Human Intestinal Enteroids as a Model System of Shigella Pathogenesis.

The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface.

New developments in rabies vaccination.

Current rabies vaccines are safe and, when administered properly, they are highly effective. In addition, they elicit long-lasting immunity, with virus-neutralising antibody titres persisting for years after vaccination. However, current regimens require multiple doses to achieve high neutralising titres and they are costly, which means that it is difficult for developing countries, where rabies deaths are highest, to implement widespread vaccination. New innovations are the only way to reduce rabies disease to acceptable rates. Numerous preclinical and clinical studies are under way, testing novel vaccines, adjuvants and injection methods. Research into the use of live vaccines and alternative vaccine vectors is ongoing, while attempts to develop DNA vaccines have so far failed to match the immunogenicity and neutralising capability of traditional vaccines. The development of molecular adjuvants that induce faster, stronger immune responses with less antigen has yielded exciting preclinical results and appears to edge us closer to a better rabies vaccine. However, steep challenges remain: molecular adjuvants require administration with live vaccines, and differences in species specificity of immune molecules complicate development. Over all, the array of research undertaken over the past decade is impressive and encouraging, but most new vaccines have yet to be tested in clinical trials, and the viability of such experimental vaccines in the global market remains to be seen. Only a vaccine that outperforms currently available vaccines in every area will have a chance at widespread adoption. Nevertheless, the authors are confident that some vaccine candidates will meet these criteria.

Les vaccins actuels contre la rage sont sûrs et très efficaces lorsqu'ils sont administrés correctement. En outre, ils confèrent une immunité durable, avec le maintien de titres neutralisants d'anticorps plusieurs années après la vaccination. Néanmoins, les régimes actuels nécessitent l'administration de plusieurs doses pour obtenir des titres élevés d'anticorps neutralisants et ils sont onéreux, de sorte que la vaccination à grande échelle est difficile à mettre en oeuvre dans les pays en développement, pourtant les plus touchés par la mortalité par rage. Seule l'adoption de solutions innovantes permettra de ramener l'incidence de la rage à un niveau acceptable. De nombreuses études précliniques et cliniques sont en cours, visant à tester les innovations en matière de vaccins, de modes d'injection et d'adjuvants. La recherche sur l'utilisation de vaccins à virus vivant et sur de nouveaux vecteurs vaccinaux se poursuit, alors que les tentatives de développement de vaccins à ADN n'ont pas réussi jusqu'à présent à obtenir un effet immunogène ou des capacités de neutralisation virale équivalents à ceux des vaccins traditionnels. Les résultats d'essais précliniques sur de nouveaux adjuvants moléculaires induisant une réponse immune plus rapide et plus puissante avec moins d'antigène sont extrêmement prometteurs et semblent annoncer l'imminence de meilleurs vaccins contre la rage. Il subsiste toutefois d'importantes difficultés : les adjuvants moléculaires ne peuvent être administrés qu'avec des vaccins vivants et les différences de spécificité d'espèce des molécules immunes rendent le développement plus complexe. Globalement, les efforts déployés depuis une décennie par la recherche sont impressionnants et encourageants mais la plupart des nouveaux vaccins doivent encore être soumis à des essais cliniques ; d'autre part la viabilité de ces vaccins expérimentaux dans le marché mondial reste à démontrer. Seul un vaccin capable de surpasser les performances des vaccins actuels dans chaque domaine aura une chance d'être largement adopté. Les auteurs estiment cependant que certains vaccins candidats pourront satisfaire à ces exigences.

