A hyperacute immune map of ischaemic stroke patients reveals alterations to circulating innate and adaptive cells.

Systemic immune changes following ischaemic stroke are associated with increased susceptibility to infection and poor patient outcome due to their role in exacerbating the ischaemic injury and long-term disability. Alterations to the abundance or function of almost all components of the immune system post-stroke have been identified, including lymphocytes, monocytes and granulocytes. However, subsequent infections have often confounded the identification of stroke-specific effects. Global understanding of very early changes to systemic immunity is critical to identify immune targets to improve clinical outcome. To this end, we performed a small, prospective, observational study in stroke patients with immunophenotyping at a hyperacute time point (< 3 h) to explore early changes to circulating immune cells. We report, for the first time, decreased frequencies of type 1 conventional dendritic cells (cDC1), haematopoietic stem and progenitor cells (HSPCs), unswitched memory B cells and terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA). We also observed concomitant alterations to human leucocyte antigen D-related (HLA-DR), CD64 and CD14 expression in distinct myeloid subsets and a rapid activation of CD4+ T cells based on CD69 expression. The CD69+ CD4+ T cell phenotype inversely correlated with stroke severity and was associated with naive and central memory T (TCM) cells. Our findings highlight early changes in both the innate and adaptive immune compartments for further investigation as they could have implications the development of post-stroke infection and poorer patient outcomes.

The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans.

A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.

A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans.

This paper describes the fabrication and use of a microfluidic device for performing whole-animal chemical screens using non-invasive electrophysiological readouts of neuromuscular function in the nematode worm, C. elegans. The device consists of an array of microchannels to which electrodes are attached to form recording modules capable of detecting the electrical activity of the pharynx, a heart-like neuromuscular organ involved in feeding. The array is coupled to a tree-like arrangement of distribution channels that automatically delivers one nematode to each recording module. The same channels are then used to perfuse the recording modules with test solutions while recording the electropharyngeogram (EPG) from each worm with sufficient sensitivity to detect each pharyngeal contraction. The device accurately reported the acute effects of known anthelmintics (anti-nematode drugs) and also correctly distinguished a specific drug-resistant mutant strain of C. elegans from wild type. The approach described here is readily adaptable to parasitic species for the identification of novel anthelmintics. It is also applicable in toxicology and drug discovery programs for human metabolic and degenerative diseases for which C. elegans is used as a model.

Photosynthetic pathway and ecological adaptation explain stomatal trait diversity amongst grasses.

• The evolution of C(4) photosynthesis in plants has allowed the maintenance of high CO(2) assimilation rates despite lower stomatal conductances. This underpins the greater water-use efficiency in C(4) species and their tendency to occupy drier, more seasonal environments than their C(3) relatives. • The basis of interspecific variation in maximum stomatal conductance to water (g(max) ), as defined by stomatal density and size, was investigated in a common-environment screening experiment. Stomatal traits were measured in 28 species from seven grass lineages, and comparative methods were used to test for predicted effects of C(3) and C(4) photosynthesis, annual precipitation and habitat wetness on g(max) . • Novel results were as follows: significant phylogenetic patterns exist in g(max) and its determinants, stomatal size and stomatal density; C(4) species consistently have lower g(max) than their C(3) relatives, associated with a shift towards smaller stomata at a given density. A direct relationship between g(max) and precipitation was not supported. However, we confirmed associations between C(4) photosynthesis and lower precipitation, and showed steeper stomatal size-density relationships and higher g(max) in wetter habitats. • The observed relationships between stomatal patterning, photosynthetic pathway and habitat provide a clear example of the interplay between anatomical traits, physiological innovation and ecological adaptation in plants.

Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes.

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to d-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source.

Chemistry and the worm: Caenorhabditis elegans as a platform for integrating chemical and biological research.

