The influence of cultural conditions on the expression in Salmonella typhimurium of an antigen related to cholera toxin.

The choice of strain, culture conditions, composition of medium and size of inoculum all affected the expression of a cholera-toxin-related antigen (CTRA) in Salmonella typhimurium. A previous study had shown that the number of organisms expressing CTRA in Casamino acid Yeast Extract (CYE) medium decreased between 4 h and 6 h in uninterrupted culture. In the present experiments, organisms harvested at 4-5 h were subcultured into fresh CYE medium and incubated for a further 2 h; the total number of organisms increased, and the decrease in the proportion of organisms expressing CTRA was reduced. Use of Hartley Digest Broth in place of CYE medium increased the proportion of organisms expressing CTRA in all strains tested, in both the uninterrupted and the subculture procedures. The higher the initial inoculum, the lower was the proportion of organisms expressing CTRA. The presence of the antigen in cells remained constant for about 18 h after transfer from 37 degrees C to 4 degrees C. These data have important implications for the production and purification of CTRA: they show that it was expressed during log-phase of growth, and they suggest that expression was regulated by a non-growth-limiting factor. Moreover, some avirulent strains were better producers of the antigen than virulent ones. The significance of the data is discussed in relation to the in-vivo situation.

Studies on early association of Salmonella typhimurium with intestinal mucosa in vivo and in vitro: relationship to virulence.

The abilities of six strains of Salmonella typhimurium to associate with rabbit ileal mucosa have been measured in vitro. Two were "virulent" strains (TML and W118 which are invasive and inducers of fluid secretion in rabbit ileal loops); four were "avirulent" (LT7, M206 and SL1027 which are invasive but induce negligible fluid secretion, and Thax-1 which is neither invasive nor an inducer of fluid secretion). A special organ-culture apparatus was designed to expose only the luminal surface of the mucosa to organisms. Viable counts of washed homogenised tissue taken 30 min after challenge showed that virulent strains TML and W118 and avirulent strains LT7 and M206 could not be distinguished from each other. Avirulent strain SL1027 associated less well than the other four strains, and Thax-1 associated less well than SL1027; both these strains were non-motile whereas the other four were motile. Thus, early association with gut mucosa did not discriminate all avirulent strains from the virulent strains. Qualitative examination of tissues by scanning electronmicroscopy did not detect strains LT7 and M206 on the mucosal surface whereas strains TML and W118 were readily seen, suggesting that the nature of association of virulent and avirulent strains was different. Qualitative examination by transmission electronmicroscopy of tissues challenged in vivo for 120 min showed virulent strains TML and W118 invading epithelial cells; similar events were reproduced after 120-min challenge in vitro. In contrast, invasion by avirulent strains was observed only very rarely.

Quantification of the leucocyte influx into rabbit ileal loops induced by strains of Salmonella typhimurium of different virulence.

Leucocyte influx into rabbit ileal loops, induced by strains of Salmonella typhimurium of different virulence, was assessed with 111Indium-labelled leucocytes. Strains fell into two groups on the basis of their leucotactic potential: "virulent" strains (which induced fluid secretion) caused a dose-dependent leucocyte influx; strains which did not induce fluid secretion failed to induce a significant leucocyte influx. Fluid secretion was never observed in the absence of leucocyte influx, but leucocyte influx per se did not induce fluid secretion. The phenotype of the challenge inoculum influenced fluid secretion; young log-phase organisms induced fluid secretion with a higher frequency than overnight cultures. These findings support earlier evidence implicating leucocytes in an interactive but not exclusive role in the genesis of salmonella-induced fluid secretion. They suggest, though do not prove, that interaction of leucocytes with the appropriate phenotype of organisms results in the release of a host-derived or bacterial secretagogue, or both. The bacterial factor may or may not be the antigen related to cholera toxin, described previously.

The role of leucocytes in the induction of fluid secretion by Salmonella typhimurium.

Nitrogen mustard (N2M) treatment of rabbits induced neutropenia, and, in ligated ileal loops, it inhibited fluid secretion induced by salmonella or by cholera toxin (CT). Pretreatment of rabbits with indomethacin almost abolished salmonella-induced fluid secretion and significantly reduced that induced by CT. Similar effects of N2M and indomethacin on fluid secretion induced by salmonella, but not by CT, have been reported by other workers and used to implicate prostaglandins, from the salmonella-induced inflammation, as mediators of fluid secretion. In contrast, we show that N2M treatment, in addition to reducing CT-induced secretion, caused severe morphological alterations to ileal mucosa. Irradiation techniques were developed for inducing neutropenia, but they did not totally inhibit salmonella-induced leucocyte influx into ileal mucosa. We propose an alternative mechanism for the inhibitory effect of N2M on salmonella- and CT-induced secretion, based on the known anti-mitotic activity of N2M. Also, the anti-secretory effect of indomethacin cannot be attributed uniquely to its anti-inflammatory activity because it depressed CT-induced secretion as well as salmonella-induced secretion. These results support the concept of pathophysiological secretion in infectious diarrhoea, developed previously for rotavirus and extended to bacterial infections.