Las actuales vacunas antirrábicas son seguras y, si se administran debidamente, muy eficaces. Además, inducen inmunidad duradera, con títulos de anticuerpos neutralizantes que subsisten años después de la vacunación. Sin embargo, los regímenes actuales resultan costosos y exigen dosis múltiples para lograr títulos de neutralización elevados, lo que dificulta a los países en desarrollo, que son los más golpeados por la rabia, la implantación generalizada de la vacunación. El único camino para reducir la rabia a niveles aceptables pasa por la innovación. Están en marcha numerosos estudios preclínicos y clínicos en los que se ensayan vacunas, adyuvantes y métodos de inyección novedosos. También sigue adelante la investigación sobre el uso de vacunas vivas y vectores vacunales alternativos, mientras que ninguna de las tentativas realizadas hasta la fecha con vacunas de ADN ha deparado niveles de inmunogenicidad y capacidad de neutralización equiparables a los de las vacunas tradicionales. La obtención de adyuvantes moleculares que inducen una respuesta inmunitaria más rápida y vigorosa en presencia de menos cantidad de antígeno ha dado resultados preclínicos muy interesantes y poco a poco parece acercarnos al logro de una mejor vacuna antirrábica. Subsisten, empero, arduas dificultades: los adyuvantes moleculares solo funcionan si se administran con vacunas vivas, y las diferencias existentes entre las especies en cuanto a la especificidad de las moléculas inmunitarias complican las labores de desarrollo. Globalmente, el conjunto de investigaciones emprendidas en el último decenio es impresionante y alentador, pero la mayoría de las nuevas vacunas aún deben pasar por la fase de ensayo clínico, y está por ver qué viabilidad tienen estas vacunas experimentales en el mercado mundial. Solo una vacuna que supere a las actuales en todos los aspectos tiene posibilidades de ser adoptada a gran escala. Pese a todo, los autores expresan su confianza en que algunas de las vacunas candidatas cumplan estos criterios.

Role of intracellular carbon metabolism pathways in Shigella flexneri virulence.

Shigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular S. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkAB and pykAF mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene eda resulted in small plaques, but the double eda edd mutant formed normal-size plaques. This suggested that the plaque defect of the eda mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular S. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type S. flexneri also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of S. flexneri in vitro, suggesting that it may be a preferred carbon source inside host cells.

Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth.

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

Mussel growth supported by methane as sole carbon and energy source.

Symbioses between chemoautotrophic bacteria and several specialized marine invertebrates are well documented. However, none of these symbioses have been demonstrated to provide sufficient energy and carbon to the host to enable it to grow. Growth rates of seep mussels collected from hydrocarbon seeps off the coast of Louisiana were measured in a controlled environment where methane was the sole carbon and energy source. The growth rates increased to a maximum of 17.2 micrometers per day in response to methane and approached zero in the absence of methane. These mussels contain methanotrophic symbiotic bacteria in their gills, which suggests that these bacteria provide their hosts with a net carbon flux originating from methane.

Stable carbon isotopic evidence for carbon limitation in hydrothermal vent vestimentiferans.

Stable carbon isotope composition (delta(13)C values) can be used to evaluate an animal's source of nutritional carbon. Most animals with chemoautotrophic endosymbionts have quite negative tissue delta(13)C values due to discrimination against (13)C associated with chemoautotrophic assimilation of inorganic carbon. However, the delta(13)C values of hydrothermal vent (HTV) vestimentiferans are significantly higher than the values reported for non-HTV vestimentiferans or other invertebrates with chemoautotrophic endosymbionts. Tissue delta(13)C values of two species of HTV vestimentiferans increase with increasing size of the animals. This relation supports the hypothesis that the relatively high delta(13)C values are the result of inorganic carbon limitation during carbon fixation. A more favorable relation between gas exchange and carbon fixation in the smaller individuals is expected, due to differences in the geometric scaling of gas-exchange surfaces and trophosome volume.

A methanotrophic marine molluscan (bivalvia, mytilidae) symbiosis: mussels fueled by gas.

