Peter H. Williams

INTESTINAL PERMEABILITY AND INFLAMMATION IN RHEUMATOID ARTHRITIS: EFFECTS OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

The suggestion that the intestinal mucosa may be abnormally permeable and a site of absorption of antigens in rheumatoid arthritis was tested by the use of a 51Cr-EDTA (edetic acid) absorption test. 24 patients with rheumatoid arthritis excreted significantly more 51Cr-EDTA than did 34 controls. Intestinal permeability was normal in untreated patients but almost invariably abnormal in patients treated with non-steroidal anti-inflammatory drugs. Studies in patients with osteoarthritis showed that the permeability abnormalities were due to an effect of NSAIDs on both the proximal and the distal intestine and that the effect was systemically mediated. Indium-111-labelled leucocyte scans showed ileocaecal inflammation in 6 of 9 patients on or recently on NSAIDs. Although increased intestinal permeability does not seem to be important in the pathogenesis of rheumatoid arthritis, the administration of NSAIDs may lead to loss of intestinal integrity, thus facilitating antigenic absorption and perhaps contributing to persistence of the disease.

Effect of non-steroidal anti-inflammatory drugs and prostaglandins on the permeability of the human small intestine.

Intestinal permeability was estimated in healthy subjects after ingestion of aspirin (1.2+1.2 g), ibuprofen (400+400 mg) and indomethacin (75+50 mg) at midnight and an hour before starting a 51chromium labelled ethylenediaminetetraacetate absorption test. Intestinal permeability increased significantly from control levels following each drug and the effect was related to drug potency to inhibit cyclooxygenase. Intestinal permeability increased to a similar extent after oral and rectal administration of indomethacin showing that the effect is systemically mediated. Prostaglandin E2 decreased intestinal permeability significantly but failed to prevent the indomethacin induced increased intestinal permeability. These studies show that non-steroidal anti-inflammatory drugs disrupt the intestinal barrier function in man and suggest that the morphological correlates of the damage may reside at the level of the intercellular junctions.

Virulence of Enteropathogenic Escherichia coli, a Global Pathogen

SUMMARY Enteropathogenic Escherichia coli (EPEC) remains an important cause of diarrheal disease worldwide. Research into EPEC is intense and provides a good virulence model of other E. coli infections as well as other pathogenic bacteria. Although the virulence mechanisms are now better understood, they are extremely complex and much remains to be learnt. The pathogenesis of EPEC depends on the formation of an ultrastructural lesion in which the bacteria make intimate contact with the host apical enterocyte membrane. The formation of this lesion is a consequence of the ability of EPEC to adhere in a localized manner to the host cell, aided by bundle-forming pili. Tyrosine phosphorylation and signal transduction events occur within the host cell at the lesion site, leading to a disruption of the host cell mechanisms and, consequently, to diarrhea. These result from the action of highly regulated EPEC secreted proteins which are released via a type III secretion system, many genes of which are located within a pathogenicity island known as the locus of enterocyte effacement. Over the last few years, dramatic increases in our knowledge of EPEC virulence have taken place. This review therefore aims to provide a broad overview of and update to the virulence aspects of EPEC.

The Mammalian Neuroendocrine Hormone Norepinephrine Supplies Iron for Bacterial Growth in the Presence of Transferrin or Lactoferrin

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [(3)H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.

HIGH-DENSITY LIPOPROTEIN AND CORONARY RISK FACTORS IN NORMAL MEN

Serum high-density-lipoprotein cholesterol (H.D.L.) was measured in 2568 men attending a screening centre and registered with a medical practitioner in Great Britain. Serum-total-cholesterol (T.C.), serum-triglyceride, serum-glucose, systolic and diastolic blood-pressures, electrocardiogram, chest X-ray, height, weight, cigarette and alcohol history, and a brief assessment of physical activity were also recorded. H.D.L. was inversely related to cigarette-smoking, relative weight, and serum-triglyceride level, and directly related to physical activity, total-cholesterol level, and alcohol consumption. The ratio of H.D.L. to T.C. (H.D.L./T.C.) showed similar significant relationships to the above variables (except that the ratio was negatively correlated with T.C.). In addition the ratio was inversely related to age. These relationships were independent of the other measured variables. Both the H.D.L. and H.D.L./T.C. were inversely related to coronary risk rating. It is suggested that, despite methodological problems, H.D.L. is a useful biochemical measurement to add to a coronary risk profile.

Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis.