Expression of an antigen in strains of Salmonella typhimurium which reacts with antibodies to cholera toxin.

Six strains of Salmonella typhimurium (W118, TML, SL1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1-4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.

Novel C-2 substituted carbapenem derivatives. Part II. Synthesis and structure-activity relationships of isoxazolin-2-yl, isoxazolidin-2-yl and 2-pyrazolin-2-yl carbapenems generated using 1,3-dipolar cycloaddition chemistry.

A series of carbapenems containing novel C-2 semisaturated heterocyclic substituents were synthesised by 1,3 dipolar cycloaddition reactions of nitrile oxides, nitrile imines and a nitrone to 2-vinylcarbapenem. The isoxazoline and isoxazolidine compounds showed potent antibacterial activity but moderate stability to human dehydropeptidase 1 (DHP-1). Stability to DHP-1 was improved by methyl substitution in the isoxazoline ring, but at the expense of antibacterial activity. The pyrazolines exhibited excellent stability to DHP-1, but reduced potency against Gram-negative organisms.

Novel C-2 substituted carbapenem derivatives. Part IV. Synthesis and biological activity of five membered heteroaromatic derivatives.

The synthesis, antibacterial activity, and stability to human dehydropeptidase-1 (DHP-1) of a novel series of (5R,6S)-6-[(1R)-1-hydroxyethyl]-2-heterocyclylcarbapen-2-em-3-carb oxylates are described. Of the compounds investigated 1,5-disubstituted pyrazol-3-yl and 3-substituted isoxazol-5-yl derivatives have the best combination of antibacterial activity and stability to DHP-1. They are particularly active against community-acquired respiratory tract pathogens and have stabilities to DHP-1 superior to that of meropenem.

In defense of deinstitutionalization.

Political and economic decisions contributing to deinstitutionalization had widespread support from psychiatrists, social reformers, and civil libertarians. The fortuitous advent of Medicaid and SSI abetted movement out of institutions, but these federal programs impede efforts to reform remaining state hospitals--yet progress has been achieved.

Enhancement of relaxation rate with paramagnetic contrast agents in NMR imaging.

Enhancement of spin-lattice relaxation using oral ferric chloride and using inhaled oxygen is illustrated. A 0.06% solution of ferric chloride reduced the spin-lattice relaxation time in the fundus of the stomach from 730 ms to 285 ms. Ferric chloride may be useful as a bowel-labeling agent. In 5 volunteers inhalation of 100% oxygen decreased the mean observed spin-lattice relaxation time of blood within the left ventricular cavity. Although the change was small, it may have important uses in monitoring metabolic processes.

Kinetics, tissue specificity and pathological changes in murine rotavirus infection of mice.

Mice that did not contain antibodies to rotavirus were orally infected with murine rotavirus (EDIM strain) and observed over 7 days. As judged by ELISA, only the small intestine was infected, not the colon. The infection was biphasic, viral antigen peaks being observed at 48 h and approximately 120 h post-infection. Clinically evident diarrhoea was maximal at 72 h. Virus in the upper, middle and lower regions of the small intestine was mainly tissue-associated; most virus was found in the middle small intestine. Two peaks (48 h and 120 h post-infection) of virus antigen were observed in the colon, but these corresponded to luminal, not tissue-associated viral antigen. Only enterocytes in the upper two-thirds of villus epithelia were infected as judged by fluorescent-antibody analysis and transmission electron microscopy. Scanning electron microscopy revealed morphological appearances not hitherto correlated with the progress of the infection: villus tips were convoluted, corresponding to the shedding of virus-infected cells but the lower regions of infected villi were shrunken and considerably narrowed compared to tips.

An electron microscopic investigation of time-related changes in the intestine of neonatal mice infected with murine rotavirus.

