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Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660–Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur.

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.

Objectives: The objective of this study was to demonstrate the reproducibility of a standardized photographic technique for recording fluorosis when used by a group of epidemiologists as part of a large multicentred European study. Methods: Studies were first carried out to develop the equipment specification and photographic method. The author (JAC) was then trained and calibrated in this method. She was then responsible for the training and calibration of examiners from a further six European study sites. The method involved taking two transparencies of the permanent maxillary central incisors of 8-year-old children, the first after 8 s while the teeth were still wet and the second after 105 s when the teeth had been allowed to dry out naturally. Data were collected at a central location during a training/calibration exercise and subsequently, during the conduct of a large study to measure fluorosis prevalence, at the seven sites. Intra- and interexaminer reproducibility of the photographic method were measured by grading the transparencies produced by all the examiners according to the DDE and TF indices. Results: The time period in which the transparencies were taken was to within 4 s among the examiners. Transparencies scored according to the TF index gave a range of Kappa values of 0.45-0.66 for intraexaminer reliability and 0.32-0.55 for interexaminer reliability. When using the DDE index Kappa values ranged from 0.43 to 0.70 for intraexaminer reliability and from 0.34 to 0.69 for interexaminer reliability. Conclusion: The photographic method was mostly robust and reproducible when used by epidemiologists from seven European study sites.

Objectives: To assess the impact of enamel fluorosis in three of the communities examined in ‘Project FLINT’, it was decided to conduct a structured telephone interview with the parents of some of the children who took part in the study. Methods: Three communities involved in this project were able to conduct this investigation: Reykjavik (Iceland), Cork (Ireland) and Knowsley (England). The aim was to interview the parents of children with a range of Thylstrup and Fejerskov (TF) index grades selected from each participating centre with respect to the appearance of their child's permanent maxillary central incisors. Interviewers were blinded as to the TF grade of the subject. Results: Interviews were conducted with parents of 215 children: 69 with TF grade 0; 70 with TF grade 1; 60 with TF grade 2 and 16 with TF grade 3 or greater. There was a trend towards more parents being unhappy with the appearance of their child's teeth with increasing TF grade. However, the main reasons given by parents for being unhappy with the appearance of their child's teeth was tooth alignment followed by the more yellow colour of permanent compared with primary teeth. Only with a TF grade of 3 was any appreciable concern expressed about fluorosis. Conclusion: It would appear that there is a public awareness of both developmental defects and enamel fluorosis although this is not always expressed as dissatisfaction. Further research is required into the clinical impact of both fluorosis and other developmental defects of enamel.

Objectives: The objectives of this study were to report on the prevalence of enamel opacities from seven European study sites using a standardized photographic method, and to investigate the importance of variables responsible for enamel fluorosis. Methods: The sample comprised a randomly selected group of 300 8-year-old children in each of the study areas. One examiner from each area was trained and calibrated in the use of a standardized photographic technique. Two transparencies were taken of each child's permanent maxillary central incisor teeth; one to represent the teeth 'wet' and one when the teeth had been allowed to dry out naturally for 105 s. The transparencies were viewed 'blind' by the author (JAC) and scored using the DDE and TF indices. Data relating to variables considered to be associated with enamel fluorosis were also collected. Results: The prevalence of diffuse opacities ranged from 61% in fluoridated Cork (Ireland) to 28% in Athens (Greece). The percentage of subjects with a TF score of three or more ranged from 4% in Cork and nonfluoridated Haarlem (the Netherlands) to zero in Oulu (Finland) and Athens. Fluoridated water and the prolonged use of fluoride tablets were found to be significant contributory factors to fluorosis. Conclusions: The prevalence of fluorosis was found to be highest in fluoridated Cork. The prolonged use of fluoride supplements was also found to be a significant risk indicator associated with fluorosis. © Blackwell Munksgaard, 2004.

