Defective macrophage handling of Escherichia coli in Crohn's disease.

BACKGROUND AND AIM: Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. METHODS: Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). RESULTS: Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CONCLUSIONS: CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

Distinct microbial populations exist in the mucosa-associated microbiota of sub-groups of irritable bowel syndrome.

BACKGROUND: There is increasing evidence to support a role for the gastrointestinal microbiota in the etiology of irritable bowel syndrome (IBS). Given the evidence of an inflammatory component to IBS, the mucosa-associated microbiota potentially play a key role in its pathogenesis. The objectives were to compare the mucosa-associated microbiota between patients with diarrhea predominant IBS (IBS-D), constipation predominant IBS (IBS-C) and controls using fluorescent in situ hybridization and to correlate specific bacteria groups with individual IBS symptoms. METHODS: Forty-seven patients with IBS (27 IBS-D and 20 IBS-C) and 26 healthy controls were recruited to the study. Snap-frozen rectal biopsies were taken at colonoscopy and bacterial quantification performed by hybridizing frozen sections with bacterial-group specific oligonucleotide probes. KEY RESULTS: Patients with IBS had significantly greater numbers of total mucosa-associated bacteria per mm of rectal epithelium than controls [median 218 (IQR - 209) vs 128 (121) P = 0.007], and this was chiefly comprised of bacteroides IBS [69 (67) vs 14 (41) P = 0.001] and Eubacterium rectale-Clostridium coccoides [52 (58) vs 25 (35) P = 0.03]. Analysis of IBS sub-groups demonstrated that bifidobacteria were lower in the IBS-D group than in the IBS-C group and controls [24 (32) vs 54 (88) vs 32 (35) P = 0.011]. Finally, amongst patients with IBS, the maximum number of stools per day negatively correlated with the number of mucosa-associated bifidobacteria (P < 0.001) and lactobacilli (P = 0.002). CONCLUSIONS & INFERENCES: The mucosa-associated microbiota in patients with IBS is significantly different from healthy controls with increases in bacteroides and clostridia and a reduction in bifidobacteria in patients with IBS-D.

Molecular characterization of the bacteria adherent to human colorectal mucosa.

AIMS: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. METHODS AND RESULTS: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. CONCLUSIONS: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. SIGNIFICANCE AND IMPACT OF THE STUDY: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.

In vitro exposure effects of cyclosporin A and bis(tri-n-butyltin)oxide on lymphocyte proliferation, cytokine (receptor) mRNA expression, and cell surface marker expression in rat thymocytes and splenocytes.

Rat thymocytes and splenocytes were exposed in vitro to the model compounds Cyclosporin A (CsA), an immunosuppressive drug, and bis(tri-n-butyltin)oxide (TBTO), an immunotoxic environmental contaminant. The lymphocyte transformation test (LTT), cytokine (receptor) mRNA expression (RT-PCR and dot blot hybridisation), and flow cytometry were evaluated as assays for in vitro immunotoxicity, at dose levels that did not show effects on viability, this being the aim of the study. LTT and RT-PCR proved useful assays. Lymphocyte transformation was suppressed by both compounds, while IL-2 mRNA expression was suppressed by CsA but not by TBTO, and both compounds suppressed IL-2R mRNA expression in splenocytes but not in thymocytes. Furthermore, the data obtained suggest that antiproliferative effects may be more relevant than apoptosis induction for TBTO induced thymus atrophy.

Information needs of workers.

In Britain, the law places duties on employers and suppliers to provide information to ensure health and safety of employees, so far as is reasonably practicable, and there are regulations for the appointment of employees' safety representatives which employers are required to consult. A recent survey by HSE has shown that suppliers are the most important source of information on substances. However, the flow of information is often obstructed by barriers between the suppliers and the companies, and within organisations. Safety representatives, where they exist, are often better informed than employers, and in companies with safety representatives accident rates are lower. Information from suppliers can be inappropriate for the end use, and the goal-setting approach which has dominated in recent years may not help the non-expert employer. We welcome HSE's move to more specific control guidance for chemicals.

