The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct.

The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide > thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.     

Use of Two-Part Regression Calibration Model to Correct for Measurement Error in Episodically Consumed Foods in a Single-Replicate Study Design: EPIC Case Study

In epidemiologic studies, measurement error in dietary variables often attenuates association between dietary intake and disease occurrence. To adjust for the attenuation caused by error in dietary intake, regression calibration is commonly used. To apply regression calibration, unbiased reference measurements are required. Short-term reference measurements for foods that are not consumed daily contain excess zeroes that pose challenges in the calibration model. We adapted two-part regression calibration model, initially developed for multiple replicates of reference measurements per individual to a single-replicate setting. We showed how to handle excess zero reference measurements by two-step modeling approach, how to explore heteroscedasticity in the consumed amount with variance-mean graph, how to explore nonlinearity with the generalized additive modeling (GAM) and the empirical logit approaches, and how to select covariates in the calibration model. The performance of two-part calibration model was compared with the one-part counterpart. We used vegetable intake and mortality data from European Prospective Investigation on Cancer and Nutrition (EPIC) study. In the EPIC, reference measurements were taken with 24-hour recalls. For each of the three vegetable subgroups assessed separately, correcting for error with an appropriately specified two-part calibration model resulted in about three fold increase in the strength of association with all-cause mortality, as measured by the log hazard ratio. Further found is that the standard way of including covariates in the calibration model can lead to over fitting the two-part calibration model. Moreover, the extent of adjusting for error is influenced by the number and forms of covariates in the calibration model. For episodically consumed foods, we advise researchers to pay special attention to response distribution, nonlinearity, and covariate inclusion in specifying the calibration model.     

Heterologous Stacking of Prion Protein Peptides Reveals Structural Details of Fibrils and Facilitates Complete Inhibition of Fibril Growth