An undescribed mussel (family Mytilidae), which lives in the vicinity of hydrocarbon seeps in the Gulf of Mexico, consumes methane (the principal component of natural gas) at a high rate. The methane consumption is limited to the gills of these animals and is apparently due to the abundant intracellular bacteria found there. This demonstrates a methane-based symbiosis between an animal and intracellular bacteria. Methane consumption is dependent on the availability of oxygen and is inhibited by acetylene. The consumption of methane by these mussels is associated with a dramatic increase in oxygen consumption and carbon dioxide production. As the methane consumption of the bivalve can exceed its carbon dioxide production, the symbiosis may be able to entirely satisfy its carbon needs from methane uptake. The very light (delta(13)C = -51 to -57 per mil) stable carbon isotope ratios found in this animal support methane (delta(13)C = -45 per mil at this site) as the primary carbon source for both the mussels and their symbionts.

Deep-sea hydrocarbon seep communities: evidence for energy and nutritional carbon sources.

Mussels, clams, and tube worms collected in the vicinity of hydrocarbon seeps on the Louisiana slope contain mostly "dead" carbon, indicating that dietary carbon is largely derived from seeping oil and gas. Enzyme assays, elemental sulfur analysis, and carbon dioxide fixation studies demonstrate that vestimentiferan tube worms and three clam species contain intracellular, autotrophic sulfur bacterial symbionts. Carbon isotopic ratios of 246 individual animal tissues were used to differentiate heterotrophic (8(13)C = -14 to -20 per mil), sulfur-based (8(13)C = -30 to -42 per mil), and methane-based (8(13)C = <-40 per mil) energy sources. Mussels with symbiotic methanotrophic bacteria reflect the carbon isotopic composition of the methane source. Isotopically light nitrogen and sulfur confirm the chemoautotrophic nature of the seep animals. Sulfur-based chemosynthetic animals contain isotopically light sulfur, whereas methane-based symbiotic mussels more closely reflect the heavier oceanic sulfate pool. The nitrogen requirement of some seep animals may be supported by nitrogen-fixing bacteria. Some grazing neogastropods have isotopic values characteristic of chemosynthetic animals, suggesting the transfer of carbon into the background deep-sea fauna.

Maple syrup urine disease in Mennonites. Evidence that the Y393N mutation in E1 alpha impedes assembly of the E1 component of branched-chain alpha-keto acid dehydrogenase complex.

Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex. This causes a tyrosine to asparagine substitution at position 393 (Y393N). To assess the functional significance of this missense mutation, we have carried out transfection studies using E1 alpha-deficient MSUD lymphoblasts (Lo) as a host. The level of E1 beta subunit is also greatly reduced in Lo cells. Efficient episomal expression in lymphoblasts was achieved using the EBO vector. The inserts employed were chimeric bovine-human cDNAs which encode mitochondrial import competent E1 alpha subunit precursors. Transfection with normal E1 alpha cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1 alpha and E1 beta subunits were markedly increased. Introduction of Y393N mutant E1 alpha cDNA failed to produce any measurable decarboxylation activity. Mutant E1 alpha subunit was expressed at a normal level, however, the E1 beta subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1 alpha with E1 beta into a stable alpha 2 beta 2 structure, resulting in the degradation of the free E1 beta subunit.

A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom).

