phagocytose apoptotic PMNs, and capacity to prevent apoptosis in PMNs are impaired by smoke. To test these hypotheses, AMs were harvested by bronchoalveolar lavage from sheep before and after the animal was exposed to cotton smoke. The two populations of AMs were incubated with Pseudomonas aeruginosa (PSA) in vitro. Normal AMs (NAMs) phagocytosed a mean of 99 ± 11% of the PSA placed in their wells, whereas smoke-exposed AMs (SAMs) ingested only 60 ± 8%. NAMs killed 80 ± 8% of PSA ingested, whereas SAMs killed only 56 ± 16% (P < 0.05). When sheep PMNs, allowed to undergo apoptosis, were incubated with the two AM populations, 66 ± 3% of the NAMs and 40 ± 6% of the SAMs demonstrated phagocytosis of these apoptotic PMNs (P < 0.05). Fresh sheep PMNs were incubated in unconditioned media, NAM and SAM-conditioned media, and followed over 48 hr for the development of apoptosis and maintenance of viability. The NAM-conditioned media markedly prevented apoptosis and augmented PMN survival relative to the unconditioned and SAM-conditioned media (P < 0.05). The poor antimicrobial function known to be characteristic of apoptotic PMNs, together with the directly impaired antimicrobial function of AMs, may contribute to the infectious complications of Sll. If the PMNs recruited to the lung in Sll are not properly supported by the AMs following smoke injury, large numbers may undergo apoptosis. If not properly disposed of by these SAMs, the apoptotic PMNs could eventually lyse, releasing tissue toxins, resulting in escalation of lung injury and leading to ARDS. © 1995 Wiley-Liss, Inc.     

Neutrophils activate alveolar macrophages by producing caspase-6-mediated cleavage of IL-1 receptor-associated kinase-M

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R-associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-α fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α. CASP-6(-/-) mice subjected to cecal ligation and puncture have impaired TNF-α production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6-mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces.     

Leukocytic cell sources of airway tissue kallikrein

Lung tissue kallikrein (TK) is a serine proteinase that putatively plays a role in the pathophysiology of asthma by generating kallidin and bradykinin, mediators that contribute to airway hyperresponsiveness. In previous studies we observed biphasic increases in TK activity in bronchoalveolar lavage fluid following airway allergen challenge in allergic sheep. Although glandular TK is likely a major source of the initial increase in TK, the sources of the late increases in TK that are associated with the development of airway hyperresponsiveness may be dependent on activated resident and recruited inflammatory cells including alveolar macrophages (AMs) and neutrophils (PMNs). These cells increase concomitantly with the late increases in TK activity. To test this hypothesis, we obtained AMs from bronchoalveolar lavage fluid and PMNs and monocytes (precursors of AMs) from sheep blood and determined whether these cells contained TK and whether these same cells could release TK upon activation. Using confocal microscopy, immunocytochemical techniques, and enzyme activity assays, we found that all three cell types contained and secreted TK. All three cell types demonstrated basal release of TK, which could be increased after stimulation with zymosan. In addition, PMNs also released TK in the presence of phorbol ester, suggesting multiple secretory pathways in these cells. Furthermore, we showed that human monocytes also contain and secrete TK. We conclude that in the airways, monocytes, PMNs, and AMs may contribute to increased TK activity. Knowing the sources of TK in the airways could be important in understanding the mechanisms of inflammation that contribute to the pathophysiology of asthma and may help in the development of new therapies to control the disease.     

The activities of elastase and other lysosomal enzymes in professional phagocytes and mechanism of their release

Differences in specific activities of elastase, myeloperoxidase, and lysozyme were found between polymorphonuclear leukocytes (PMNs) and macrophages of various species, between PMNs derived from different anatomic sites and between macrophages isolated in different ways. The percentage of extracellular release of lysosomal enzymes during immunological phagocytosis depends on the receptors in the phagocyte membrane which were stimulated by binding of a ligand. These differences may reflect various functional activities of separate populations of phagocytes.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Early antibodies to human serum albumin formed in germfree piglets

Immunization of germfree piglets with HSA at the time of birth results in formation of antibodies of IgM, IgG and IgA classes in spite of the fact that prior to immunization only IgG and IgA immunoglobulins are detectable in their sera. The possible significance of this finding is discussed. Early IgG antibodies formed in these piglets differ from late and adult pig antibodies by the presence of lower molecular weight constituents and their molecular heterogeneity corresponds to that of IgG of nonimmunized newborn precolostral piglets suggesting that small amounts of immunoglobulins formed in piglets during the intrauterine life are of antibody nature. Dedicated to Prof. F. Patočka on the occasion of his 70th birthday The work was supported by a WHO grant.     

Protective effects of nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ-free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ-free piglets. Three-week-old germ-free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole-body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non-irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush-border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM-pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.     

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.     

The gut microbiota modulates host amino acid and glutathione metabolism in mice

The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV‐R) and germ‐free (GF) mice using gene expression data and tissue‐specific genome‐scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue‐specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV‐R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N‐acetylated AAs in the hepatic portal vein of CONV‐R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV‐R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice.     

The oral gold compound auranofin triggers arachidonate release and cyclooxygenase metabolism in the alveolar macrophage

We examined the effect of in vitro incubation with the oral gold compound auranofin (AF) on arachidonic acid (AA) release and metabolism by rat alveolar macrophages (AMs). AF stimulated dose- and time-dependent release of 14C-AA from prelabeled AMs, which reached 4.7 +/- 0.3% (mean +/- SEM) of incorporated radioactivity at 10 micrograms/ml for 90 min, as compared to 0.5 +/- 0.1% release following control incubation for 90 min (p less than 0.001). Similar dose- and time-dependent synthesis of thromboxane (Tx) A2 (measured as TxB2) and prostaglandin (PG) E2 was demonstrated by radioimmunoassay of medium from unlabeled cultures, reaching 18-fold and 9-fold, respectively, of the control values at 10 micrograms/ml AF for 90 min (p less than 0.001 for both). AF-induced TxB2 and PGE2 synthesis was inhibited by indomethacin as well as by pretreatment with methylprednisolone. No increase in the synthesis of immunoreactive leukotrienes (LT) B4 or C4 was noted at any dose or time of AF. High performance liquid chromatographic separation of 14C-eicosanoids synthesized by prelabeled AMs confirmed that AF induced the release of free AA and its metabolism to cyclooxygenase, but not 5-lipoxygenase, metabolites. The ability of AF to trigger macrophage AA metabolism may be relevant to the exacerbation of certain inflammatory processes which sometimes accompany gold therapy.