A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever.

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.     

Barriers to the adoption of electronic health records: using concept mapping to develop a comprehensive empirical model

The study attempts to unify prior research and develop a comprehensive, empirically based conceptual model of the barriers to EHR adoption among community physicians. The model uses concept mapping, which taps the shared expertise of a group and provides reliable estimates with relatively small sample sizes. The methodology includes brainstorming of barrier statements and sorting and rating of issue statements. The model illuminates the larger structure of barriers as well as the finer details of constituent issues. Core issues are standardization and interoperability; also important are technical issues and the cost-benefit of adopting EHRs. However, psychosocial issues, the main focus of diffusion research, seem relatively peripheral. We believe the development of this model is an important first step in creating effective and measurable interventions that enhance the adoption of EHRs in healthcare.     

High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma

Renal cell carcinoma (RCC) accounts for 85% of primary renal neoplasms, and is rarely curable when metastatic. Approximately 70% of RCCs are clear-cell type (ccRCC), and in >80% the von Hippel-Lindau (VHL) gene is mutated or silenced. We developed a novel, high-content, screening strategy for the identification of small molecules that are synthetic lethal with genes mutated in cancer. In this strategy, the screen and counterscreen are conducted simultaneously by differentially labeling mutant and reconstituted isogenic tumor cell line pairs with different fluorochromes and using a highly sensitive high-throughput imaging-based platform. This approach minimizes confounding factors from sequential screening, and more accurately replicates the in vivo cancer setting where cancer cells are adjacent to normal cells. A screen of ~12,800 small molecules identified homoharringtonine (HHT), an FDA-approved drug for treating chronic myeloid leukemia, as a VHL-synthetic lethal agent in ccRCC. HHT induced apoptosis in VHL-mutant, but not VHL-reconstituted, ccRCC cells, and inhibited tumor growth in 30% of VHL-mutant patient-derived ccRCC tumorgraft lines tested. Building on a novel screening strategy and utilizing a validated RCC tumorgraft model recapitulating the genetics and drug responsiveness of human RCC, these studies identify HHT as a potential therapeutic agent for a subset of VHL-deficient ccRCCs.     

The steroidogenic acute regulatory protein,StAR, works only at the outer mitochondrial membrane

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol into mitochondria to initiate steroidogenesis, but its site of action on the mitochondria has been uncertain. One model states that StAR has a fairly rigid structure and functions in the intramembranous space (IMS) where it transports cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM); another model states that StAR works solely on or in the OMM and undergoes a partially open molten globule conformation while picking up and discharging cholesterol. We designed, built and tested a series of StAR fusion proteins designed to immobilize StAR on the OMM, the IMS, or the matrix side of the IMM. Only the constructs at the OMM were active, either in vivo or in vitro. As these data indicated that StAR acts at or in the OMM we hypothesized that StAR' s activity would be proportional to the amount of time it spends on the OMM. To test this hypothesis, we built a series of StAR proteins with altered mitochondrial leaders designed to speed or slow StAR's mitochondrial entry. Cell transfections showed that the constructs that slowed entry had more activity and those designed to speed entry had less activity. Analysis of import kinetics in vitro confirmed that these constructs accelerated import inversely proportional to their activity. These data show that StAR works only on the OMM, providing an unusual example of a protein that exerts its biological activity in a cellular location it occupies only transiently, rather than in the location (the matrix) to which it is targeted.     

Why is pseudomonas the colonizer and why does it persist?

Summary Pseudomonas aeruginosa is currently the major cause of morbidity and mortality in cystic fibrosis. Studies to understand why this particular organism is a problem and why host defenses fail to clear it, are beginning to provide some answers. Implicit in any working hypothesis are the prerequisites that: (i)P. aeruginosa should have a tropism for the respiratory tract; (ii) there should be a physical clearance defect; and (iii) there should be an acquired immune clearance defect. Studies from many laboratories support these contentions. This organism exhibits its tropism by adhering to tracheal cells and to tracheobronchial mucins by means of pili or the mucoid exopolysac-charide of mucoid strains. The receptors on both cells and mucins contain sialic acid as the dominant sugar moiety. Many factors contribute to its persistence, chief among which is the failure of phagocytic defenses caused by microbial or host enzymes and even by mucins which inhibit the opsonophagocytosis ofP. aeruginosa. Injury to the mucociliary system, again caused by microbial or host factors, is also a prominent factor in the persistence ofP. aeruginosa. We hypothesize that this organism is the dominant pathogen because of the existence of receptors in the respiratory tract for it and that it persists because bacteria in stagnant mucus cannot be cleared physically or immunologically. We are doubtful that conventional vaccination approaches will yield a solution to this problem.