Objectives: To develop a standardized method for measuring the variables affecting fluoride ingestion from toothpaste in young children between the ages of 1.5 and 3.5 years, and to use the method at seven European sites. Methods: Random samples of children were invited to take part in the study. Parents who gave consent were visited at home. The children brushed their teeth using the toothpaste brand and toothbrush type currently in use. The difference between the fluoride dispensed onto the toothbrush and the fluoride recovered after accounting for losses was deemed to be the fluoride ingested. Details of other oral health-care habits were collected by questionnaire. For each child, the fluoride concentration of the toothpaste used was measured in the laboratory, from which an estimate of total daily fluoride ingestion was made. Results: There was considerable variation between countries in the types of toothpaste used and in the amounts of toothpaste applied and ingested. The amount of fluoride ingested ranged from 0.01 to 0.04 mg fluoride per kg of body weight per day. Conclusion: The amount of fluoride ingested that is likely to be a risk factor for the development of dental fluorosis during tooth formation is equivocal and was found to vary widely between European countries. There appears to be a need for clearer health messages regarding the use of fluoridated toothpaste by young children. © Blackwell Munksgaard, 2004.

An important challenge encountered in this multicentred project was the need to take account of the different cultural and legal differences between the seven sites when agreeing the protocol. Examples such as access to registers of births and subject consent dictated that there were some differences in the methods used in the different sites. The data presented showed that it was possible to train and calibrate a number of examiners in a standardized photographic method for recording enamel fluorosis. This method has a number of important advantages for the objective monitoring of enamel fluorosis over time. There were considerable differences between the seven sites in the formulations of the toothpaste used and in the pattern of their use. The results indicate that it is possible to agree and adopt a standardized method for measuring fluoride ingestion from toothpaste. The aesthetic impact of enamel fluorosis seemed low in the populations included in this project, but further work is required on this issue. © Blackwell Munksgaard, 2004.

Objectives: The aim of this study was to collate data on national policies for the use of fluoride in the seven European countries participating in the FLINT project. Methods: Policies on the use of fluoride were obtained for each of the study areas. Data collected included the presence of water fluoridation and regulations governing fluoride toothpaste and fluoride supplements. Results: In Ireland 74% of the population had a fluoridated water supply but in all the other countries fluoride toothpaste was the principal form of delivering fluoride, usually recommended as a dose of a pea-sized amount. Fluoride supplement use varied considerably between countries. The Netherlands had the clearest regulations covering the use of fluoride supplements and definition of at-risk individuals. Most countries, even if they recognized particular caries-risk did not define the term clearly. In Iceland all children were regarded as being at high risk of developing caries. Conclusion: Considerable variation exists between European countries in their policies for fluoride use and no clear definitions of high-caries-risk individuals were found. The results show that there is even a lack of coherent thought and planning within the different countries, let alone between them. © Blackwell Munksgaard, 2004.

Objectives: The aim of this study was to determine the effects of rinsing and spitting on fluoride ingestion from toothpaste during normal oral-hygiene procedures of younger children, and hence to make recommendations on rinsing during toothbrushing. Methods: The brushing habits of 166 Dutch and 185 Irish children between 1.5 and 3.5 years were observed during home visits. The weight of the toothpaste tube was determined before and after use. After brushing, the toothbrush and any associated expectorate and rinses, combined with any toothpaste spilled during the brushing procedures, were collected. The amounts of fluoride retained on the toothbrush and in the associated expectorate and rinses were measured. Results: Over 90% of the Dutch children used a special toddlers' toothpaste with ≤500 ppm F. Eleven per cent of the younger (

Objectives: The objective of this study was to demonstrate the reproducibility of a standardized photographic technique for recording fluorosis when used by a group of epidemiologists as part of a large multicentred European study. Methods: Studies were first carried out to develop the equipment specification and photographic method. The author (JAC) was then trained and calibrated in this method. She was then responsible for the training and calibration of examiners from a further six European study sites. The method involved taking two transparencies of the permanent maxillary central incisors of 8-year-old children, the first after 8 s while the teeth were still wet and the second after 105 s when the teeth had been allowed to dry out naturally. Data were collected at a central location during a training/calibration exercise and subsequently, during the conduct of a large study to measure fluorosis prevalence, at the seven sites. Intra-and interexaminer reproducibility of the photographic method were measured by grading the transparencies produced by all the examiners according to the DDE and TF indices. Results: The time period in which the transparencies were taken was to within 4 s among the examiners. Transparencies scored according to the TF index gave a range of Kappa values of 0.45-0.66 for intraexaminer reliability and 0.32-0.55 for interexaminer reliability. When using the DDE index Kappa values ranged from 0.43 to 0.70 for intraexaminer reliability and from 0.34 to 0.69 for interexaminer reliability. Conclusion: The photographic method was mostly robust and reproducible when used by epidemiologists from seven European study sites. © Blackwell Munksgaard, 2004.