Role of interleukin-10 in the lung response to silica in mice.

There is evidence that, following exposure to crystalline silica, the release of several proinflammatory cytokines contributes to the induction of unbalanced inflammatory reaction leading to lung fibrosis. We have examined the potential contribution of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the development of silicosis. In a mouse model of inflammatory lung reaction induced by intratracheal instillation of silica (0.5 mg and 5 mg DQ12/mouse), the levels of IL-10 protein (determined by ELISA) both in cells obtained after bronchoalveolar lavage (BAL) and in lung tissue homogenates were significantly increased when compared with controls. After in vitro lipopolysaccharide (LPS) stimulation (1 microg/ml), BAL cells obtained from silica-treated animals produced significantly more IL-10 protein and mRNA than cells obtained from control animals. To examine the role of IL-10 in the lung reaction induced by silica, IL-10-deficient animals were instilled with 5 mg of silica. Twenty-four hours after treatment, the amplitude of the inflammatory response (lactate dehydrogenase [LDH], protein and number of inflammatory cells in BAL) was significantly greater in IL-10-deficient animals than in the wild type. In contrast, the fibrotic response, evaluated by measuring lung hydroxyproline content and by histopathologic analysis 30 days after silica, was significantly less important in IL-10-deficient than in wild-type mice. Together, these data suggest that increased IL-10 synthesis induced by silica can limit the amplitude of the inflammatory reaction, but also contributes to amplify the lung fibrotic response.

Immunotoxicology: extrapolation from animal to man--estimation of the immunotoxicologic risk associated with TBTO exposure.

Bis(tri-n-butyltin)oxide (TBTO) has been shown to be immunotoxic in rodents, resulting in decreased resistance to infections. The no-effect level assessed by estimating effects on host resistance in rats has been found to lie between 0.5 and 5.0 mg TBTO/kg food (0.025 and 0.25 mg/kg body weight). For risk assessment such animal data need to be extrapolated to the human situation. In risk assessment procedures uncertainty factors are used to account for interspecies variation (extrapolation from animal to man) and for variation within the human species. For both factors a value of 10 is often used, based on international guidelines. Hence, exposures below 0.00025 mg/kg body weight should not pose a risk for the human population. In the present study we have taken an alternative approach. We have produced dose-response curves for the effect of TBTO exposure on resistance to Trichinella spiralis. To extrapolate this curve to the human situation, we produced additional dose response data concerning in vitro effects of TBTO exposure on the mitogen responsiveness of both rat lymphoid cells and human blood cells. Using regression analyses of these dose-response data, we calculated a factor that accounts for interspecies variation (IEV) and a factor that accounts for intraspecies variation (IAV) within the human samples. Using these factors, we estimated the dose that decreases resistance in man to an infection. We choose 10% increase of the infectious load as a reference point which in our view is of biological significance. Based on these considerations, we estimated the dose that may affect resistance in adult humans at 0.04 mg/kg body weight. Pre- and postnatal exposure will probably result in effects at lower concentrations, due to the vulnerability of the developing immune system.

Modulation of cytokine mRNA expression in differentiated human monocytic cell lines following exposure to inorganic particles.

In these preliminary studies we have examined the effects of in vitro challenge with inorganic dusts on cytokine gene expression by undifferentiated cells, cells undergoing phorbol 12-myristate 13-acetate (PMA) induced differentiation, and on differentiated cells. We have used three different human promonocytic cell lines, U937, THP1 and HL60 in an undifferentiated state and following differentiation with PMA. We found that cytokine specific mRNA levels could be modulated by inorganic dusts with known toxic effects, whereas those which are classified as biologically inert had no measurable effects on cytokine gene expression. These findings were only observed on fully differentiated cells with no modulation seen in undifferentiated cells or those undergoing differentiation.