Trauma is well recognized to result in the immediate and sustained release of stress-related neurochemicals such as the catecholamine norepinephrine. Past work has shown that in addition to their ability to function as neurotransmitters, catecholamines can also directly stimulate the growth of a number of pathogenic bacteria. The development of trauma-associated sepsis has often been linked to the ability of otherwise normal commensal bacteria to invade and penetrate the gut mucosal barrier. Therefore, the aim of our study was to examine whether catecholamines could also stimulate the growth of commensal Escherichia coli strains of the type present in the intestinal tract at the time of a traumatic event. Herein we report that the growth of a range of non-pathogenic isolates of E. coli of human and environmental origin was significantly increased in the presence of catecholamines. A primary mechanism by which catecholamines increase bacterial growth was shown to be iron removal from lactoferrin and transferrin and subsequent acquisition by bacteria. The 3,4-dihydroxybenzoyl (catechol) structure of the catecholamines was further demonstrated to be critical to iron acquisition. The synthetic catecholamine inotropes dobutamine and isoprenaline, as well as norepinephrine metabolites that retained the catechol structure were also active, whereas norepinephrine metabolites in which the catechol moiety had been modified were not. A role for catecholamine-mediated bacterial iron supply in the pathophysiology of gut-derived sepsis due to trauma is proposed.

Differentiated caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli

A collection of 44 Campylobacter isolates (37 C. jejuni and seven C. coli) from children with colitis (21 strains) or watery diarrhoea (23 strains) was analysed for toxin production, association with HeLa cells, and invasion of differentiated Caco-2 cell cultures. There was no obvious association of clinical symptoms with species, biotype or enterotoxin production. All colitis strains and most of the isolates from watery diarrhoea were cytotoxic for Chinese hamster ovary cells. Measurements of bacterial association indices with HeLa cells varied with time, and were considered to be unreliable for discriminating between isolates from the two diagnostic groups. Statistically significant differences were observed between the two groups (all colitis strains and 65% of strains from non-inflammatory diarrhoea) with respect to invasion of both HeLa and Caco-2 cell monolayers. However, among the strains from non-inflammatory diarrhoea that did invade, numbers of internalised bacteria were similar to the range observed for colitis strains. Of the colitis strains, 86% were able to transcytose through polarised Caco-2 monolayers grown on filters, compared with 48% of isolates from non-inflammatory disease. We propose the use of Caco-2 cells as a model for studying invasion of intestinal epithelia by C. jejuni and C. coli.

Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes

BACKGROUND Bacterial colonisation of indwelling medical devices by coagulase-negative staphylococci is a prevalent risk in intensive-care units. Factors determining biofilm formation and progression to catheter- related infection are incompletely understood. We postulated that administration of inotropic agents via indwelling intravenous catheters may stimulate growth and formation of biofilms by Staphylococcus epidermidis. METHODS Inocula representing physiologically relevant infecting doses of S epidermidis were incubated in a minimum medium supplemented with fresh human plasma in the presence or absence of pharmacological concentrations of norepinephrine or dobutamine. Biofilm formation on polystyrene and medical-grade silicone was examined. After incubation, cultures were assessed for growth and formation of biofilms by colony counting and scanning electronmicroscopy. The production of exopolysaccharide, a major constituent of S epidermidis biofilms, was also assessed by use of immunofluorescence microscopy. FINDINGS Incubation of S epidermidis with catecholamine inotropes in the presence of human plasma resulted in a significant increase in growth compared with control on both polystyrene and silicone surfaces, with pronounced increases in biofilm formation as visualised by scanning electronmicroscopy. Experiments with transferrin labelled with radioactive iron showed the ability of catecholamine inotropes to facilitate acquisition of iron by S epidermidis. Immunofluorescence microscopy revealed extensive exopolysaccharide production associated with S epidermidis biofilms. INTERPRETATION The ability of catecholamine inotropic drugs to stimulate bacterial proliferation and biofilm formation may be an aetiological factor in the development of intravascular catheter colonisation and catheter-related infection. The removal of iron from transferrin for subsequent use by S epidermidis is a possible mechanism by which catecholamine inotropes stimulate bacterial growth as biofilms.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni

Background:Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. Aim: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. Methods:C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 μM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. Results: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. Conclusion: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

Resuscitation of Salmonella enterica Serovar Typhimurium and Enterohemorrhagic Escherichia coli from the Viable but Nonculturable State by Heat-Stable Enterobacterial Autoinducer

ABSTRACT Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the σs subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.