Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.     

Structure, dynamics and binding characteristics of the second PDZ domain of PTP-BL

The PDZ domains of the protein tyrosine phosphatase PTP-BL mediate interactions by binding to specific amino acid sequences in target proteins. The solution structure of the second PDZ domain of PTP-BL, PDZ2, displays a compact fold with six β strands and two α-helices. A unique feature of this domain compared to the canonical PDZ fold is an extended flexible loop at the base of the binding pocket, termed L1, that folds back onto the protein backbone, a feature that is shared by both the murine and human orthologues. The structure of PDZ2 differs significantly from the orthologous human structure. A comparison of structural quality indicators clearly demonstrates that the PDZ2 ensemble is statistically more reasonable than that of the human orthologue. The analysis of ¹⁵N relaxation data for PDZ2 shows a normal pattern, with more rigid secondary structures and more flexible loop structures. Close to the binding pocket, Leu85 and Thr88 display greater mobility when compared to surrounding residues. Peptide binding studies demonstrated a lack of interaction between murine PDZ2 and the C terminus of the murine Fas/CD95 receptor, suggesting that the Fas/CD95 receptor is not an in vivo target for PDZ2. In addition, PDZ2 specifically binds the C termini of both human Fas/CD95 receptor and the RIL protein, despite RIL containing a non-canonical PDZ-interacting sequence of E-x-V. A model of PDZ2 with the RIL peptide reveals that the PDZ2 binding pocket is able to accommodate the bulkier side-chain of glutamic acid while maintaining crucial protein to peptide hydrogen bond interactions.     

Identification of a host-associated species complex using molecular and morphometric analyses,with the description of Gyrodactylus rugiensoides n. sp. (Gyrodactylidae,Monogenea)

Gyrodactylus rugiensis was originally described as a parasite occurring on the marine gobies Pomatoschistus minutus and Pomatoschistus microps. In our preliminary survey this species was also frequently found on Pomatoschistus pictus and Pomatoschistus lozanoi. Subsequent molecular analysis of the internal transcribed spacers rDNA region revealed that this parasite actually represents a complex of two apparently cryptic species, one restricted to P. microps and the other shared by P. minutus, P. lozanoi and P. pictus. Morphometric analyses were conducted on 17 features of the opisthaptoral hard parts of specimens collected from all four host species. Standard discriminant analysis showed a clear separation of both genotypes by significant differences in marginal hook and ventral bar features. Statistical classifiers (linear discriminant analysis and nearest neighbours) resulted in an estimated misclassification rate of 4.7 and 3.1%, respectively. Based on molecular, morphological and statistical analyses a new species, Gyrodactylus rugiensoides is described. This species seems to display a lower host-specificity than generally observed for Gyrodactylus species as it infects three sympatric host species.However, seasonal and host-dependent morphometric variation is shown for G. rugiensoides collected on P. pictus. Host-switching and gene flow might be important factors preventing speciation on closely related and sympatric host species. The presence of host associated species complexes in this Gyrodactylus-Pomatoschistus system is also confirmed by the presence of two host-dependent genotypes within G. micropsi found on P. minutus and P. lozanoi, and P. microps, respectively. By comparing host and parasite phylogeny, phylogenetic and ecological factors influencing host-specificity are discussed.     

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule

 The generation of cytotoxic effector T cells requires delivery of two signals, one derived from a specific antigenic epitope and one from a costimulatory molecule. A phase I clinical trial was conducted with a non-replicating canarypoxvirus (ALVAC) constructed to express both human carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. This was the first study in cancer patients to determine if the delivery of costimulation with a tumor vaccine was feasible and improved immune responses. Three cohorts of six patients, each with advanced CEA-expressing adenocarcinomas, were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine (4.5 × 10⁶, 4.5 × 10⁷, and 4.5 × 10⁸ plaque-forming units, PFU). Patients were vaccinated by intramuscular injection every 4 weeks for 3 months and monitored for side-effects, tumor growth and anti-CEA immune responses. ALVAC-CEA- B7.1 at doses up to 4.5 × 10⁸ PFU was given without evidence of significant toxicity or autoimmune reactions. Three patients experienced clinically stable disease that correlated with increasing CEA-specific precursor T cells, as shown by in vitro interferon-γ enzyme-linked immunoassay spot tests (ELISPOT). These three patients underwent repeated vaccination resulting in augmented CEA-specific T cell responses. This study represents the first use of costimulation to enhance antitumor vaccines in cancer patients. This approach resulted in CEA-specific immunity associated with stable diseases in three patients. This study also demonstrated that CEA-specific T cell responses could be sustained by repeated vaccinations. Although the number of patients was small, the addition of B7.1 to virus-based vaccines may improve immunological and stable diseases to vaccination against tumor-associated antigens with tolerable toxicity. Received: 6 May 2000 / Accepted: 13 July 2000     

Comparison of three Listeria monocytogenes strains in a guinea-pig model simulating food-borne exposure

Three different Listeria monocytogenes strains, LO28 (a laboratory strain with truncated InlA), 4446 (a clinical isolate) and 7291 (a food isolate), were compared in a guinea-pig model designed to mimic food-borne exposure. The objectives were (1) to verify the applicability of the animal model for distinguishing between Listeria with different virulence properties and (2) to explore whether it was possible to reduce the required number of animals by dosing with mixed cultures instead of monocultures. Consistent with in vitro observations of infectivity in Caco-2 cells, faecal densities and presence in selected organs were considerably lower for LO28 than for the other two strains. Additionally, the animal study revealed a difference in prevalence in faeces as well as in internal organs between the clinical isolate and the food isolate, which was not reproduced in vitro . Dosage with monocultures of Listeria strains gave similar results as dosage with a mixture of the three strains; thus, the mixed infection approach was a feasible way to reduce the number of animals needed for determination of listerial virulence.     