This Review discusses the potential usefulness of the worm Caenorhabditis elegans as a model organism for chemists interested in studying living systems. C. elegans, a 1 mm long roundworm, is a popular model organism in almost all areas of modern biology. The worm has several features that make it attractive for biology: it is small (<1000 cells), transparent, and genetically tractable. Despite its simplicity, the worm exhibits complex phenotypes associated with multicellularity: the worm has differentiated cells and organs, it ages and has a well-defined lifespan, and it is capable of learning and remembering. This Review argues that the balance between simplicity and complexity in the worm will make it a useful tool in determining the relationship between molecular-scale phenomena and organism-level phenomena, such as aging, behavior, cognition, and disease. Following an introduction to worm biology, the Review provides examples of current research with C. elegans that is chemically relevant. It also describes tools-biological, chemical, and physical-that are available to researchers studying the worm.

Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans.

This article describes the fabrication of a microfluidic device for the liquid culture of many individual nematode worms (Caenorhabditis elegans) in separate chambers. Each chamber houses a single worm from the fourth larval stage until death, and enables examination of a population of individual worms for their entire adult lifespans. Adjacent to the chambers, the device includes microfluidic worm clamps, which enable periodic, temporary immobilization of each worm. The device made it possible to track changes in body size and locomotion in individual worms throughout their lifespans. This ability to perform longitudinal measurements within the device enabled the identification of age-related phenotypic changes that correlate with lifespan in C. elegans.

Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devices.

This paper describes a method for prefabricating screw, pneumatic, and solenoid valves and embedding them in microfluidic devices. This method of prefabrication and embedding is simple, requires no advanced fabrication, and is compatible with soft lithography. Because prefabrication allows many identical valves to be made at one time, the performance across different valves made in the same manner is reproducible. In addition, the performance of a single valve is reproducible over many cycles of opening and closing: an embedded solenoid valve opened and closed a microfluidic channel more than 100,000 times with no apparent deterioration in its function. It was possible to combine all three types of prefabricated valves in a single microfluidic device to control chemical gradients in a microfluidic channel temporally and spatially.

Using ratchets and sorters to fractionate motile cells of Escherichia coli by length.

This paper describes the fabrication of a composite agar/PDMS device for enriching short cells in a population of motile Escherichia coli. The device incorporated ratcheting microchannels, which directed the motion of swimming cells of E. coli through the device, and three sorting junctions, which isolated successively shorter populations of bacteria. The ratcheting microchannels guided cells through the device with an average rate of displacement of (32 +/- 9) microm s(-1). Within the device, the average length of the cells decreased from 3.8 microm (Coefficient of Variation, CV: 21%) at the entrance, to 3.4 microm (CV: 16%) after the first sorting junction, to 3.2 mum (CV: 19%) after the second sorting junction, to 3.0 mum (CV: 19%) after the third sorting junction.

Prognosis in patients with transient ischaemic attack (TIA) and minor stroke attending TIA services in the North West of England: the NORTHSTAR Study.

BACKGROUND: The ABCD2 score predicts stroke risk within a few days of transient ischaemic attack (TIA). It is not clear whether the predictive value of the ABCD2 score can be generalised to UK TIA services, where delayed presentation of TIA and minor stroke are common. We investigated prognosis, and the use of the ABCD2 score, in patients attending TIA services in the North West of England with a diagnosis of TIA or minor stroke. METHODS: 711 patients with TIA or minor stroke were prospectively recruited from five centres (median duration from index event to recruitment 15 days). The primary outcome was the composite of incident TIA, stroke, acute coronary syndrome or cardiovascular death at the 3 month follow-up. Prognostic factors were analysed using Cox proportional hazards regression. RESULTS: The primary outcome occurred in 126 (18%) patients. Overall, there were 30 incident strokes. At least one incident TIA occurred in 100 patients (14%), but only four had a subsequent stroke. In multifactorial analyses, the ABCD2 score was unrelated to the risk of the primary outcome, but predicted the risk of incident stroke: score 4-5: hazard ratio (HR) 3.4 (95% CI 1.0 to 12); score 6-7: HR 4.8 (1.3 to 18). Of the components of the ABCD2 score, unilateral motor weakness predicted both the primary outcome (HR 1.8 (1.2 to 2.8)) and stroke risk (HR 4.2 (1.3 to 14)). CONCLUSIONS: In patients attending typical NHS TIA services, the risk of incident stroke was relatively low, probably reflecting delays to assessment. Current provision of TIA services, where delayed presentation to "rapid access" TIA clinics is common, does not appear to provide an appropriate setting for urgent evaluation, risk stratification or timely secondary prevention for those who may be at highest risk.