We report the features of a new syndrome of aromatase deficiency due to molecular defects in the CYP19 (P450arom) gene in a 46,XX female. At birth, the patient presented with a nonadrenal form of female pseudohermaphrodism. At 17 months of age, laparotomy revealed normal female internal genital structures; the histological appearance of the ovaries was normal. FSH concentrations were markedly elevated at 9.4 ng/mL LER 869, and estrone and estradiol levels were undetectable (< 37 pmol/L). By 14 yr of age, she had failed to exhibit breast development. The clitoris had enlarged to 4 x 2 cm, and pubic hair was Tanner stage IV. The plasma concentration of testosterone was elevated at 3294 pmol/L, as was androstenedione at 9951 pmol/L. Plasma estradiol levels were below 37 pmol/L. ACTH and dexamethasone tests indicated a nonadrenal source of testosterone and androstenedione. Plasma gonadotropin levels were in the castrate range. Pelvic sonography and magnetic resonance imaging showed multiple 4- to 6-cm ovarian cysts bilaterally. Despite increased circulating androgens and clitoral growth, the bone age was 10 yr at chronologic age 14 2/12 yr. Estrogen replacement therapy resulted in a growth spurt, breast development, menarche, suppression of gonadotropin levels, and resolution of the cysts. The clinical findings suggested the diagnosis of P450arom deficiency. Analyses of genomic DNA from ovarian fibroblasts demonstrated two single base changes in the coding region of the P450arom gene, one at 1303 basepairs (C-T), R435C, and the other at 1310 basepairs (G-A), C437Y, in exon 10. The molecular genetic studies indicate that the patient is a compound heterozygote for these mutations. Expression of these mutations showed that the R435C mutation had 1.1% the activity of the wild-type P450arom enzyme, whereas the C437Y mutation demonstrated no activity. The cardinal features of this syndrome are a consequence of P450arom deficiency: 1) the fetal masculinization in this syndrome can be ascribed to defective placental conversion of C19 steroids to estrogens, leading to exposure of the female fetus to excessive amounts of testosterone; 2) the pubertal failure, mild virilization, multicystic ovaries, and hyperstimulation of the ovaries by FSH and LH are the result of the inability of the ovary to aromatize testosterone and androstenedione to estrogens; and 3) the striking delay in bone age at 14 2/12 yr supports the notion that estrogens, in contrast to androgens, are the major sex steroid driving skeletal maturation during puberty. Familial P450arom deficiency, although rare, may be more common than previously suspected.(ABSTRACT TRUNCATED AT 400 WORDS)

Trends in Medicare enrollee use of physician and supplier services, 1983-86.

Beginning in 1984, the long-term trend of increasing utilization of inpatient hospital care by Medicare enrollees reversed. As Medicare patients increasingly received care in outpatient hospital facilities, ambulatory surgical centers, and physicians' offices, the structure of charges for physicians' services changed significantly. Medical services by physicians in inpatient hospitals declined rapidly. Surgical care for less life-threatening illnesses, such as eye conditions, moved from inpatient hospitals to outpatient facilities and physicians' charges derived from inpatient care was offset primarily by the increased proportion derived from physician care in outpatient facilities, mostly for surgery.

Physician charges for surgical services under Medicare, by medical specialty: 1980 and 1985.

Since 1980, a number of Medicare practice and utilization patterns have changed as a result of payment reform, certification of new types of providers, and changes in technology. The shift in physician surgical charges by specialty and by setting is examined in this article.

Trends in total hospital financial performance under the prospective payment system.

In this article, the author examines trends in determinants of total hospital facility revenues, expenses, and net profits during the period 1977-89. Measures of change in transaction prices are developed, which enable an analysis of trends in real hospital outputs and total factor productivity. The main source of hospital spending growth in excess of the gross national product is identified as growth in hospital employee compensation.

Hospital and Medicare financial performance under PPS, 1985-90.

Although an increasing number of hospitals are reporting net losses from the Medicare prospective payment system (PPS) for inpatient care, overall hospital facility profit rates remain stable. Hospitals that reported net profits in the Medicare inpatient PPS sector in PPS 7 (1990) had smaller increases in Medicare expenses than hospitals that reported PPS losses in PPS 7. Medicare PPS inpatient net losses in PPS 7 were more than offset by non-Medicare net profits. Even though Medicare PPS revenues grew more slowly than the gross domestic product from 1985 to 1990, other hospital revenues grew more rapidly.

Differences by age groups in health care spending.

This paper presents differences by age in health care spending by type of expenditure and by source of funds through 1978. Use of health care services generally increases with age. The average health bill reached $2,026 for the aged in 1978, $764 for the intermediate age group, and $286 for the young. Biological, demographic, and policy factors determine each age group's share of health spending. Public funds financed over three-fifths of the health expenses of the aged, with Medicare and Medicaid together accounting for 58 percent. Most of the health expenses of the young age groups were paid by private sources.