High ABCC2 and Low ABCG2 Gene Expression Are Early Events in the Colorectal Adenoma-Carcinoma Sequence

Development of colorectal cancer (CRC) may result from a dysfunctional interplay between diet, gut microbes and the immune system. The ABC transport proteins ABCB1 (P-glycoprotein, Multidrug resistance protein 1, MDR1), ABCC2 (MRP2) and ABCG2 (BCRP) are involved in transport of various compounds across the epithelial barrier. Low mRNA level of ABCB1 has previously been identified as an early event in colorectal carcinogenesis (Andersen et al., PLoS One. 2013 Aug 19;8(8):e72119). ABCC2 and ABCG2 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 106 adenoma cases (12 with severe dysplasia, 94 with mild-moderate dysplasia) and from 18 controls with normal endoscopy.We found significantly higher level of ABCC2 in adenomas with mild to moderate dysplasia and carcinoma tissue compared to the levels in unaffected tissue from the same individual (P = 0.037, P = 0.037, and P<0.0001) and in carcinoma and distant unaffected tissue from CRC cases compared to the level in the healthy individuals (P = 0.0046 and P = 0.036). Furthermore, ABCG2 mRNA levels were significantly lower in adenomas and carcinomas compared to the level in unaffected tissue from the same individuals and compared to tissue from healthy individuals (P<0.0001 for all). The level of ABCB2 in adjacent normal tissue was significantly higher than in tissue from healthy individuals (P = 0.011).In conclusion, this study found that ABCC2 and ABCG2 expression levels were altered already in mild/moderate dysplasia in carcinogenesis suggesting that these ABC transporters are involved in the early steps of carcinogenesis as previously reported for ABCB1. These results suggest that dysfunctional transport across the epithelial barrier may contribute to colorectal carcinogenesis.     

Methodological Framework for World Health Organization Estimates of the Global Burden of Foodborne Disease

Background

The Foodborne Disease Burden Epidemiology Reference Group (FERG) was established in 2007 by the World Health Organization to estimate the global burden of foodborne diseases (FBDs). This paper describes the methodological framework developed by FERG''s Computational Task Force to transform epidemiological information into FBD burden estimates.

Methods and Findings

The global and regional burden of 31 FBDs was quantified, along with limited estimates for 5 other FBDs, using Disability-Adjusted Life Years in a hazard- and incidence-based approach. To accomplish this task, the following workflow was defined: outline of disease models and collection of epidemiological data; design and completion of a database template; development of an imputation model; identification of disability weights; probabilistic burden assessment; and estimating the proportion of the disease burden by each hazard that is attributable to exposure by food (i.e., source attribution). All computations were performed in R and the different functions were compiled in the R package ''FERG''. Traceability and transparency were ensured by sharing results and methods in an interactive way with all FERG members throughout the process.

Conclusions

We developed a comprehensive framework for estimating the global burden of FBDs, in which methodological simplicity and transparency were key elements. All the tools developed have been made available and can be translated into a user-friendly national toolkit for studying and monitoring food safety at the local level.     

Inflammatory Bowel Disease and Risk of Adverse Pregnancy Outcomes

Background and Objectives

Existing data on pregnancy complications in inflammatory bowel disease (IBD) are inconsistent. To address these inconsistencies, we investigated potential associations between IBD, IBD-related medication use during pregnancy, and pregnancy loss, pre-eclampsia, preterm delivery, Apgar score, and congenital abnormalities.

Methods

We conducted a cohort study in >85,000 Danish National Birth Cohort women who were pregnant in the period 1996-2002 and had information on IBD, IBD-related medication use (systemic or local corticosteroids, 5-aminosalicylates), pregnancy outcomes and potential confounders. We evaluated associations between IBD and adverse pregnancy/birth outcomes using Cox regression and log-linear binomial regression.

Results

IBD was strongly and significantly associated with severe pre-eclampsia, preterm premature rupture of membranes and medically indicated preterm delivery in women using systemic corticosteroids during pregnancy (hazard ratios [HRs] >7). IBD was also associated with premature preterm rupture of membranes in women using local corticosteroid medications (HR 3.30, 95% confidence interval [CI] 1.33-8.20) and with medically indicated preterm delivery (HR 1.91, 95% CI 0.99-3.68) in non-medicated women. Furthermore, IBD was associated with low 5-minute Apgar score in term infants (risk ratio [RR] 2.19, 95% CI 1.03-4.66). Finally, Crohn’s disease (but not ulcerative colitis) was associated with major congenital abnormalities in the offspring (RR 1.85, 95% CI 1.06-3.21). No child with a congenital abnormality born to a woman with IBD was exposed to systemic corticosteroids in utero.

Conclusion

Women with IBD are at increased risk of severe pre-eclampsia, medically indicated preterm delivery, preterm premature rupture of membranes, and delivering infants with low Apgar score and major congenital malformations. These associations are only partly explained by severe disease as reflected by systemic corticosteroid use.     

Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.