Salmonella Paratyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever.

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers.

The Escherichia coli gene pool.

Wild Escherichia coli are superbly adapted to survive in the intestines of their mammalian hosts and in the environment. E. coli K12 derivative (MG1655) encodes 4288 potential genes that provide the background genetic framework of this species. Particular E. coli clonal types encode additional chromosomal and extrachromosomal genes that facilitate the ability of E. coli to adapt to new environments. These additional genes are often clustered, have related functions (for example, virulence-associated genes in pathogenicity islands) and may be integrated at specific sites on the E. coli chromosome.

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

Effect of low phosphorus diets on intestinal calcium absorption and the concentration of calcium-binding protein in intact and parathyroidectomized pigs.

The effect of changing the dietary concentration of phosphorus on the intestinal absorption of calcium has been studied in conscious pigs each prepared with a Thiry--Vella loop of jejunum. A reduction in the percentage of phosphorus in the diet from 0.7 to 0.3% caused an increase in the efficiency of absorption of calcium from the fluid used to perfuse the jejunal loop in both intact and parathyroidectomized animals. There was a marked increase in the amount of calcium-binding protein (CaBP) in the small intestine of pigs fed the low phosphrous diet. Parathyroidectomy did not affect the amount of CaBP in the small intestine when either the normal or the low phosphorus diets were fed.

Measurement of parathyroid hormone-related protein in extracts of fetal parathyroid glands and placental membranes.

A radioimmunoassay based on an antiserum to human parathyroid hormone-related protein PTHrP(1-16) was used with PTHrP(1-34) standard to measure the concentration of immunoreactive PTHrP in extracts of fetal parathyroid glands from lambs and calves and also placental membranes obtained from several species, including man. Dilution curves from these sources were parallel to those obtained for PTHrP(1-34) standard. It was demonstrated that this parallelism was not the result of tracer damage caused by enzymic activity in the tissue extracts. Extracts of human placental membranes were subjected to high-pressure liquid chromatography with a linear acetonitrile gradient. Co-elution of cytochemical biological activity with 125I-labelled PTHrP(1-34) was noted. These results provide further evidence for both the fetal parathyroid glands and the placenta containing material resembling PTHrP which may be responsible for sustaining the activity of the placental calcium pump which maintains the fetus hypercalcaemic relative to its mother.

The transfer of calcium during perfusion of the placenta and intact and thyroparathyroidectomized sheep.

Placental perfusions were carried out in six ewes during the last two weeks of gestation. Perfusions were carried out using autologous fetal blood and the flow rates adjusted to give a perfusion pressure of 50--70 mmHg. Perfusion plasma calcium concentrations rose steadily throughout the perfusions achieving a mean increase of 1.65 mmol/1 above initial concentration within 100 minutes. A further three ewes in the last two weeks of gestation were thyroparathyroidectomized and normal plasma calcium concentration maintained by an intravenous infusion of calcium borogluconate. After three days, placental perfusions were carried out as before. The mean perfusion plasma calcium concentration achieved by those three ewes in a period of 100 minutes showed an increase of 1.25 mmol/1. It is concluded that the presence of the fetus is not necessary for the continued active transfer of calcium across the placenta from mother to fetus. The reduced rate of accumulation of calcium in the perfusate in TXPTX ewes is attributed to a decline in 1,25-DHCC concentrations in both maternal and fetal circulations. The implications of these results in relation to fetal calcium homeostasis and the placental transfer of calcium are discussed.

The effect of fetal thyroparathyroidectomy on the transport of calcium across the ovine placenta to the fetus.

The ovine fetal placenta has been perfused with autologous fetal blood under controlled conditions in eleven experiments in which the fetus was first removed. Eight of these experiments involved four pairs of twins, one lamb of which had been thyroparathyroidectomized (TXPTX) three to seven days earlier. By this time the normal placental calcium gradient from mother to fetus had either decreased or been reversed. The mean rate of transport of calcium from the mother was unchanged by previous fetal TXPTX, but the final calcium gradient achieved from the mother to the perfusing blood was significantly less than with placentae from intact fetuses. No significant alteration in fetal plasma I,25-dihydroxyvitamin D (I,25(OH)2D) concentration was observed as a result of the fetal TXPTX, indicating that hypocalcaemia can compensate for the lack of PTH in fetal production of I,25(OH)2D. Fetal thyroidectomy with replacement of thyroxine did not lead to reversal of the placental calcium gradient, indicating that calcitonin was not involved. It is suggested that in the ovine fetus, parathyroid hormone promotes the active transport of calcium from mother to fetus, so that in its absence the fetus must obtain its calcium for growth by reducing its calcaemia and thereby allow net diffusion of calcium to replace the action of the placental calcium pump. The price paid for this compensation is marked hypocalcaemia and defective calcification of osteoid.