To develop a standardized method for measuring the variables affecting fluoride ingestion from toothpaste in young children between the ages of 1.5 and 3.5 years, and to use the method at seven European sites.Random samples of children were invited to take part in the study. Parents who gave consent were visited at home. The children brushed their teeth using the toothpaste brand and toothbrush type currently in use. Variables measured were: type of toothpaste used, fluoride concentration of toothpaste used, weight of toothpaste used, frequency of brushing and body weight of the child.It was not possible to follow the agreed protocol in all seven countries and in three countries appropriate alternative methods were employed. There was considerable variation between countries in the variables investigated. Use of children's toothpaste ranged from 69% in Ireland to 98% in Portugal. In the Netherlands up to 60% of the children were using toothpaste containing400 ppm F and in Finland up to 27% of children were using toothpaste containing1200 ppm F. Over half of the children used0.25 g of toothpaste per brushing and the majority of children brushed once or twice per day.Although adherence to the agreed protocol was not possible at all study sites there was a clear picture of considerable variation in the oral hygiene practices of young children throughout Europe.

Objectives: To develop a standardized method for measuring the variables affecting fluoride ingestion from toothpaste in young children between the ages of 1.5 and 3.5 years, and to use the method at seven European sites. Methods: Random samples of children were invited to take part in the study. Parents who gave consent were visited at home. The children brushed their teeth using the toothpaste brand and toothbrush type currently in use. Variables measured were: type of toothpaste used, fluoride concentration of toothpaste used, weight of toothpaste used, frequency of brushing and body weight of the child. Results: It was not possible to follow the agreed protocol in all seven countries and in three countries appropriate alternative methods were employed. There was considerable variation between countries in the variables investigated. Use of children's toothpaste ranged from 69% in Ireland to 98% in Portugal. In the Netherlands up to 60% of the children were using toothpaste containing 1200 ppm F. Over half of the children used

When an American company is considering building a research facility in a foreign country, the question is frequently asked: "Why not expand facilities in the U.S. instead?" Certainly, by any objective measure of cost-effectiveness, to build a clone of a U.S. research (as opposed to development) laboratory in another industrialized country does not make much sense. Such action can only be justified by defining ways in which the foreign laboratory can be differentiated from that in the parent country and deciding how this differentiation can be used to augment the effectiveness of the overall research effort. This, in turn, may be related to specific aspects of the culture of the country where the laboratory will be located. Japan provides an excellent example in many areas of both the opportunities and challenges posed to a U.S. company by cultural differences. I shall address only one of these areas here: the gathering and dissemination of information. The term "Information Society" has been applied to Japan with good reason because the accumulation of information is much more actively pursued in Japan than elsewhere in the industrialized world (1). There is a culture of storing information that has no obvious application in the present but may have unforeseen advantages in the future (2). It is probable that this attitude was purposely cultivated at the time of the Meiji Restoration and the ensuing modernization of Japan (3). It seems likely that the Japanese were the first nation to really understand the importance of information as a national resource. In most Japanese companies, information gathering is considered to be an integral part of the job of all technical employees at any level. In many companies, this is formalized and each staff member is assigned a specific area or areas in which he or she is expected to be constantly aware of new developments (2). It should be emphasized that, as suggested above, this goes well beyond keeping up with the literature on one's current research project. In fact, it may involve gathering information on a topic of no current interest to the company but one that may, conceivably, become important in the future. The sources of this information can be very diverse and the priorities of various types of access quite different from elsewhere. In the U.S. and Europe, the most widely used sources of new technical information are journal publications and scientific databases. In Japan, on the other hand, personal exchange of information through a human network is at least as, and probably more, important. Much more so than in the West, Japanese technical people keep close and constant contact with former fellow students and professors, technical persons within their extended family, and so on, in order to learn about new developments. It is vital that Western companies be plugged into this human network if they are to learn, in a timely manner, of technical advances made by Japanese industry (4). Furthermore, it has been estimated by the European Community that there are approximately 300,000 significant technical publications published per year in Japan, excluding the very large number of patent applications, patents and related materials. Of these, only about 200,000 are retrievable by non-Japanese using conventional sources. This leaves some 100,000 publications that are accessible to Japanese companies but are lost to those Westerners who have no effective information-gathering capability in Japan. A simple example of the value of access to this human network is the following: A division of W.R. Grace had the opportunity to become a supplier of a product but could not find a source for a reactive resin that would yield a product with the required specifications. Using personal contacts of the staff at Grace's Japan Research Center, a company in Japan supplying such a resin was located. Since this company neither advertised the product nor had literature in English, it is unlikely that it would have been located in any other way. …