Secretory and accessory cell functions of the alveolar macrophage.

We have attempted to address the requirements necessary for alveolar macrophage accessory cell function. We have also examined the in vitro and in vivo factors that must be taken into account when interpreting results from experimental studies. Differences in phenotypic expression by rat alveolar pleural and peritoneal macrophages are noted, as well as the differing expression of major histocompatibility complex (MHC) class II molecules. Furthermore, alveolar macrophages, harvested from rat lung, do not express the interleukin (IL)-1 cytokines, and lipopolysaccharide (LPS) treatment of quiescent cells (after 24-hr in vitro culture) induces low levels of expression of IL-1 alpha and IL-1 beta. Short-term inhalation of refractory ceramic fibers, however, results in markedly increased IL-1 beta expression after stimulation with LPS. We suggest that, in vivo, IL-1 beta may be involved in the initial recruitment and activation of inflammatory cells rather than in induction of immune responses. We also postulate, based on recent published evidence, that alveolar macrophages activate the dendritic cells within the respiratory epithelium. Thus alveolar macrophages would release cytokines critical for the activation of dendritic cells during the afferent limb of the immune response, and they would respond to products of sensitized T-cells such as interferon-gamma and IL-4 to interact with T-helper cells in an antigen-specific MHC-restricted manner during the efferent limb of the response.

Lithium counteracts histamine suppression of human T cell mitogenesis.

Human T cell proliferative responses to concanavalin A (conA) were suppressed by approximately 50% by histamine (100 microM). In contrast, LiCl (1 or 3 mM) potentiated T cell responses by about 50%, but 10 mM LiCl had no significant effect on T cell proliferation. Histamine suppression was not significantly affected by the presence of potentiating concentrations of LiCl, whereas 10 mM LiCl completely abrogated histamine suppression.

Stimulation of human peripheral lymphocytes by methionine enkephalin and delta-selective opioid analogues.

The opioid peptide methionine enkephalin was shown to stimulate human peripheral lymphocyte proliferation in vitro in the absence of mitogen. A study of the time course of stimulation revealed a maximum proliferative response, measured by [3H]thymidine incorporation, after 4-5 days incubation. The kinetics of the response are similar to those for in vitro T cell responses to antigen rather than via polyclonal activation through lectin or anti-CD3 triggering, suggesting a physiological basis for the phenomenon. The stimulatory influence was blocked by the delta-selective antagonist ICI-174864, suggesting the mediation of classical opioid delta receptors. The opioid receptor specificity is further demonstrated using delta- and mu-selective agonists. The delta-selective agonists DSLET and DPDPE stimulate proliferation, whereas the mu-selective agonist DAGON was without effect.

Local increase in interleukin-1-like activity following UVB irradiation of human skin in vivo.

Using an in vivo skin chamber method, we demonstrated increased release of interleukin-1 (IL-1)-like activity at the site of irradiation with 3 times the minimal erythema dose of ultraviolet B (UVB). IL-1-like activity was estimated using the mouse thymocyte amplification assay. UVB-augmented release of IL-1-like activity peaked 1 h after irradiation and levels returned to baseline by 2 h. Release of IL-1-like activity from human skin after exposure to UV radiation may account for some of the local and systemic features of the sunburn response.

Bronchoalveolar lavage cell counts as a predictor of short term outcome in pulmonary sarcoidosis.