A microfabricated array of clamps for immobilizing and imaging C. elegans.

This paper describes the fabrication of a microfluidic device for rapid immobilization of large numbers of live C. elegans for performing morphological analysis, microsurgery, and fluorescence imaging in a high-throughput manner. The device consists of two principal elements: (i) an array of 128 wedge-shaped microchannels, or clamps, which physically immobilize worms, and (ii) a branching network of distribution channels, which deliver worms to the array. The flow of liquid through the device (driven by a constant pressure difference between the inlet and the outlet) automatically distributes individual worms into each clamp. It was possible to immobilize more than 100 worms in less than 15 min. The immobilization process was not damaging to the worms: following removal from the array of clamps, worms lived typical lifespans and reproduced normally. The ability to monitor large numbers of immobilized worms easily and in parallel will enable researchers to investigate physiology and behavior in large populations of C. elegans.

Vasoactive intestinal peptide (VIP) prevents killing of virulent and phoP mutant Salmonella typhimurium by inhibiting IFN-gamma stimulated NADPH oxidative pathways in murine macrophages.

Vasoactive intestinal peptide is an immunomodulator with great potential in the treatment of inflammatory pathology. In this study, we have examined the effect of VIP on the growth dynamics of virulent Salmonella enterica. Serovar typhimurium (S. typhimurium) 14028 and 4/74 and an avirulent mutant (14028 phoP) in a murine, macrophage cell line (J774.2). In contrast to standard growth dynamics, in which phoP mutants do not survive in macrophages, we show that VIP (10(-10) M) significantly enhances phoP growth over a 24 h post-infection period even when the cells are co-cultured with IFN-gamma. We examined the effect of VIP on the generation of NADPH-induced reactive oxygen species (ROS) in Salmonella-infected/IFN-gamma cultured J774 cells. VIP inhibited gp91 mRNA levels, gp91 protein and subsequent ROS. The importance of ROS in killing of Salmonella by J774 cells was highlighted by experiments in which ROS production by J774 cells was inhibited using a conventional inhibitor, N-acetyl-L-cysteine captopril (ACC) and in which Salmonella growth significantly increased. Our findings suggest that although VIP inhibits inflammatory pathways in myeloid cells it also promotes the growth of avirulent (phoP) mutants.

Insulin glargine or NPH combined with metformin in type 2 diabetes: the LANMET study.

AIMS/HYPOTHESIS: In type 2 diabetic patients we compared 9 months of combination therapy with insulin glargine and metformin with 9 months of NPH insulin combined with metformin. The primary focus was changes in HbA(1c); secondary focus was diurnal glucose profiles and symptomatic hypoglycaemia. METHODS: In this investigator-initiated open, parallel-group clinical trial involving seven centres, 110 insulin-naive type 2 diabetic patients with poor glycaemic control (HbA(1c) >or=8.0%) on oral hypoglycaemic agents (90% using sulfonylurea plus metformin) were randomised to receive bedtime insulin glargine with metformin (G+MET) or bedtime NPH with metformin (NPH+MET) for 36 weeks. The patients were taught how to self-adjust their insulin dose and use a modem to send the results of home glucose monitoring to treatment centres. The goal was to achieve a fasting plasma glucose (FPG) of 4.0 to 5.5 mmol/l in both groups. RESULTS: During the last 12 weeks, FPGs averaged 5.75+/-0.02 and 5.96+/-0.03 mmol/l (p<0.001) and insulin doses were 68+/-5 and 70+/-6 IU/day (0.69+/-0.05 and 0.66+/-0.04 IU kg(-1) day(-1), NS) in the G+MET and NPH+MET groups, respectively. At 36 weeks, mean HbA(1c) was 7.14+/-0.12 and 7.16+/-0.14%, respectively (NS). Symptomatic, but not confirmed symptomatic, hypoglycaemia was significantly lower during the first 12 weeks in the G+MET group (4.1+/-0.8 episodes/patient-year) than in the NPH+MET group (9.0+/-2.3 episodes/patient-year, p<0.05), but not significantly different thereafter. Glucose levels before dinner were higher in the NPH+MET group (10.1+/-0.3 mmol/l) than in the G+MET group (8.6+/-0.3 mmol/l, p=0.002) throughout the 36-week study. With regard to baseline characteristics such as initial glycaemia or C-peptide, there was no difference between patients who achieved good glycaemic control (HbA(1c) <7.0%) and those who did not. Differences were seen in the following: between study centres, weight gain during the run-in period and insulin therapy, and FPG during the last 12 weeks (5.7+/-0.2 vs 6.7+/-0.3 mmol/l for patients reaching vs those not reaching target, p<0.01). CONCLUSIONS/INTERPRETATION: Good glycaemic control can be achieved with both G+MET and NPH+MET. Use of G+MET reduces symptomatic hypoglycaemia during the first 12 weeks and dinner time hyperglycaemia compared with NPH+MET.

Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge.

Salmonella enterica remains one of the most important food-borne pathogens of humans and is often acquired through consumption of infected poultry meat or eggs. Control of Salmonella infections in chicken is therefore an important public health issue. Infection with S. enterica serovar Typhimurium results in a persistent enteric infection without clinical disease in chickens of more than 3 days of age, and represents a source for contamination of carcass at slaughter and entry into the human food chain. Data presented indicate a profound effect of age at initial exposure on the persistence of infection and a lesser effect on the development of effective immunity to re-challenge. The percentage of birds positive for Salmonella was high until 8-9 weeks of age, regardless of the age at which the birds were infected (1, 3 or 6 weeks). The birds infected at 3 and 6 weeks of age produced a more rapid and higher antibody response (IgY and IgA) than those infected at 1 week of age, but in all cases infection persisted for a considerable period despite the presence of high antibody levels. Following a re-challenge infection with S. Typhimurium, all three previously-infected groups had fewer bacteria in the gut, spleen and liver compared with age-matched birds receiving a parallel primary infection. However, the birds primary infected at 3 and 6 weeks of age cleared infection more rapidly than those infected at a younger age. Interestingly older-primed birds had higher specific T lymphocyte proliferative responses and specific circulating levels of IgY antibody at time of re-challenge. Although birds initially infected at 1 week of age and those that were previously uninfected produced a stronger antibody response following re-challenge, they were slower to clear Salmonella from the gut than the older-primed groups which expressed a stronger T lymphocyte response. The data presented indicate that clearance of Salmonella from the gut is age-dependent and we propose that this relates to the increased competence of the enteric T cell response. The findings that Salmonella persists beyond 8-9 weeks, irrespective of age at exposure, has implications for the broiler sector and indicates the need to remain Salmonella free throughout the rearing period. Moreover, the re-challenge data demonstrates that infection at a young age is less effective in producing protective immunity than in older chickens. This feature of the development of protective immunity needs to be considered when developing vaccines for the broiler sector of the poultry industry.

Comparison of repaglinide vs. gliclazide in combination with bedtime NPH insulin in patients with Type 2 diabetes inadequately controlled with oral hypoglycaemic agents.