A Campylobacter jejuni gene encoding a homologue of the flagellar biosynthesis gene flhB was identified downstream of the peroxide stress defence gene ahpC. Insertional mutagenesis of the flhB gene rendered C. jejuni non-motile, with most cells aflagellate, although a small number expressed truncated flagella. The absence of FlhB also appeared to affect cell shape, as the majority of cells were straight rather than curved rods. Transcription of the flagellin gene flaA was significantly reduced in the C. jejuni flhB mutants, which also did not express significant amounts of flagellin proteins, indicating that FlhB is an essential protein for subsequent expression of flagellar genes. The transcription start site of the flhB gene, as determined by primer extension, was located 91 bp upstream of the flhB start codon, but no recognizable promoter sequence could be identified immediately upstream of this transcription start site. Transcriptional flhB::lacZ reporter gene fusions confirmed that the flhB gene has its own promoter region, is expressed at very low levels and is transcribed independently of ahpC, and that its transcription is not regulated by iron or growth phase.

Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides in C. jejuni -mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second l - glycero - d - manno -heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide. In this report we confirm experimentally that C. jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. The Campylobacter waaF gene complements an S. enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF in C. jejuni , waaF mutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM 1 , asialo-GM 1 , and GM 2 ganglioside epitopes. The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C. jejuni . The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-β(1,3) N -acetylgalactosamine. In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species.

To compare 24-hour urinary fluoride excretion in young children exposed to different fluoride regimes.Twenty-four-hour urine samples were collected from children aged between 1.8 and 5.2 years. Samples were collected from Cork, Ireland (n=19) where the water is fluoridated to a concentration between 0.8 and 1.0 mg/l; Knowsley, UK, where the water fluoride concentration is0.1 mg/l (n=22); and from children in Knowsley drinking milk containing 0.5 mg fluoride in nursery school each day (n=16). The volume of the samples was measured, they were analysed for fluoride concentration and the 24-hour urinary fluoride excretion was calculated.It was found that the mean fluoride excretion in response to usual conditions of fluoride intake in these children was 0.21 mg (SD=0.14) in non-fluoridated Knowsley; 0.36 mg (SD=0.11) in fluoridated Cork and 0.30 mg (SD=0.10) in the children drinking fluoridated school milk.The daily fluoride excretion in these children, corrected for age and fluoride ingested from toothpaste, appeared to indicate that the fluoride intake in the children drinking fluoridated school milk was somewhere between those living in an optimally fluoridated area and those in a low fluoride area.

Enteric Campylobacter species cause gastrointestinal diseases in humans. Like almost all organisms, campylobacters have an absolute requirement for iron, but are faced with variable availability of iron in the environment and host tissues. Campylobacters have developed mechanisms to scavenge sufficient iron for metabolism and growth. However, iron also participates in the formation of reactive oxygen species, and this forces pathogens to maintain intracellular iron homeostasis and to cope with oxidative stresses. The presence of two separate, but possibly overlapping iron-responsive regulatory systems, which regulate iron acquisition and oxidative stress defense, and the presence of genes encoding multiple iron acquisition and detoxification systems in Campylobacter indicate the central role that iron plays in Campylobacter gene regulation and virulence.

Infection of rabbit ileal loops with inflammatory Campylobacter jejuni strains caused elevation of cyclic AMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. Incubation of cultured Caco-2 cells with loop fluids caused elevated cellular cyclic AMP levels, an effect which was inhibited by antiserum against prostaglandin E2.

