Evaluation of serum glycoprotein biomarker candidates for detection of esophageal adenocarcinoma and surveillance of Barrett’s esophagus

Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett’s esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance for BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. Serum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and USA (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier 3 assay. The area under receiver operating characteristic curve (AUROC) was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multimarker panel for BE surveillance. Complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarkers in lectin pull-down samples for EAC across both cohorts. A panel of 10 serum glycoprotein biomarker candidates discriminated BE patients not requiring intervention [BE+/- low grade dysplasia] from those requiring intervention [BE with high grade dysplasia (BE-HGD) or EAC] with an AUROC value of 0.93. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed towards EAC, levels of serum C9 were significantly (P\u3c0.05) increased with disease progression in EPHA (erythroagglutinin from Phaseolus vulgaris) and NPL (Narcissus pseudonarcissus lectin) pull-down samples. The results confirm alteration of complement pathway glycoproteins during BE-EAC pathogenesis. Further prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted, prior to development of a first-line BE surveillance blood test

The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-κB p65

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-κB, to the host cell nucleus. NF-κB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-κB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFα and IL-1β stimulated p65 nuclear translocation and IκB degradation, NleB inhibited the TNFα pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators

The cellular redox environment alters antigen presentation

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5-10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.This work was supported, in whole or in part, by National Institutes of Health Grant R01 NS036592. This work was also supported by an infrastructure grant (Grant LE100100036) from the Australian Research Council (ARC) and a project grant from the Juvenile Diabetes Research Foundation (17-2012-134)

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Prevention of Cytotoxic T Cell Escape Using a Heteroclitic Subdominant Viral T Cell Determinant

High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2Kb to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2Kb. The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity

