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The relationship between differentiation of human myeloid cells and apoptosis remains unclear. Recent studies have shown that terminal differentiation need not necessarily lead to the apoptotic demise of myeloid cells, while other studies have shown that induction of differentiation is associated with increased resistance to apoptosis-inducing agents, such as chemotherapy and gamma-irradiation. Such results are pertinent to the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome, where differentiating agents and hemopoietic growth factors are being combined with chemotherapy to enhance response and limit toxicity. To elucidate the factors governing apoptosis in human AML blasts, we have studied the cytotoxic effect of idarubicin on HL60, U937 and KG1 cells, after incubation with all-trans retinoic acid (ATRA), 1, 25(OH)2 D3, and granulocyte-macrophage colony-stimulating factor (GM-CSF ). We show that prior incubation of human myeloid leukemic cells with ATRA or 1,25(OH)2 D3 induced resistance to idarubicin-induced apoptosis, which was modulated by coincubation with GM-CSF. The altered chemosensitivity of cells depended on the degree of G0/G1 cell-cycle arrest induced by incubation with ATRA, 1, 25(OH)2 D3, and GM-CSF and was independent of differentiation status or Bcl-2 oncoprotein expression. These findings suggest that cell-cycle arrest in human leukemic cells can be induced by exogenous agents and may promote drug resistance. Determining the mechanisms by which cell-cycle arrest is induced may permit understanding of the processes by which the cells escape cytotoxic drug-mediated apoptosis.

We characterised the population of acute myocardial infarction patients admitted to Norwegian hospitals and quantified the actual use and potential maximal use of thrombolytic therapy. Data were collected by medical record review of all acute myocardial infarction patients discharged from hospital in April and May 1993 in Health region 1. The clinical population differed significantly from the patients recruited to the thrombolytic clinical trials. Patients were more likely to have ST depression on admission (23% vs. 7%) and to be over 74 years (42% vs. 10%) than in the trials. A fifth of patients presented more than 12 h after symptom onset (or time indeterminate). Thrombolysis was given to 32% of patients, mainly utilising streptokinase. Late presentation or diagnostic difficulty appeared to be the main reasons for non- thrombolysis. Approximately 50% of the clinical population were eligible for thrombolysis. Eligibility for thrombolytic therapy was therefore severely restricted by the presenting characteristics of the clinical population. Substantial numbers of patients belonged to subgroups where the reported benefit from thrombolysis is equivocal. Uncertainty remains on the extrapolation of the trials evidence to those subgroups who were under-represented in the clinical trials.

The enteric pathogens Campylobacter jejuni and Campylobacter coli are a major cause of infectious diarrhoea. Their ability to adhere to human epithelial cells is ubiquitous and their propensity to invade cells is also well documented and requires motility and de novo protein synthesis, as well as several host factors. The molecular basis of the interaction between campylobacters and host cells is only beginning to be elucidated. The characteristics of this interaction promise to be interesting and may provide new insights into host-pathogen interactions in other enteric diseases.

Caveolae are plasma membrane invaginations found in a variety of mammalian cells and are implicated in clathrin-independent endocytosis and signal transduction. Here we show that pretreatment of Caco-2 cell monolayers with filipin III, which disrupts caveolae by chelating cholesterol, significantly reduces the ability ofCampylobacter jejunito enter these cells. Furthermore inhibitors of host protein tyrosine phosphorylation, the phosphatidylinositol-3 kinase (PI 3-kinase) inhibitor wortmannin, and cholera toxin, all significantly reduced invasion of Caco-2 cells byC. jejuni

Although the rate at which we are developing a detailed understanding of the molecular pathogenesis of the campylobacters is certainly increasing, we are still only just emerging from the ‘lag’ phase and we still have some way to go before we reach’ stationary’ phase. There is a valuable base of reported work on both the toxins and invasion of host cells by campylobacter, but much of it is controversial, non-molecular or observational in nature. In contrast, the excellent work on the flagellum has done much to characterise the genetic basis of this determinant and established its importance in pathogenesis. The objective of this workshop was to present work that addressed all stages of the process of infection by campylobacters. Our aim was to achieve a structured overview of the abstracts as an update and also to facilitate the generation of ideas and hypotheses for the many as yet unclear aspects of the infection process.

Bacterial infection strategies can be described as adaptations by the pathogen to a series of microenvironments within the host. In order to succeed, bacteria must constantly adjust their survival and growth in the surrounding conditions. This is achieved by utilising signal transduction systems, including the two-component histidine protein kinase (HK)/response regulator (RR) systems which are able to detect stimuli, process them and direct the production of adequate cellular responses.