Seven-day-old mice were infected orally with murine rotavirus (EDIM) and regions of the gut examined at 24 h intervals up to 7 days by electron microscopy. Structural changes were correlated with data on viral antigen production, thymidine kinase activity, and clinical signs of diarrhea. No pathological changes were detected in the colon. Infection and structural damage were confined to the small intestine, with middle regions showing the most pronounced changes. Constriction of villus bases, edema of the lamina propria, and vacuolation of enterocytes occurred at 24 h postinfection (PI), i.e., before evidence of major virus replication. Transient villus atrophy occurred at 48 h PI. Recovery of villus length was evident by 72 h PI accompanied by evidence of marked enterocyte replication at villus bases. Many enterocytes were damaged with little evidence for the presence of virus particles. By 96 h PI, villi had almost recovered from infection although some enterocytes were still damaged; no virus particles were detected in these cells. A second phase of villus damage and edema of the lamina propria occurred at 120 h PI; the pathology resembled that at 24-48 h PI. By 144 to 168 h PI, recovery of the mucosa from infection was virtually complete. We suggest that many of the pathological features following rotavirus infection result from rotavirus-induced ischemia of villi and that diarrhea results from malabsorption of fluid by damaged villi and hypersecretion of ions released from increased numbers of dividing cells at villus-crypt borders.

Intestinal enzyme profiles in normal and rotavirus-infected mice.

To investigate further the pathophysiology of rotavirus-induced diarrhea, changes in specific activities of eight relevant intestinal enzymes [alkaline phosphatase, thymidine kinase, lactase, maltase, sucrase, Na+,K+-adenosine triphosphatase (ATPase), adenylate and guanylate cyclases] were measured following infection of suckling mice with murine rotavirus (epizootic diarrhea of infant mouse strain) and compared with age-matched control mice. The concentration of lactose within the lumen of the gastrointestinal tract during infection was also measured. During the course of infection, activities of alkaline phosphatase and lactase decreased, whilst the activity of thymidine kinase increased. Precocious maturation profiles of sucrase and maltase enzymes were observed. No significant changes were detected in the activities of Na+,K+-ATPase or the adenylate and guanylate cyclases. These results are discussed in relation to existing and novel hypotheses on the pathogenesis of rotavirus-induced diarrhea.

Nuclear magnetic resonance imaging of the liver: initial experience.

Nuclear magnetic resonance (NMR) scans of the liver were obtained in 12 normal volunteers and 32 patients using a whole-body machine developed by Thorn-EMI Ltd., and the results were compared with x-ray computed tomography (CT). Two types of NMR scan, saturation-recovery and inversion-recovery, were performed in order to obtain values for the spin-lattice relaxation time, T1. Although the saturation-recovery scans show little soft-tissue detail, the inversion-recovery scans demonstrated the interlobar fissure, hepatic veins, portal veins, bile ducts, and gallbladder. In comparison with CT (Siemens Somatom 2), both types of NMR scan showed some blurring due to respiratory movement but much less linear artifact across the liver from the air-fluid interface in the stomach. Focal disease within the liver was demonstrated by both CT and NMR, although an area of focal atrophy and another of hepatic infarction were only recognized with NMR. In diffuse disease the pattern varied. In steatosis CT was virtually diagnostic, while NMR showed no specific features. In hemochromatosis, hepatitis, eight cases of cirrhosis, and one of Wilson disease, both techniques showed abnormalities of varying specificity. In two cases of cirrhosis and one of primary biliary cirrhosis, only the NMR scan was abnormal. Nuclear magnetic resonance images are now sufficiently anatomically detailed to permit serious comparisons with technically advanced computed tomography. The information revealed is fundamentally different and can be expected to have some diagnostic utility.

Magnetic resonance properties of hydrogen: imaging the posterior fossa.

Posterior fossa scans were performed on five healthy volunteers using a nuclear magnetic resonance (NMR) machine constructed by Thorn-EMI Ltd. Three different NMR scanning sequences were used. In the first, a type of saturation-recovery technique was used to produce images strongly dependent on the density of hydrogen nuclei, but with some dependence on the spin-lattice relaxation time (T1). In the second, an inversion-recovery technique was used to produce images with a stronger dependence on the spin-lattice relaxation time. In the third, a spin-echo technique was used to obtain images with a dependence on the spin-spin relaxation time (T2). All three types of NMR image were unaffected by bone artifact. Visualization of brain adjacent to the skull base was obtained without loss of detail due to partial-volume effect from bone. The saturation-recovery images highlighted arteries and veins that were clearly visible without the use of contrast agents. The inversion-recovery images showed remarkable gray-white matter differentiation enabling internal structure to be seen within the brainstem and cerebellum. The trigeminal nerve and ganglion were also seen outside the brain. Experience with the spin-echo technique is limited, but the images at the base of the brain show considerable soft-tissue detail. The NMR images of the posterior fossa in this study were comparable in quality to those obtained from a new rotate-rotate x-ray computed tomography machine and were superior in several respects.