Optical measurement of three-dimensional collagen gel constructs by elastic scattering spectroscopy.

Analysis of the formation and organization of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology. We have analyzed early-stage responses in collagen lattices, using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions. The method is based on an optical system in which an optical fiber delivers white light onto the tissue and the back-scattered light is collected for spectroscopy by another optical fiber. Results show correlation between changes in the spectral signatures with changes in the collagen gel contraction or internal organization in all three models of collagen construct analyzed. Therefore elastic scattering spectroscopy is a promising tool to monitor tissue-engineered constructs or early repair in collagenous tissues.

Neutralisation of immunologically distinct staphylococcal delta haemolysins by antibodies.

Antibodies against delta-haemolysin from Staphylococcus strain CN4108 (Newman) did not neutralise delta-haemolysin from the canine strain of S. aureus CN7450 to the same extent as delta-haemolysin prepared from S. aureus strain CN4108. This is additional evidence for the immunological distinctness of delta-haemolysin from these two S. aureus strains of human and canine origin.

Diagnosis of breast cancer using elastic-scattering spectroscopy: preliminary clinical results.

We report on the first stages of a clinical study designed to test elastic-scattering spectroscopy, mediated by fiberoptic probes, for three specific clinical applications in breast-tissue diagnosis: (1) a transdermal-needle (interstitial) measurement for instant diagnosis with minimal invasiveness similar to fine-needle aspiration but with sensitivity to a larger tissue volume, (2) a hand-held diagnostic probe for use in assessing tumor/resection margins during open surgery, and (3) use of the same probe for real-time assessment of the "sentinel" node during surgery to determine the presence or absence of tumor (metastatic). Preliminary results from in vivo measurements on 31 women are encouraging. Optical spectra were measured on 72 histology sites in breast tissue, and 54 histology sites in sentinel nodes. Two different artificial intelligence methods of spectral classification were studied. Artificial neural networks yielded sensitivities of 69% and 58%, and specificities of 85% and 93%, for breast tissue and sentinel nodes, respectively. Hierarchical cluster analysis yielded sensitivities of 67% and 91%, and specificities of 79% and 77%, for breast tissue and sentinel nodes, respectively. These values are expected to improve as the data sets continue to grow and more sophisticated data preprocessing is employed. The study will enroll up to 400 patients over the next two years.

Measuring the effectiveness of cataract surgery: the reliability and validity of a visual function outcomes instrument.

AIMS: To assess test-retest reliability and validity of the "TyPE" patient self assessed visual function questionnaire, as part of a study in two hospitals measuring the effectiveness of cataract surgery. The American TyPE questionnaire had minor adaptations made for use in Britain. METHODS: Test-retest reliability was assessed on 63 out of 378 adult cataract surgery patients in the study, using Spearman correlation coefficients and kappa coefficients of agreement. "Construct" validity was evaluated by comparing the association between changes in visual function questionnaire scores after surgery, with patients' perception of change in visual function obtained by independent interview of 24 patients. RESULTS: The TyPE questionnaire items showed very good test-retest reliability. Average Spearman and kappa coefficients for 39 patients from hospital 1 were 0.93 and 0.84 respectively. Spearman and kappa coefficients of 0.9 and 0.81 were obtained for those nine patients in hospital 2 where both the test and retest questionnaires were filled in by the same people. However, for the 15 patients from hospital 2, where the questionnaire was filled in by different people in the retest, reliability was less good: the Spearman coefficients were still high, average 0.72, but the kappa coefficients were poor, 0.27. Good construct validity was exhibited, with a correlation of 0.79 between change in distance vision score from the questionnaires and the independent interview. CONCLUSIONS: The adapted TyPE questionnaire is both very reliable and has good construct validity. The kappa coefficient should be used wherever possible to evaluate reliability. The test-retest reliability and validity and practicability of other visual function questionnaires have not been assessed adequately, and further development should be carried out of all such questionnaires, so that they may be introduced into routine clinical care.

Detection of cervical intranodal metastasis in oral cancer using elastic scattering spectroscopy.

While histopathology remains the gold standard for tissue diagnosis, several new diagnostic techniques are being developed that rely on physical and biochemical changes that mirror or precede malignant change within tissue. The aim of this study was to compare findings of elastic scattering spectroscopy (ESS) with histopathology on formalin fixed neck dissection specimens to see if this technique could be used as an adjunct or alternative to histopathology in defining nodal involvement. The technique involves the use of Mie scattering and is a simple non-invasive method of tissue interrogation. One hundred and thirty lymph nodes were examined from 13 patients who underwent neck dissection. The nodes were formalin fixed, bivalved and examined by ESS using a pulsed xenon lamp. The intensity of the spectrum at 4 points was considered for comparison; at 360, 450, 630 and 690 nm. The nodes were then routinely processed and haematoxylin and eosin-stained sections examined histopathologically, and the results compared. Using this technique, a sensitivity of 98% and a specificity of 68% were obtained.