To consider the feasibility of using school milk as a vehicle to deliver fluoride to children, suffering from high rates of dental disease, in socially deprived districts.The legal aspects of adding fluoride to milk and availability of milk subsidies were updated. The organisational requirements of using school milk as a vehicle for fluoride were investigated and the consultation process established. The uptake of fluoridated milk was monitored and the fluoridated milk was subjected to rigorous quality control. The costs involved in running a scheme were calculated.Fluoridated milk can now be called milk with added fluoride and to date the product has attracted subsidies from the European Economic Community and from the Welfare Foods Section in the Department of Health. The demonstration scheme in St. Helens, Merseyside, generated interest from neighbouring health authorities leading to the subsequent expansion of the programme. By working with the dairy, recommendations to improve the quality of school milk have been developed. The main organisations involved in running school based milk fluoridation schemes have been encouraged by the low costs involved.The UK programme has demonstrated that it is feasible to use school milk as a vehicle to deliver fluoride on a community basis. Attention must be given to improving the quality and particularly the temperature control of school milk.

Twenty–four–hour urine samples were collected from each of thirteen 5– to 6–year–old children both under customary conditions of fluoride intake (i.e. usual diet including milk containing 0.5 mg fluoride and usual oral hygiene procedures with fluoride toothpaste) and during a 4–day study period in which their customary fluoride intake was replaced with standard fluoride doses. The 24–hour fluoride excretion under customary conditions of fluoride intake was 0.30 mg and in response to fluoride doses in the range of 0.5–2.0 mg it was in the range 0.26–0.61 mg. Using data obtained during the study period for all subjects combined, the fluoride intake from all sources under customary conditions was estimated as 0.76 mg, representing a fractional fluoride excretion of 39%. It is concluded that these children are possibly receiving suboptimal amounts of fluoride.

A gene encoding a putative 2[4Fe--4S] ferredoxin (FdxA) was identified upstream of, and divergent to the peroxide stress defense gene ahpC of the microaerophilic pathogen Campylobacter jejuni. The transcription start site of fdxA was located 27 and 28 bp upstream of the fdxA start codon. Transcriptional fusions of the fdxA promoter to a lacZ reporter gene demonstrated that expression of fdxA is iron-induced, and thus oppositely regulated to the iron-repressed ahpC gene. Insertional mutagenesis of the fdxA gene did not affect microaerobic growth of C. jejuni, but significantly reduced aerotolerance of C. jejuni. The fdxA gene is the first reported iron-induced gene of C. jejuni, and encodes a novel component of its oxidative stress defense.

A lacZ-based reporter gene system was used to identify the promoter of the Campylobacter jejuni iron-responsive gene regulator Fur. In other Gram-negative bacteria, the fur promoter is usually located directly upstream of the fur gene and is often autoregulated in response to iron. In this study we demonstrate that expression of the C. jejuni fur gene is controlled from two promoters located in front of the first and second open reading frames upstream of fur. Neither of these promoters was iron-regulated, and the presence of both promoters in front of fur gives higher expression of the lacZ reporter than with either promoter alone. Expression from two distal promoters might be a mechanism for regulating the level of the C. jejuni Fur protein in response to unknown stimuli.

Alterations in the response of leukaemic cells to apoptosis-inducing stimuli may account for resistance to chemotherapy and treatment failure, either by disruption of the apoptotic pathway itself or by altered DNA repair; quiescent cells and those with disrupted cell-cycle checkpoints may also display decreased apoptosis. Quiescence can be induced by the differentiation of myeloid cells, and this led us to investigate whether the modulation of drug-induced apoptosis associated with differentiation might be a model for quiescence-associated resistance generally. We have demonstrated that resistance to idarubicin-induced apoptosis increased with greater duration of incubation of HL60 and U937 cells with ATRA and 1,25(OH)2 D3 and that this protective effect correlated with the degree of G0/G1 accumulation. In addition, the cytoprotective effects held for other classes of cytotoxic drugs with different mechanisms of action to idarubicin. Prolonged exposure to idarubicin or vinblastine was associated with diminution of the protective effect and re-entry of cells into cycle. The full cytoprotective effect was restored by resupplementation with ATRA or 1,25(OH)2 D3 during exposure to idarubicin, with concomitant persistence of G0/G1 accumulation. Differentiating agents prevented the accumulation of leukaemic cells at the G2/M checkpoint in response to low concentrations of idarubicin. Understanding how differentiating agents modulate these cell-cycle checkpoints, and how quiescent cells evade apoptosis, may allow the development of therapeutic strategies to limit such apoptosis-inhibiting effects and maximise cell kill from chemotherapy.