Workshop A1: Detection and Culture. Workshop A2: Epidemiology, Clinical Aspects and Treatment of Helicobacters. Workshop A3: Genetics Methods. Workshop B1: Pathogenic Mechanisms of Campylobacters Workshop B3: Epidemiology of Campylobacters. Workshop C2: Immunology and Vaccines of Helicobacters. Workshop C3: Poultry Infections and Their Control. Workshop D1: Clinical Aspects of Campylobacters. Workshop E1: Veterinary Infections. Workshop E3: Detection of Campylobacters in Foods and the Environment. Workshop F3: Antimicrobial Resistance. Workshop G1: Immunology and Vaccine of Campylobacters. Workshop G2: New and Emerging Pathogens. Workshop G3: Speciation and Subtyping. Indexes.

Some strains of C. jejuni are able to enter a variety of eukaryotic cells in culture and this characteristic is thought to reflect the ability of the organism to invade host cells in the gut. This ability is believed to be an important determinant of virulence for these organisms4,6,7,9. In order to study the mechanisms by which campylobacters enter into eukaryotic cells several groups have used a number of different cell lines and strains of C. jejuni. Evidence has been presented for an involvement in endocytosis of microfilaments6, microtubules and clathryn-coated pits7 and an energy dependent mechanism involving none of these host components9. Nothing is currently known of the signal transduction pathways involved in induced uptake of cell associated C. jejuni into eukaryotic cells.

In response to stress, bacteria upregulate specific groups of proteins which maintain the integrity of the cell; one such protein is HtrA, a serine protease. HtrA production in different bacteria appears to be induced by distinct environmental stress conditions, eg. in Escherichia coli the htrA gene is essential for bacterial survival at high temperatures7, whereas a Salmonella typhimurium htrA mutant was temperature insensitive, but susceptible to oxidative stress5. A Bruceila abortus htrA mutant is both temperature and oxidative stress sensitive4. The HtrA family of stress-response proteins may also be important in bacterial pathogenicity as the S. typhimurium and B. abortus mutants are attenuated for mouse virulence4,5. The mutant’s susceptibility to oxidative stress suggests increased sensitivity to oxidative killing within macrophages.

Campylobacter upsaliensis is fast becoming recognised as a significant human pathogen, causing, amongst other symptoms, diarrhoea, bacteraemia and respiratory problems. The organism was first described by Sandstedt et al. 8 from dogs with or without diarrhoea and later from asymptomatic cats by Fox et al. 1. In subsequent studies, C. upsaliensis was isolated from human faecal samples3,6,9,11,12, human blood5,7,11, a breast abscess2 and foetoplacental tissues4. However, relatively little is known about the virulence determinants of C. upsaliensis, and the mechanisms by which it causes disease remain unclear.

The mechanisms of the pathogenesis of Campylobacter jejuni/coli remain largely unknown. Current approaches to the identification of virulence and colonisation factors frequently involve producing a gene knockout mutant, which then requires testing in in vivo or in vitro models. Unfortunately, there is to date no model of human campylobacteriosis. However, the avian gastro-intestinal tract is considered the natural environment of the thermophilic campylobacters. The true importance of these genes can then be seen by testing the deletion mutants in vivo. The chick, therefore, represents an appropriate model for testing genetically-engineered mutants for their ability to colonise and survive in vivo.

OBJECTIVES--To identify and rank the factors that currently limit the use of thrombolytic treatment in patients admitted to hospital with acute myocardial infarction. DESIGN--Weighted sampling study with retrospective data retrieval from clinical records. SETTING--All hospitals within the Trent region providing acute general medical services. PATIENTS--Random sample of 420 patients admitted during February-April 1993 who had acute myocardial infarction as the main discharge diagnosis. MAIN OUTCOME MEASURES--Treatment odds ratios (and 95% confidence intervals (CI)) for the use of thrombolysis in patient groups defined by relevant clinical characteristics. RESULTS--The patient population was older and less likely to have ST segment elevation on the initial electrocardiogram than patients entered into the randomised trials of thrombolysis. Thrombolytic treatment was given to 49% of patients (SE 2.4%). After controlling for negative associations with a history of stroke (treatment odds ratio 0.18 (95% CI 0.04 to 0.53)) and peptic ulcer (odds ratio 0.52 (95% CI 0.26 to 1.01)) use of thrombolysis decreased with increasing patient age. This was particularly noticeable for those aged > 74 years (odds ratio 0.17 (95% CI 0.05 to 0.51)) relative to those aged < 65 years. Thrombolysis was less likely to be used in patients with ST depression (odds ratio 0.22 (95% CI 0.11 to 0.41)) or bundle branch block (odds ratio 0.18 (95% CI 0.07 to 0.44)) than in those with ST elevation on the initial electrocardiogram. Delay from symptom onset to admission was more than 12 h in 15% of patients. CONCLUSIONS--The patient population admitted to hospital with acute myocardial infarction differs in several respects from the samples that have been included in the trials of thrombolysis. The main factors limiting wider use of thrombolysis are diagnostic uncertainty at admission and delayed presentation. Perceived clinical contraindications to treatment are of lesser importance. There is evident reluctance to use thrombolytic treatment in older patients, who were substantially under-represented in the clinical trials.