AIMS: This open-label randomized controlled clinical trial compared the effect on glycaemic control and weight gain of repaglinide vs. gliclazide combined with bedtime NPH insulin in patients with Type 2 diabetes inadequately controlled with oral hypoglycaemic therapy [HbA1c>7.0% (DCCT aligned assay, normal range 4.6-6.2%)]. METHODS: Eighty subjects with Type 2 diabetes were randomized to 13 weeks' open-label treatment with repaglinide 4 mg t.i.d. or gliclazide 160 mg b.i.d. in combination with bedtime NPH insulin (initial dose 0.5 units/kg). The fasting blood glucose (FBG) target was < or =6.0 mmol/l. RESULTS: Baseline characteristics were similar for age, sex, weight, BMI, FBG and HbA1c. Glycaemic control improved similarly in both groups-insulin/gliclazide by (mean) 1.0%, from 9.2 to 8.2% (P=0.001) and by 0.9%, from 9.4 to 8.5% in the insulin/repaglinide group (P=0.005) (P=0.83 between groups). Weight gain averaged (mean +/- sem) 4.1 +/- 0.5 and 3.4 +/- 0.4 kg in the insulin/gliclazide and insulin/repaglinide groups, respectively (P<0.0001 for both groups from baseline) (P=0.29 between groups). The mean number of hypoglycaemic episodes experienced per patient was 2.95 +/- 0.82 (insulin/gliclazide) and 2.3 +/- 0.52 (insulin/repaglinide) (P=0.81 between groups). Both treatments were associated with significant improvements in Diabetes Treatment Satisfaction [Diabetes Treatment Satisfaction Questionnaire-potential range 0 (min) to 36 (max)]; in the insulin/gliclazide group, by 4.9 +/- 1.1 points to 33.3 +/- 0.6 (P<0.0001) and by 3.0 +/- 0.9 points to 34.6 +/- 0.4 (P=0.0006) in the insulin/repaglinide group (P=0.29 between groups). CONCLUSIONS: Over 13 weeks, both repaglinide and gliclazide, when combined with bedtime NPH insulin produce similar significant improvements in glycaemic control (-1%) and similar weight gain.

Induction of antimicrobial pathways during early-phase immune response to Salmonella spp. in murine macrophages: gamma interferon (IFN-gamma) and upregulation of IFN-gamma receptor alpha expression are required for NADPH phagocytic oxidase gp91-stimulated oxidative burst and control of virulent Salmonella spp.

The effect of gamma interferon (IFN-gamma) on elevation of reactive oxygen species and the viability of virulent wild-type and avirulent mutants of Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis was studied in a murine macrophage cell line (J774.2 cells). S. enterica serovar Typhimurium 14028 phoP and a rough lipopolysaccharide mutant of S. enterica serovar Infantis 1326/28 (phi(r)) (avirulent mutants) induced NADPH phagocytic oxidase gp91 (gp91(phox)) activity and a significant (P < 0.05) elevation of reactive oxygen species within 12 h without coculture with IFN-gamma. This coincided with reduced survival of S. enterica serovar Typhimurium14028 phoP or stasis of S. enterica serovar Infantis phi(r). Fluorometric studies indicated that expression of IFN-gamma on infected J774.2 cells was not significantly (P > 0.05) elevated. However, studies with the virulent S. enterica serovar Typhimurium strains showed that a comparable level of control of bacterial numbers could only be achieved by coculture with IFN-gamma. This coincided with significant upregulation of IFN-gamma receptor alpha expression on the surface of J774.2 cells and was completely abolished by N-acetyl-L-cysteine captopril (an inhibitor of reactive oxygen species). Delay in reactive oxygen species induction due to a requirement for IFN-gamma and upregulation of IFN-gamma receptor alpha in macrophages infected with virulent salmonellae may result in greater dissemination of virulent salmonellae in host tissue.

Contribution of proton-translocating proteins to the virulence of Salmonella enterica serovars Typhimurium, Gallinarum, and Dublin in chickens and mice.

We investigated the attenuating effects of a range of respiratory chain mutations in three Salmonella serovars which might be used in the development of live vaccines. We tested mutations in nuoG, cydA, cyoA, atpB, and atpH in three serovars of Salmonella enterica: Typhimurium, Dublin, and Gallinarum. All three serovars were assessed for attenuation in their relevant virulence assays of typhoid-like infections. Serovar Typhimurium was assessed in 1-day-old chickens and the mouse. Serovar Gallinarum 9 was assessed in 3-week-old chickens, and serovar Dublin was assessed in 6-week-old mice. Our data show variation in attenuation for the nuoG, cydA, and cyoA mutations within the different serovar-host combinations. However, mutations in atpB and atpH were highly attenuating for all three serovars in the various virulence assays. Further investigation of the mutations in the atp operon showed that the bacteria were less invasive in vivo, showing reduced in vitro survival within phagocytic cells and reduced acid tolerance. We present data showing that this reduced acid tolerance is due to an inability to adapt to conditions rather than a general sensitivity to reduced pH. The data support the targeting of respiratory components for the production of live vaccines and suggest that mutations in the atp operon provide suitable candidates for broad-spectrum attenuation of a range of Salmonella serovars.