To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae: this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone.

Microaerophiles likeCampylobacter jejunimust resist oxidative stresses during transmission or infection. Growth ofC. jejuni81116 under iron limitation greatly increased the expression of two polypeptides of 26 and 55 kDa. The identification of these proteins by N-terminal amino acid sequencing showed both to be involved in the defense against oxidative stress. The 55-kDa polypeptide was identical toC. jejunicatalase (KatA), whereas the N terminus of the 26-kDa polypeptide was homologous to a 26-kDaHelicobacter pyloriprotein. The gene encoding theC. jejuni26-kDa protein was cloned, and the encoded protein showed significant homology to the small subunit of alkyl hydroperoxide reductase (AhpC). The upstream region ofahpCencoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences containedflhBandmotBhomologs, which are involved in flagellar motility. There was no evidence for an adjacent homolog ofahpF, encoding the large subunit of alkyl hydroperoxide reductase. Reporter gene studies showed that iron regulation ofahpCandkatAis achieved at the transcriptional level. Insertional mutagenesis of theahpCgene resulted in an increased sensitivity to oxidative stresses caused by cumene hydroperoxide and exposure to atmospheric oxygen, while resistance to hydrogen peroxide was not affected. TheC. jejuniAhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.

Salmonella typhimurium strains harboring independent, defined mutations in aroA , invA , ssrA , or msbB were assessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ileal loops. Three wild-type strains of S. typhimurium , TML, HWSH, and SL1344, and two mutant strains, S. typhimurium SL1344 ssrA and S. typhimurium SL1344 msbB , consistently induced fluid accumulation in the lumen of loops and inflammation of loop-associated tissues. In contrast, three different S. typhimurium aroA strains and an invA mutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops. However, the S. typhimurium aroA strains did induce an inflammatory infiltrate and some local villus-associated damage, but the invA mutant did not. Histologically, wild-type S. typhimurium , S. typhimurium SL1344 ssrA , and S. typhimurium SL1344 msbB demonstrated more severe effects on villus architecture than S. typhimurium aroA strains, whereas S. typhimurium invA -infected loops showed no detectable damage. This suggests that villus damage most likely contributes to fluid accumulation within the loop.

Summary: A region encoding ORFs with homology to known lipopolysaccharide (LPS) biosynthesis genes was isolated from two strains of Campylobacter jejuni. One of the strains produces LPS, but the second strain is reported to produce only lipooligosaccharide (LOS) and therefore lacks the O-chain. The two strains shared six predicted ORFs, but an additional ORF, orfE, of unknown function was identified in the LOS-producing strain. Mutation of the shared wbeE (rfbE) homologue (orfF) or deletion of five of the seven genes reduced core reactivity with specific antiserum without affecting O-chain production. Mutation of either the capD homologue (orfG) or the unique orfE had no detectable effect on LOS or LPS production. The presence or absence of orfE in 36 isolates of C. jejuni did not correlate with LOS/LPS phenotype or serotype. However, after insertion of orfE into a LPS-producing orfE-negative strain the O-chain ladder was no longer detectable on Western blots. We were not able to disrupt the wbaP (rfbP) homologue (orfC) in C. jejuni.