Medicare episodes of illnesses: a study of hospital, skilled nursing facility, and home health agency care.

This paper analyzes charges incurred under the Medicare program for inpatient hospital, skilled nursing facility (SNF), and home health agency (HHA) care for 1976. This research was made possible through the construction of a new data set which links a beneficiary's use of these three services. Summary highlights reveal that an overwhelming majority of the 7.5 million Medicare episodes of illness do not involve post-hospital SNF or HHA care. Those episodes of illness that use only hospital care are substantially (53%) cheaper than all other episodes. A large percentage of these charge differences reflect the greater number of hospital days of care associated with post-hospital care services. However, an analysis of the beneficiaries' demographic characteristics suggests that persons who use post-hospital care generally differ from those who receive only hospital care. We found that persons who use post-hospital SNF or HHA, or both types of care are likely to be female, to have cancer, diabetes, fractured bones or a central nervous or vascular system disease, and to be older than persons who do not use these types of care. The data also show that a beneficiary's area of residence greatly influences the amount and types of care received. Persons who reside in the New England, Middle Atlantic and Pacific Divisions are more likely to receive post-hospital care services than person who live elsewhere in the United States. These persons also incur among the highest per capita institutional charges in the United States. Part of this variation in institutional charges per capita is explained by the high input price index found in these areas, and in some cases by the high quantity of services index.

Physician charges and utilization trends.

A synopsis of charge and payment trends of Medicare physicians and other noninstitutional suppliers of goods and services is provided in this article. Included are longitudinal variations in charges for services, trends in program expenditures, and patterns in beneficiary liabilities.

Medicare physician and hospital utilization and expenditure trends.

During the period 1983-86, the period directly following implementation of the Medicare prospective payment system, inpatient hospital care declined. Concurrently, fee-for-service utilization rates for physicians and other noninstitutional suppliers of medical goods and services and for outpatient facility care rose. Medicare expenditures for physicians and other suppliers and for outpatient facility care paralleled changes in utilization. In 1987, the proportion of Medicare patients receiving inpatient hospital care stabilized, but the proportion receiving outpatient hospital care continued to increase.

Tubeworm succession at hydrothermal vents: use of biogenic cues to reduce habitat selection error?

Species colonizing new deep-sea hydrothermal vents along the East Pacific Rise show a distinct successional sequence: pioneer assemblages dominated by the vestimentiferan tubeworm Tevnia jerichonana being subsequently invaded by another vestimentiferan Riftia pachyptila, and eventually the mussel Bathymodiolus thermophilus. Using a manipulative approach modified from shallow-water ecological studies, we test three alternative hypotheses to explain the initial colonization by T. jerichonana and its subsequent replacement by R. pachyptila. We show that R. pachyptila and another vestimentiferan, Oasisia alvinae, colonized new surfaces only if the surfaces also were colonized by T. jerichonana. This pattern does not appear to be due to restricted habitat tolerances or inferior dispersal capabilities of R. pachyptila and O. alvinae, and we argue the alternative explanation that T. jerichonana facilitates the settlement of the other two species and is eventually outcompeted by R. pachyptila. Unlike the classic model of community succession, in which facilitating species promote their own demise by modifying the environment to make it more hospitable for competitors, we suggest that T. jerichonana may produce a chemical substance that induces settlement of these competitors. This process of selecting habitat based on biogenic cues may be especially adaptive and widespread among later-successional species that occupy a physically variable and unpredictable environment. In these cases, the presence of weedy species implies some integrated period of environmental suitability, whereas an instantaneous assessment of physical habitat conditions, such as water temperature for vent tubeworms, provides a poorer predictor of long-term habitat suitability.

Mutagenicity of actinomycin D in Neurospora crassa.

Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent RNA and protein synthesis. We report here the induction by actinomycin D of purple adenine-requiring mutants (ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 106 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X ray-induced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl-N'-nitro-N-nitrosoguanidine, agents known to induce mainly base-pair substitutions.