Fibrils play an important role in the pathogenesis of amyloidosis; however, the underlying mechanisms of the growth process and the structural details of fibrils are poorly understood. Crucial in the fibril formation of prion proteins is the stacking of PrP monomers. We previously proposed that the structure of the prion protein fibril may be similar as a parallel left-handed β-helix. The β-helix is composed of spiraling rungs of parallel β-strands, and in the PrP model residues 105–143 of each PrP monomer can contribute two β-helical rungs to the growing fibril. Here we report data to support this model. We show that two cyclized human PrP peptides corresponding to residues 105–124 and 125–143, based on two single rungs of the left-handed β-helical core of the human PrP^Sc fibril, show spontaneous cooperative fibril growth in vitro by heterologous stacking. Because the structural model must have predictive value, peptides were designed based on the structure rules of the left-handed β-helical fold that could stack with prion protein peptides to stimulate or to block fibril growth. The stimulator peptide was designed as an optimal left-handed β-helical fold that can serve as a template for fibril growth initiation. The inhibiting peptide was designed to bind to the exposed rung but frustrate the propagation of the fibril growth. The single inhibitory peptide hardly shows inhibition, but the combination of the inhibitory with the stimulatory peptide showed complete inhibition of the fibril growth of peptide huPrP-(106–126). Moreover, the unique strategy based on stimulatory and inhibitory peptides seems a powerful new approach to study amyloidogenic fibril structures in general and could prove useful for the development of therapeutics.Transmissible spongiform encephalopathies are neurodegenerative disorders in a wide range of mammalian species, including Creutzfeldt-Jacob disease in man, scrapie in sheep, and bovine spongiform encephalopathy in cattle. The deposition of aggregated prion protein fibrils on and in neurons is regarded to be the source of these neurodegenerative diseases and is frequently associated with occurrence of Congo red positivity (1–3). The fibrils are formed by the conformational change of the prion protein (PrP^c)² into the scrapie form (PrP^Sc). The misfolded conformer of the prion protein (PrP^Sc) is considered as the causative agent in these diseases according to the protein-only hypothesis (4). Studies have shown the toxicity of fibrils of the full-length recombinant mammalian prion protein as well as soluble β-rich oligomers to cultured cells and primary neurons (5).It is still unknown how much of the whole PrP^Sc molecule is involved in the fibril growth. It is shown that the N-terminal part of PrP, specifically residues 112–141, can go through conformational changes involving β-strand formation, which subsequently triggers fibril growth (6–8), and solid state NMR studies showed that residues 112–141 are part of the highly ordered core of huPrP-(23–144) (9). It was previously shown that peptides based on the 89–143 region of the human PrP protein can form fibrils rich in β-sheet structure which are biologically active in transgenic mice (10). Within this region it is the huPrP-(106–126) peptide that is the smallest known region of PrP that forms fibrils that are toxic and resemble the physiological properties of PrP^Sc (11–16). The formation of PrP^Sc is considered to be a two-step event; first, there is the binding between PrP^c and PrP^Sc and subsequently the conformational conversion from PrP^c into PrP^Sc occurs. Mutation studies in a prion-infected neuroblastoma cell line showed that in mouse PrP the regions 101–110 and 136–158 are crucial for the binding and conversion events, respectively (17). Because prevention of fibril growth is the prime therapeutic target, detailed structural knowledge of the fibril is essential for understanding the mechanism of fibril growth. However, structural analysis of amyloid fibrils is hampered by insolubility, isomorphism, and aggregation. X-ray diffraction of several amyloid fibrils revealed a so-called cross-β diffraction pattern which indicates that the fibrils contain β-strands perpendicular to the fibril axis and hydrogen bonds in parallel (18, 19). Thus, for fibril growth the β-strands have to stack on top of each other. Several structures have been suggested to explain the structure of the stacked β-strands; e.g. a parallel in register organization of stacked β hairpins (24) or the comparable dry steric zipper structure (25). Previously, we and other groups suggested that the β-sheet structures in the PrP^Sc fibril may be similar to the topologically most simple class of β-sheets; that is, the parallel left-handed β-helix (Fig. 1A) (6, 20, 21). The left-handed β helix is formed by triangular progressive coils (rungs) of 18–20 residues. Each rung is formed by three hexapeptide motifs, which results in an approximate 3-fold symmetry. Backbone-backbone hydrogen bonding and stacking of the side chains in adjacent rungs contribute to the folding of β-helical rungs. We suggested that each PrP^Sc monomer contributes two left-handed β-helical rungs to the fibril, comprising residues 105–124 and 125–143 (Fig. 1A). This two-rung structural model was recently confirmed for amyloid fibrils of the HET-s prion by NMR analysis (22). In contrast to fibrils which are composed of homologous stacks of identical peptides, e.g. the Aβ peptide (23), the PrP^Sc fibril is more complex because it is composed of heterologous stacks of at least two peptides. For homologous stacking of two identical peptides, the complementarity issue is relatively simple because the identical side chains are in register (e.g. Ile-Ile, Val-Val stacking, and Asn ladders). However, in the case of heterologous stacking, the side chains of the additional heterologous peptide needs to be complementary with the other peptide to allow fibril growth.Open in a separate windowFIGURE 1.A, theoretical model of the fibrillogenic core of PrP^Sc. In the PrP^Sc model based on the left-handed β-helix structure, each PrP^Sc monomer contributes two stacked rungs to the fibril (different color for each monomer). The protofibril is formed by consecutive stacking of the two windings. The stack of two rungs provides enough elevation to accommodate the remaining part (residues ∼ 146–253) of the PrP^Sc molecule (20). B, the left-handed β-helix structure of LpxA-based on x-ray crystallography. In the left-handed β-helix structure of LpxA (PDB code 1LXA) rungs 6 and 7 are indicated (red) that were used for the heterologous stacking studies. Linear and cyclized peptides based on rung 6 and rung 7 were modified to satisfy the ideal left-handed β-helix motif (see “LpxA Peptides” under “Results”) and tested for their intrinsic and cooperative fibrillogenicity. C, left-handed β-helical rung based on rung 6 of LpxA. The rung is formed by three hexapeptide motifs, which results in an approximate 3-fold symmetry. A left-handed β-helical rung can be cyclized by a disulfide bridge after the introduction of a cysteine at position 2 of the first hexapeptide and position 1 of the fourth hexapeptide (according to the numbering used for the hexapeptide repeats in the left-handed β-helix).To investigate whether the suggested rungs 105–123 and 125–143 from human PrP could be complementary (20), we studied the homologous stacking and the heterologous stacking of linear and cyclized prion protein peptides comprising the huPrP-(105–143) region (KTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGS). Qualitative and semiquantitative analysis were done by electron microscopy and Congo red staining. The quantification of the fibril formation was assessed by thioflavin S staining, in which the addition of polyanions (e.g. heparin) enhance the β-sheet formation of peptides comprising the 82–143 region of PrP and improve the reproducibility of the fibril growth (24). This study provides first evidence of heterologous stacking by two isolated putative β-strand layers (or rungs) of the human prion protein with fibril formation as a result. The left-handed β-helix structure provided insight for the “stack-and-stop” approach. With this approach a mix of a stimulatory peptide and an inhibitory peptide could completely block fibril formation. The stimulatory peptide was based on the 125–143 region that was optimized to serve as a folding template for the consecutive stacking of the 106–126 peptide. This cooperative fibril growth was completely inhibited by the inhibitory peptide based on peptides 106–126 with strategic d-amino acid and/or proline substitutions. The findings in this study support models in which the sequential strands in a fibril must somehow spiral up- or downward along the fibril axis, e.g. like the hypothetical left-handed β-helical structure of PrP^Sc fibrils (20). Furthermore, it allows the development of well defined small protein modules which can be used for structure studies of the 82–143 domain of PrP^Sc and the development of therapeutics.     