Aims Each year approximately 65,000 young people present to Emergency Departments (ED) as a result of alcohol intoxication. There is a large body of evidence in the written literature, detailing ways in which ED9s can use these presentations to help identify young people in need and intervene to reduce future alcohol related presentations. We performed a service evaluation to explore how EDs in London determine if a young person presenting intoxicated is at risk from their drinking, or drinking because of other co-morbidities, and what interventions are available to support the young person. We asked the paediatric lead for each of the 32 ED9s in the Greater London area to complete a short paper questionnaire. We hypothesised that the 16-17 year old group may be particularly at risk due to age thresholds between paediatric and adult services, and specifically asked questions to assess this risk. Results 22/32 ED9s completed the questionnaire (69%). Assessment of risk Alcohol screening tools were more common in adults (55%) than children under 16 (9%). Structured psychosocial histories were routinely taken in 7% units. Safeguarding practices 41% of ED9s always discuss patients under 16 in safeguarding meetings compared to 23% for patients aged 16-17 years. For under 169s, 71% of ED9s shared information with the patients GP, compared to 64% for 16-179s. For patients less than 16, 71% shared information with school nurses; 43% for those 16-17. Treatment/support Information about alcohol services was available in 73% ED9s. Referral pathways to drug and alcohol services were similar between adults and young people, 41-45%. Conclusion There appears to be inadequate screening and discussion of young people who present intoxicated to the ED and vulnerable young people could be missed. In particular we noted that the 16-17 age groups are potentially vulnerable as they fall in the gap between adult and child services. Where they exist, policies are variable and we call for college level guidelines to be developed and disseminated.

The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions. We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V. cholerae O1 strains. However, if the donor V. cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR. The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.

Little is known about incorporation of new knowledge from randomised clinical trials into clinical practice. Thrombolytic therapy was shown to reduce the mortality of acute myocardial infarction in several large trials published during 1986-88. To examine the effect of these data on clinical practice, we analysed the supply of thrombolytic drugs in a representative English region (population 4·7 million) in 1987-92. During the study period there were over 10 000 hospital admissions per year in the region for acute myocardial infarction. From a very low initial level, thrombolytic drug use rose slowly for several years after publication of the trial results and reached a plateau in 1991-92. Rates of use per 1000 patients admitted with myocardial infarction varied almost six-fold between districts in 1989-90 and over two-fold in 1991-92. Level of use attained by districts in the latter period was strongly associated with the extent of their previous participation in multicentre trials of thrombolysis (p=0·003); we estimate that 35-50% of patients admitted with acute myocardial infarction were receiving thrombolytics. The full potential of thrombolytic treatment has still not been achieved in routine care and the limiting factors need to be defined.

Attenuated Vibrio cholerae O1 vaccine strains lacking the gene encoding the A subunit of cholera toxin have proven efficacious in preventing experimental cholera. As these strains move from closed, contained testing environments to large-scale field trials, a readily assayable phenotypic trait to distinguish a vaccine strain from wild-type V. cholerae O1 is desirable. We have constructed three derivatives of the attenuated V. cholerae strain CVD 103 which carry a mercury resistance or urease marker in the hlyA gene. CVD 103-HgR was constructed using a protracted marker-exchange procedure; this strain was found to have somewhat lowered colonisation efficiency in infant mice in comparison to its parent strain, CVD 103. The insertion of the resistance marker was repeated using a suicide vector system; CVD 103-HgR2 was found to colonise infant mice as efficiently as CVD 103. Strain CVD 103-UR, in which sequences encoding urease were inserted using a suicide vector, also colonised infant mice as well as CVD 103. The genetically marked strains CVD 103-HgR, CVD 103-HgR2 and CVD 103-UR form the basis for a generation of defined oral vaccines that may give single-dose, long-lasting protection to populations at risk from cholera.

Summary Four strains of Campylobacter jejuni isolated from children with inflammatory diarrhoea were assayed in the rabbit ileal loop model of infectious diarrhoea. All caused inflammatory reactions with severe macroscopic and microscopic damage in infected rabbit ileal tissue similar to that observed in the patients by endoscopy and histological analysis of colonic biopsies. Haemoglobin and other proteins were observed in loop fluids, consistent with leakage of serum from damaged mucosa. Loop fluids also contained significant bicarbonate concentrations, indicative of an active secretory component similar to that in control loops inoculated with cholera toxin. However, although three of the four clinical strains produced small amounts of a protein immunologically related to cholera toxin in vitro, none such was detected in either tissues or fluids of infected ileal loops. We propose instead that host-derived mediators of secretion may be important in pathogenesis. A mutant strain of C. jejuni with impaired motility, obtained from the National Collection of Type Cultures, did not induce tissue damage or fluid secretion in rabbit ileal loops.

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.