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Ursachen für die Besiedelung durch Pseudomonas und seine Persistenz

Zusammenfassung Pseudomonas aeruginosa ist zur Zeit die wichtigste Krankheits-und Todesursache bei Patienten mit zystischer Fibrose. Studien, die sich mit den Problemen befassen, die dieser spezielle Erreger verursacht, und mit der Frage, warum er durch Abwehrmechanismen des Wirtes nicht eliminiert wird, geben die ersten Antworten zu diesem Thema. Die Arbeitshypothesen gehen davon aus, daß 1.P. aeruginosa eine besondere Neigung hat, sich im Respirationstrakt anzusiedeln; 2. ein Defekt in der physikalischen Klärfunktion besteht und es 3. einen erworbenen Defekt in der immunologischen Komponente der Erregeradikation gibt. Diese Annahmen werden durch Laboruntersuchungen gestützt. Die Adhärenz des Erregers an Trachealzellen und an tracheobronchialen Muzinen mittels Pili oder dem mukoiden Exopolysaccharid mukoider Stämme ist die Grundlage für seinen Tropismus zum Tracheobronchialbaum. Die Rezeptoren beider Zellen und Muzine enthalten Sialinsäure als dominierende Zuckerverbindung. Zahlreiche Faktoren begünstigen die Persistenz des Erregers; am bedeutsamsten ist das Versagen der phagozytären Abwehr unter dem Einfluß von Enzymen, die vom Mikroorganismus oder dem Wirt selbst sezerniert werden, und sogar auch der Muzine, die die Opsonophagozytose vonP. aeruginosa hemmen. Eine Schädigung des mukoziliaren Systems, die wiederum durch mikrobielle oder Wirtsfaktoren verursacht sein kann, ist ebenfalls für die Erregerpersistenz von Bedeutung. Wir stellen die Hypothese auf, daß dieser Mikroorganismus deshalb der dominierende Erreger ist, weil der Respirationstrakt Rezeptoren für ihn besitzt; die Ursache für seine Persistenz liegt darin, daß die Bakterien im stagnierenden Schleim weder physikalisch noch immunologisch beseitigt werden können. Wir bezweifeln, daß sich mit konventionellen Versuchen einer Vakzination eine Lösung für dieses Problem finden läßt.

     

Inhibition of human non-pancreatic phospholipases A2 by retinoids and flavonoids. Mechanism of action

The interaction of retinoids and flavonoids with phospholipases A2 (PLA2) was studied to assess the mechanism of inhibition. Retinoids, such as retinal, retinol, retinoic acid and retinol acetate, and flavonoids, such as quercetin, rutin, morin and sciadopitysin, inhibit Ca2+-dependent PLA2 activity of human synovial fluid (HSF) in vitro in a dose-dependent fashion; ID20S ranged from 2-8 microM. Retinal inhibited neutral active Ca2+-dependent PLA2S from human platelets, human plasma, human polymorphonuclear leukocytes and Naja mossambica mossambica venom in a dose-dependent manner while quercetin inhibits extracellular PLA2 activities of human plasma, HSF and N. m. mossambica venom in a dose-dependent manner but not PLA2 activity derived from human platelets and polymorphomonuclear leukocytes. Inhibition of PLA2 activity by both flavonoid and retinoids were independent of Ca2+ or Na+. Increasing substrate concentration (9-144 nmols) relieved the inhibition of HSF-PLA2 activity by quercetin indicating probable interaction with the substrate. The inhibition by retinal is independent of substrate concentration suggesting that inhibition by retinal is probably due to direct interaction with the enzyme. both retinal and quercetin quenched the relative fluorescent intensity of N. m. mossambica PLA2 and in a dose-dependent manner in the same concentration range at which they inhibit in vitro PLA2 activity. Retinal and quercetin shift the thermotropic phase transition of distearoylphosphatidylethanolamine (DSPE) liposomes. Both compounds broadened the transition peak, shifted the Tm to lower temperature, and decreased enthalpy significantly. These findings indicate that inhibition of non-pancreatic human PLA2S by retinoids and flavonoids can be mediated by interaction with enzyme and/or substrate.     

Contribution of wood smoke to in vivo formation of N-nitrosothiazolidine-4-carboxylic acid: initial studies

The effect of administering liquid smoke or smoked food products to rats on in vivo formation of N-nitrosamino acids was investigated. Rats treated by gavage with either cysteine, formaldehyde or nitrite excreted urine containing no detectable levels of N-nitrosothiazolidine-4-carboxylic acid (NTCA). All three precursor compounds were required for NTCA formation. Two liquid smokes, when administered to rats in combination with cysteine and nitrite also produced measurable quantities of NTCA. Ascorbate inhibited in vivo formation of NTCA by approximately 90% when given to rats simultaneously with cysteine, formaldehyde, and nitrite. alpha-Tocopherol was much less effective than ascorbate in blocking NTCA formation. When smoked bacon, smoked Swiss cheese, and chicken barbecued with a sauce containing smoke flavouring were fed to rats along with nitrate, NTCA was again detected in the urine.