An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.     

Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.     

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.     

Small Sample Properties of the Mantel-Haenszel Test Statistic for Density Follow-Up Studies

For the analysis of combinations of 2×2 non-contingency tables as obtained from density follow-up studies (relating a number of events to a number of person-years of follow-up) an analogue of the Mantel-Haenszel test for 2×2 contingency tables is widely used. In this paper the small sample properties of this test, both with and without continuity correction, are evaluated. Also the improvement of the test-statistic by using the first four cumulants via the Edgeworth expansion was studied. Results on continuity correction agree with similar studies on the Mantel-Haenszel statistic for 2×2 contingency tables: Continuity correction gives a p-value which approximates the exact p-value better than the p-value obtained without this correction; both the exact test and its approximations show considerable conservatism in small samples; the uncorrected Mantel-Haenszel test statistic gives a p-value that agrees more with the nominal significance level, but can be anti-conservative. The p-value based on the first four cumulants gives a better approximation of the exact p-value than the continuity corrected test, especially when the distribution has marked skewness.     

Health Gain by Salt Reduction in Europe: A Modelling Study

Excessive salt intake is associated with hypertension and cardiovascular diseases. Salt intake exceeds the World Health Organization population nutrition goal of 5 grams per day in the European region. We assessed the health impact of salt reduction in nine European countries (Finland, France, Ireland, Italy, Netherlands, Poland, Spain, Sweden and United Kingdom). Through literature research we obtained current salt intake and systolic blood pressure levels of the nine countries. The population health modeling tool DYNAMO-HIA including country-specific disease data was used to predict the changes in prevalence of ischemic heart disease and stroke for each country estimating the effect of salt reduction through its effect on blood pressure levels. A 30% salt reduction would reduce the prevalence of stroke by 6.4% in Finland to 13.5% in Poland. Ischemic heart disease would be decreased by 4.1% in Finland to 8.9% in Poland. When salt intake is reduced to the WHO population nutrient goal, it would reduce the prevalence of stroke from 10.1% in Finland to 23.1% in Poland. Ischemic heart disease would decrease by 6.6% in Finland to 15.5% in Poland. The number of postponed deaths would be 102,100 (0.9%) in France, and 191,300 (2.3%) in Poland. A reduction of salt intake to 5 grams per day is expected to substantially reduce the burden of cardiovascular disease and mortality in several European countries.     

Do Intensive Care Data on Respiratory Infections Reflect Influenza Epidemics?

Objectives

Severe influenza can lead to Intensive Care Unit (ICU) admission. We explored whether ICU data reflect influenza like illness (ILI) activity in the general population, and whether ICU respiratory infections can predict influenza epidemics.

Methods

We calculated the time lag and correlation between ILI incidence (from ILI sentinel surveillance, based on general practitioners (GP) consultations) and percentages of ICU admissions with a respiratory infection (from the Dutch National Intensive Care Registry) over the years 2003–2011. In addition, ICU data of the first three years was used to build three regression models to predict the start and end of influenza epidemics in the years thereafter, one to three weeks ahead. The predicted start and end of influenza epidemics were compared with observed start and end of such epidemics according to the incidence of ILI.

Results

Peaks in respiratory ICU admissions lasted longer than peaks in ILI incidence rates. Increases in ICU admissions occurred on average two days earlier compared to ILI. Predicting influenza epidemics one, two, or three weeks ahead yielded positive predictive values ranging from 0.52 to 0.78, and sensitivities from 0.34 to 0.51.

Conclusions

ICU data was associated with ILI activity, with increases in ICU data often occurring earlier and for a longer time period. However, in the Netherlands, predicting influenza epidemics in the general population using ICU data was imprecise, with low positive predictive values and sensitivities.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.