Sixty seven patients with biopsy proven pulmonary sarcoidosis were prospectively studied to determine whether single point bronchoalveolar lavage cell counts were a useful indicator of functional outcome and whether repeated lavage helped in management. The mean follow up period was 25 (range 13-37) months. No patient was having corticosteroid treatment at the time of initial bronchoalveolar lavage. "High intensity alveolitis" (lymphocyte count greater than or equal to 28%) was present at the initial lavage in 42 patients. These patients showed a significant improvement in their pulmonary function and chest radiographs over the follow up period whereas patients with "low intensity alveolitis" did not. Of the 42 patients with high intensity alveolitis, 31 had chronic sarcoidosis (duration over two years, mean 80 months). These patients showed a significant improvement in FVC but not in TLCO. Corticosteroids resulted in greater functional and radiological improvement in the patients with high intensity alveolitis than in those with low intensity alveolitis. Repeat bronchoalveolar lavage in 34 patients, mean 8.4 months after the original lavage, showed a weak inverse relation between a reduced lymphocyte count and change in forced vital capacity and isotope uptake on a gallium scan. These correlations were too weak to make repeated cell counts useful in management. Our results suggest that high intensity alveolitis may be a favourable prognostic factor for lung function in pulmonary sarcoidosis, even in patients with chronic disease, but that repeat lavage adds little to the management of the individual patient.

Methionine-enkephalin stimulates in vitro proliferation of human peripheral lymphocytes via delta-opioid receptors.

The influence of methionine-enkephalin (Met-Enk) on in vitro proliferation of human peripheral lymphocytes was investigated. Met-Enk, in the concentration range 10(-12) to 10(-4) M enhanced the proliferative response to suboptimal concentrations of concanavalin A of human peripheral lymphocytes and to cells in the absence of mitogen. The response to Met-Enk in the absence of mitogen was not influenced by the presence of fetal calf serum: similar levels of enhancement were seen in cultures supplemented with 10% autologous serum. Enhancement of proliferation was blocked in a concentration-dependent manner by both naloxone and the delta specific antagonist ICI-174864. The sensitivity to the antagonistic influence of ICI-174864 suggests strongly that the stimulatory influence of Met-Enk on human lymphocyte proliferation in the absence of mitogen is mediated via the delta-opioid receptor.

Nasal histamine release following hyperosmolar and allergen challenge.

Allergic rhinitis is characterised by symptoms of sneezing, itching of the nose with watery secretions, and nasal obstruction. We have previously shown that patients can have the diagnosis of allergic rhinitis confirmed by nasal provocation tests and assessment of nasal inspiratory peak flow (NIPF) after specific allergen or hyperosmolar challenge. We now show that histamine is released into the nasal lavage fluid in response to such challenges. Saline lavage alone results in detectable histamine levels in the order of 5 ng/ml, but in the presence of allergen (HDM) there is a significant increase in histamine release in atopics but not in control subjects. With hyperosmolar challenge, atopics showed a biphasic response in that histamine release was increased with 1.8% and 3.6% saline but returned to baseline with 5.4% and 7.2% saline, then showing a further increase with 9.0% saline. This raises the possibility of two populations of responsive mast cells. Hyperosmolar challenge leads to symptoms of nasal itch and sneezing as well as histamine release in atopics but not in controls. This suggests that hyperosmolar challenge can be used as a simple diagnostic test for allergic rhinitis and may provide a model for nasal hyper-reactivity.

Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from mast cells from various locations.

Nedocromil sodium and sodium cromoglycate inhibited histamine release from rat peritoneal mast cells. Tachyphylaxis was observed with both drugs. The 2 compounds were extremely selective in their action, being less active against peritoneal mast cells from the hamster and completely ineffective against mast cells from the mouse. Human basophil leucocytes, tissue mast cells of the guinea-pig and rat intestinal mast cells were also unresponsive. Both drugs inhibited immunological histamine release from human pulmonary mast cells obtained by bronchoalveolar lavage (BAL) and, less effectively, from lung parenchyma. Nedocromil sodium was about 1 order of magnitude more potent than sodium cromoglycate in each case. Tachyphylaxis was observed with the dispersed lung, but not with the cells obtained by BAL, and the degree of inhibition varied inversely with the magnitude of the secretory response. The possible clinical significance of these observations is discussed.

Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from human lung mast cells.