Empirical evidence from many countries, obtained from sampling populations of patients admitted to hospital with acute myocardial infarction, has confirmed that elderly patients are significantly less likely to receive thrombolytic therapy. This difference persists after controlling for confounding factors such as admission delay and contraindications to thrombolysis. However, evidence supporting the efficacy of thrombolysis in reducing mortality after acute myocardial infarction is less clear cut in patients aged 75 years or above than in younger patients. These older patients are substantially under-represented in the clinical trials although they constitute one third of the clinical population. Observational studies indicate that older patients are at slightly higher risk than younger patients of experiencing haemorrhagic stroke after thrombolysis. It is, however, unlikely that efficacy and tolerability considerations alone account for the low use of thrombolytics in the elderly as similar trends are seen for other modalities of treatment of acute myocardial infarction. Since older patients have the highest mortality risk after myocardial infarction, they have the greatest potential gain from thrombolytic treatment, assuming a uniform treatment effect across age. The estimated cost effectiveness (cost per quality-adjusted life-year gained) improves with increasing age. It is concluded that patient age should not influence the treatment decision concerning thrombolysis. To ensure that elderly patients receive maximum benefit from this therapeutic advance requires attention to referral patterns from the community, speed of assessment in hospital and a clear treatment policy without age constraints. The effectiveness of these measures should be routinely audited.

Non-inpatient care is becoming an increasingly attractive option in many therapeutic areas, including the treatment of infections in patients with haematological malignancies. The choice of antibiotics for this care is governed by infection patterns and experience within particular institutions. There is an increase in infections caused by Gram-positive pathogens due to the long-term use of tunnelled catheters and intensive, punishing chemotherapy regimens. Patients suitable for non-inpatient care include both long- and short-term patients who fulfil specific clinical and social criteria. Haematological malignancy patients are often suitable for this type of care. Benefits for patients include improved quality of life, while benefits for the clinician include effective, safe care as well as reduced costs and greater bed availability. The glycopeptide teicoplanin has been assessed for use in non-inpatient care, and is particularly suitable due to its long half-life, no need for monitoring and its activity against Gram-positive pathogens. A comparative study of a teicoplanin-ciprofloxacin combination was conducted in inpatients, followed by a cohort study in non-inpatients. This combination was found to be clinically and microbiologically effective, suitable for non-inpatient administration and generated cost savings due to early discharge. The organization of a non-inpatient service demands dedicated team members, with well-defined roles and a designated treatment area. The day ward is the focus of care, which can then take place in the day ward or in the patient's home. Good communication between immediate and wider team members, and patient education are cornerstones of a successful service.

Long-term beta-blockade reduced mortality after acute myocardial infarction by about a quarter in a series of published trials. Representative data on beta-blocker use for secondary prevention are scanty but indicate wide variations. We have analysed European practice, and sources of variation, by regional sampling of acute myocardial infarction patients admitted to hospital in 11 countries during the period January 1993-June 1994.Treatment data for 4035 representative patients were collected for the hospital phase and 6 months after discharge. A logistic regression model was developed to describe the predictors of beta-blocker use. In the 11 regional samples, 6-38% (20% overall) of patients had no recorded contraindications but were discharged without a beta-blocker. In the absence of perceived contraindications, there was a strong, independent negative association between age and odds of treatment (P0.001), and women were less likely to be treated than men (adjusted odds ratio 0.76, 95% CI 0.58-0.99). Discontinuation of beta-blocker treatment by 6 months was significantly less likely in regions where the proportion given such treatment at discharge was high. In contrast, use of antithrombotic agents in the samples was consistently high.There is persisting low use of beta-blocker secondary prophylaxis, particularly in the elderly and in women, not attributable to perceived contraindications or intolerance. Considerable regional variations persist despite shared trials evidence. Discharge treatment strongly influences long-term medication.

Publisher Summary This chapter describes the existing data on the molecular biology of C. jejuni and C. coli, discusses strategies and techniques currently available to investigate the molecular genetic basis of the pathogenesis of C. jejuni, and indicates possible future directions. Further, the chapter explains cloning of campylobacter genes. C. jejuni has a genome of approximately 1700 kb, with an unusually high A+T content of 70%. This compares to a genome size of approximately 4600 kb with an A+T content of 50% in E. coli. The identification and characterization of C. jejuni genes has been severely hampered by the fact that one of the most common strategies used in the study of other bacterial pathogens, transposon mutagenesis, has so far not been successful with C. jejuni. Currently, the cloning and sequencing of around 60 C. jejuni genes is described. Common strategies for the cloning and identification of C. jejuni genes are also explained.