Development of Attenuated Salmonella Strains that Express Heterologous Antigens.

The ability of attenuated strains of Salmonella to induce humoral, secretory, and cellular immune responses following oral ingestion has made them attractive as a system for delivering foreign antigens to the mammalian immune system. DNA capable of driving the expression of heterologous antigens can be introduced into Salmonella vaccine strains using a variety of approaches. In general, there are two common methods of expressing a foreign antigen in salmonellae: from plasmid vectors or from the bacterial chromosome. Since there are many similarities in the cellular and molecular biology of Escherichia coli and Salmonella, most of the genetic manipulations required to construct expression cassettes can be carried out in E. coli. The resulting constructs can then be introduced into the vaccine strains using simple transformatron or other similar techniques. However, the laboratory manipulation of Salmonella strains should be undertaken using techniques that do not lead to the accumulation of undefined genetic lesions, which may compromise the immunogenicity of Salmonella growing in vivo. With this in mind, we will describe appropriate techniques for manipulating Salmonella with the aim of constructing effective oral vaccines.

Do out-of-hours co-operatives improve general practitioners' health?

General practitioners (GPs) have been found to have a higher level of anxiety and depression then hospital managers and consultants. In 1995 and 1998, we surveyed GPs in Buckinghamshire. We found that the development of out-of-hours co-operatives was an important factor in the improvement in GPs' health status.

The measurement of transplacental magnesium fluxes in the sheep.

Bi-directional transport rates of magnesium across the placenta of the ewe have been measured towards the end of pregnancy. The results indicated that approximately 0.042 and Mg/h/kg fetal body weight passed from ewe to fetus and that about 0.012 mg Mg/h/kg fetal body weight was transported back to the ewe. There was a significant, although small, positive gradient in plasma magnesium concentration from ewe to fetus.

The use of out of hours health services: a cross sectional survey.

OBJECTIVES: To determine the use and costs of the principal out of hours health services in Buckinghamshire. DESIGN: Prospective cross sectional survey and cost description of patient contacts with out of hours services. SETTING: Buckinghamshire during March and April 1995. SUBJECTS: General practices, accident and emergency departments, ambulance services, and community nursing services. MAIN OUTCOME MEASURE: Contacts with patients and cost of out of hours services. RESULTS: 438 patient contacts/1000 population/year were recorded at an annual incremental cost of between 4.6 m Pounds and 7.2 m Pounds (depending on the costing of general practitioner services), for a population of 660,000. Of these contacts, 21,649 (45%) were with general practitioners. Night time contacts with all services diminished sharply after 10 pm. General practitioners considered that 40% of contacts were unnecessary or could have waited until morning. Over 70% of contacts were for upper respiratory tract infections, earache, gastroenteritis, and other minor ailments. Nursing care was predominantly for elderly people, and 33% of nursing contacts were to supervise medication. Accident and emergency care was predominantely for young adults, especially men, and 41% of attendances were for medical conditions. CONCLUSIONS: New models such as multidisciplinary primary care centres with telephone advice lines and triaging are required to ensure high quality, cost effective care that is responsive to the needs of both consumers and professionals.

Molecular cloning and analysis of P. 69, a vir-controlled protein from Bordetella pertussis.

The cloning sequencing and analysis of an important antigenic component of Bordetella pertussis is described. The gene for P.69, in common with a variety of other so called "virulence" genes, (e.g., adenylate cyclase (AC), pertussis toxin (PT) and filamentous haemagglutinin (FHA)), is under control of the vir locus. The protein P.69 is externally localised on cells and protein preparations are protective as judged by the mouse intra-cerebral challenge test. The gene encoding the P.69 antigen was isolated by hybridization of mixed oligonucleotide probes against B. pertussis genomic DNA. These oligonucleotides were designed from the protein sequence data obtained from a cyanogen bromide digest of the P.69 protein. DNA sequence analysis reveals a G:C rich gene capable of encoding a protein of 910 amino acids and Mr of 93478, whose likely promoter and ribosome binding sites show little homology to their E.coli counterpart. In common with some of the genes in the PT operon the sequence 5'-CCTGG-3' was found 5' to the ATG initiation codon. At the 3', end 29 bases after the TAA stop codon, the sequence 5'GTTTTTCCT-3' was found in an equivalent position to the same sequence in the PT operon. Examination of the protein sequence reveals two regions with directly repeated elements (GGAVP)3(GGFGP)2 and (PQP)5.

Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine.

Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge. pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable. Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge. Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies. After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge. Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.