Sodium cromoglycate and nedocromil sodium produced a dose dependent inhibition of histamine secretion from human pulmonary mast cells obtained by bronchoalveolar lavage and by enzymatic dissociation of lung parenchyma. Both compounds were significantly more active against the lavage cells than against the dispersed lung cells, and nedocromil sodium was an order of magnitude more effective than sodium cromoglycate against both cell types. Tachyphylaxis was observed with the parenchymal cells but not with the lavage cells. Nedocromil sodium and sodium cromoglycate also inhibited histamine release from the lavage cells of patients with sarcoidosis and extrinsic asthma.

Nasal response to allergen and hyperosmolar challenge.

Rhinitis causes both clinical and social discomfort to patients, and in clinical practice is often underdiagnosed. We have examined a simple method for the assessment of a positive nasal provocation test to help in the diagnosis of rhinitis. In patients with histories suggestive of house dust mite (HDM) sensitivity and positive skin-prick tests or specific IgE to Dermatophagoides pteronyssinus, there was a fall in nasal inspiratory peak flow (NIPF) following nasal challenge with allergen. This was not seen in control subjects or in pollen-sensitive patients when challenged with house dust mite. Frequency of sneezing and degree of rhinorrhoea increased in these patients following challenge, and based on these findings we propose a simplified scoring system for the diagnosis of allergic rhinitis. We examined non-specific nasal reactivity using hyperosmolar solutions as a challenge system and found that allergic subjects responded with a fall in NIPF, although the clinical response was not identical to that seen with allergen. Control subjects did not respond to hyperosmolar challenge.

Serum C-reactive protein concentrations in patients with pulmonary sarcoidosis.

In a prospective study serum C-reactive protein concentrations were measured in nine patients with "active" pulmonary sarcoidosis (as assessed by bronchoalveolar lavage lymphocyte counts, gallium-67 lung scanning, and serial pulmonary function testing), and in five patients with "inactive" disease. Active pulmonary sarcoidosis was associated either with no rise or with only a modest rise in serum C-reactive protein concentrations. In contrast, serum C-reactive protein concentrations in 12 patients with sputum positive pulmonary tuberculosis were considerably raised. Serum C-reactive protein may thus provide a valuable test in the differentiation of sarcoidosis from conditions which it may mimic and which are known to induce an acute phase response.

Lack of immune deficiency in sarcoidosis: compartmentalisation of the immune response.

The original findings of peripheral anergy in sarcoidosis led to the conclusion that sarcoidosis was a disease associated with immune deficiency, but patients with sarcoidosis do not appear to suffer from repeated infections suggestive of immune suppression. With the technique of bronchoalveolar lavage it is now possible to examine the local immune response within the lung, the most commonly affected organ in sarcoidosis. In this study three different indices of cell mediated immunity (lymphocyte transformation, interleukin-2 production, and interleukin-1 production) have been examined by comparison of cells recovered by lavage with those collected from peripheral blood. It was found that in vitro anergy was confined to peripheral blood cells, where all three markers of the immune response used in this study was impaired in the 12 patients with sarcoidosis group when compared with results in the 12 controls, with the most depressed responses seen in those patients classified as having active disease (lymphocyte proliferation 45% (SD 17%); interleukin-2 production 44% (13%), and interleukin-1 production 31% (10%) of control levels). By contrast, T lymphocytes recovered from the lungs of patients with sarcoidosis showed a greater response than did those from controls in terms of lymphocyte transformation and interleukin-2 production; these differences were greatest in those with active disease (lymphocyte proliferation 209% (27%) and interleukin-2 production 202% (19%) of control levels). Interleukin-1 production by cells of the monocyte lineage recovered from the lung gave similar results to those of the control and sarcoid groups. It is concluded that the anergy seen in the peripheral blood compartment possibly reflects redistribution of T lymphocytes rather than a generalised immune deficiency.