Rapid protection of gnotobiotic pigs against experimental salmonellosis following induction of polymorphonuclear leukocytes by avirulent Salmonella enterica.

Oral inoculation of 5-day-old gnotobiotic pigs with Salmonella enterica serovar Typhimurium strain F98 resulted in severe enteritis and invasive disease. Preinoculation 24 h earlier with an avirulent mutant of Salmonella enterica serovar Infantis (1326/28) completely prevented disease for up to 14 days (when the experiment was terminated). S. enterica serovar Infantis colonized the alimentary tract well, with high bacterial counts in the intestinal lumen but with almost no invasion into the tissues. Unprotected pigs had high S. enterica serovar Typhimurium counts in the intestines, blood, and major nonintestinal organs. Recovery of this strain from the blood and major organs in S. enterica serovar Infantis-protected pigs was substantially reduced despite the fact that intestinal counts were also very high. Protection against disease thus did not involve a colonization exclusion phenomenon. Significant (P < 0.05) infiltration of monocytes/macrophages was observed in the submucosal regions of the intestines of both S. enterica serovar Infantis-protected S. enterica serovar Typhimurium-challenged pigs and unprotected S. enterica serovar Typhimurium-challenged pigs. However, only polymorphonuclear neutrophils (PMNs) were observed throughout the villus, where significant (P < 0.05) numbers infiltrated the lamina propria and the subnuclear and supranuclear regions of the epithelia, indicating that PMN induction and positioning following S. enterica serovar Infantis inoculation was consistent with rapid protection against the challenge strain. Similarly, in vitro experiments using a human fetal intestinal epithelial cell line (INT 407) demonstrated that, although significantly (P < 0.05) fewer S. enterica serovar Infantis than S. enterica serovar Typhimurium organisms invaded the monolayers, S. enterica serovar Infantis induced an NF-kappaB response and significantly (P < 0.05) raised interleukin 8 levels and transmigration of porcine PMN. The results of this study suggest that attenuated Salmonella strains can protect the immature intestine against clinical salmonellosis by PMN induction. They also demonstrate that PMN induction is not necessarily associated with clinical symptoms and/or intestinal pathology.

Evaluation of a district-wide screening programme for diabetic retinopathy utilizing trained optometrists using slit-lamp and Volk lenses.

BACKGROUND: Debate exists about the optimum way to screen for diabetic retinopathy. Cameras produce a permanent record, but offer patients less choice about when and where to be screened. Optometrists offer flexibility but sensitivity and specificity of schemes have varied considerably, perhaps because of variability in screening methodology and that there is frequently no quality assurance programme. AIMS: To audit our district-wide (population 340000) screening programme for diabetic retinopathy against national targets: sensitivity > 80%, specificity > 95% and referral to review < 3 months. METHODS: Trained optometrists performed slit-lamp examination with Volk lenses (78 dioptre) with standardized reporting. Audit was by ophthalmologist with slit-lamp and Volk lenses through dilated pupils. RESULTS: We examined 872 eyes of 439 patients; 64% were normal, 29% background diabetic retinopathy, 7% sight-threatening eye disease (STED). Sixty-three percent of patients were seen within 6 months of the original screen. Of these, sensitivity for any retinopathy was 72%, specificity 77%, positive predictive value (PPV) 53%, negative predictive value (NPV) 88%. For STED, in this group, sensitivity was 87% and specificity 91%, PPV 30%, NPV 99%. Median interval referral to ophthalmological review was 11.5 weeks with 73% reviewed in under the 13-week target. Of those referred 25% received laser therapy. Eleven patients found to have referable eye disease at their initial screen were not referred to an ophthalmologist by their GP. CONCLUSIONS: We conclude that effective district-wide screening for diabetic retinopathy by optometrists using slit-lamp and Volk lenses is possible; however, only 36% of identified people with diabetes in the district were screened over a 4-year period.