Herbst / multibracket appliance treatment in adult Class II:1 malocclusions

Das Ziel der vorliegenden Arbeit war es, das Ausmaß der dentoskelettalen und fazialen Veränderungen bei der Distalbissbehandlung (Klasse II-1) von Erwachsenen mit einer Herbst- /Multibracket-Apparatur anhand der Auswertung von Fernröntgenseitenbildern des Kopfes (FRS) zu ermitteln. Das Patientengut umfasste 23 erwachsene Probanden (4 männliche und 19 weibliche) mit einer Angle-Klasse-II-1. Das Alter der Probanden lag am Anfang der Behandlung durchschnittlich bei 21,9 Jahren. Alle Probanden wurden mit einer Herbst- und anschließender Multibracket-Apparatur behandelt. Fernröntgenseitenbilder (FRS) des Kopfes der Probanden wurden zu drei Zeitpunkten analysiert: T1: vor der Herbst-Behandlung T2: nach der Herbst-Behandlung T3: nach der anschließenden Multibracket-Behandlung Die Veränderungen der sagittalen Okklusion, der sagittalen und vertikalen Kieferrelation sowie der Gesichtshöhe, Profilkonvexität und der Lippenposition wurden ermittelt. Die kephalometrischen Veränderungen während drei Untersuchungs-zeiträumen wurden festgehalten: T2-T1: Herbst-Phase T3-T2: Multibracket-Phase (MB-Phase) T3-T1: Totaler Behandlungszeitraum Die Untersuchung lieferte folgende Ergebnisse: Der Overjet wurde während der Herbst-Phase um 9,98 mm korrigiert. Die Veränderungen waren zu 79 % dental und 21 % skelettal bedingt. Die Molarenrelation verbesserte sich um 6,82 mm. Dies war zu 69 % dental und 31 % skelettal bedingt. In der nachfolgenden MB-Phase rezidivierte der Overjet und die Molarenrelation leicht. Während des totalen Behandlungszeitraumes kam es in allen Fällen zu einer Korrektur des Distalbisses (Normalisierung des Overjets und der sagittalen Molarenrelation). Die Overjet-Korrektur von 6,75 mm teilte sich in 87 % dentale und 13 % skelettale Veränderungen. Die Korrektur der Molarenrelation von 4,11 mm bestand aus 78 % dentalen und 22 % skelettalen Veränderungen. Die sagittale Kieferrelation wurde während der Herbst-Phase signifikant (p<0,001) verändert. Vergrößert wurden die Winkel SNB (1,22°) und SNPg (0,93°), verkleinert wurden die Winkel ANB (1,22°) und ANPg (0,95°) sowie der Wits-Wert (2,30 mm). In der MB-Phase veränderten sich die Werte wieder leicht rückläufig. Während des totalen Behandlungszeitraumes kam es zu einer signifikanten (p<0,001) Vergrößerung der Winkel SNB (0,82°) und SNPg (0,70°) sowie zu einer Verkleinerung der Winkel ANB (0,70°), ANPg (0,60°) und des Wits-Wertes (1,08 mm). Der SNA-Winkel veränderte sich nicht. Die vertikale Kieferrelation (die Winkel ML/NSL, NL/NSL, ML/NL) wurde durch die Behandlung nicht beeinflusst. Die Verkleinerung des ML/NSL-Winkels ist auf normale Wachstumsveränderungen zurückzuführen. Die untere Gesichtshöhe vergrößerte sich während der Herbst-Phase signifikant (p<0,001), anterior mit einem Indexwert von 1,14 und posterior mit einem Indexwert von 1,98. Während der MB-Phase verkleinerten sich diese Indexwerte wieder leicht. Während des totalen Behandlungszeitraumes vergrößerte sich die untere Gesichtshöhe signifikant sowohl anterior mit einem Indexwert von 0,42 (p<0,05) als auch posterior mit einem Indexwert von 1,03 (p<0,01). Die Winkel zur Beschreibung der Profilkonvexität vergrößerten sich während der Herbst-Phase signifikant (p<0,001), NAPg um 2,14°, NsSnPgS um 3,92° und NsNoPgS um 2,20°. Das Hart- und Weichteilprofil wurde dadurch gerader. In der MB-Phase verkleinerten sich die Winkel wieder leicht. Während des totalen Be-handlungszeitraumes vergrößerten sich NAPg um 1,09° (p<0,01), NsSnPgS um 3,14° (p<0,001) und NsNoPgS um 1,04°(p<0,05) signifikant. Der Abstand der Oberlippe zur Esthetic-Linie wurde während der Herbst-Phase signifikant (p<0,001) um 1,63 mm größerder Abstand der Unterlippe zur Esthetic-Linie wurde signifikant (p<0,01) um 0,84 mm kleiner. In der MB-Phase vergrößerte sich der Abstand der Unterlippe wieder, der Abstand der Oberlippe veränderte sich nicht. Der Abstand der Oberlippe zur Esthetic-Linie veränderte sich während des totalen Behandlungszeitraumes signifikant (p<0,001) um 1,26 mm nach posterior, die Position der Unterlippe veränderte sich kaum. Schlussfolgernd kann festgestellt werden, dass eine Distalbissbehandlung von Erwachsenen mit der Herbst- /Multibracket-Apparatur außerordentlich erfolgreich ist und eine Alternative zu einer kieferchirurgischen Unter-kiefervorverlagerung sein könnte.The aim of this study was to assess cephalometrically the amount of dento-skeletal and facial changes during Herbst- /multibracket appliance treatment of adult Class II-11 subjects. The material consisted of 23 adult patients (4 males and 19 females) with a Class II-1. At the beginning of treatment the patients were, on average, 21,9 years old. All patients were treated with a Herbst appliance which was followed by a multibracket appliance. Lateral head films were analysed at three different times: T1: before Herbst treatment T2: after Herbst treatment T3: after multibracket treatment The changes in sagittal occlusion, sagittal and vertical jaw relationship as well as in facial height, profile convexity and lip position were assessed during three examination periods: T2-T1: Herbst phase of treatment T3-T2: multibracket phase of treatment T3-T1: total treatment period The following results were obtained: During the Herbst phase overjet correction was, on average, 9.98 mm. This was accomplished by 79 % dental and 21 % skeletal changes. Class II molar correction (6.82 mm on average) was due to 69 % dental and 31 % skeletal changes. During the following MB phase there was an insignificant relapse of the overjet and molar correction. At the end of the total treatment period, overjet and Class II molar relation were normalised in all patients. Overjet correction was, on average, 6.75 mm, due to 87 % dental and 13 % skeletal changes. Class II molar correction was, on average, 4.11 mm, resulting from 78 % dental and 22 % skeletal changes. Sagittal jaw relation was improved during the Herbst phase. An increase (p<0.001) was found in the angles SNB (1.22°) and SNPg (0.93°), thus resulting in a decrease (p<0.001) in the angles ANB (1.22°), ANPg (0.95°) and in the Wits-Appraisal (2.30 mm). During the following multibracket phase the variables relapsed insignificantly. At the end of the total treatment period an increase (p<0.001) was abserved for the angles SNB (0.82°) and SNPg (0.70°) and a decrease (p<0.001) for the angles ANB (0.70°), ANPg (0.60°) and Wits (1.08 mm). The angle SNA remained unchanged. Vertical jaw relation (ML/NSL, NL/NSL, ML/NL) did not change during the total treatment period. The reduction of ML/NSL was a result of normal growth. Lower facial height increased (p<0.001) during the Herbst phase both anteriorly (Index 1.14) and posteriorly (Index 1.98). During the MB phase the facial height relapsed insignificantly. At the end of the total treatment period lower facial height had increased significantly both anteriorly (Index 0.42, p<0.05)) and posteriorly (Index 1.03, p<0.01). The profile convexity angles increased (p<0.001) during the Herbst phase: NAPg (2.14°), NsSnPgS (3.92°) and NsNoPgS (2.20°). Thus, the profile straightened. During the MB phase, the angles relapsed insignificantly. At the end of the total treatment period the profile convexity angles had increased significantly: NAPg (1.09°, p<0.01), NsSnPgS (3.14°, p<0.001) and NsNoPgS (1.04°, p<0.05). The distance of the upper lip to the E-line increased (1.63 mm, p<0.001) during the Herbst phase, while the distance of the lower lip to the E-line decreased (0.84 mm, p<0.01). During the MB phase the position of lower lip relapsed insignificantly, the upper lip remained unchanged. At the end of the total treatment period the upper lip moved posteriorly (1.26 mm, p<0.001), while the lower lip remained unchanged. It can be concluded that treatment of adult Class II subjects with the Herbst- /multibracket appliance is successful. The treatment approach could be considered as a possible alternative to surgical mandibular advancement in borderline adult Class II subjects

A New Model for Pore Formation by Cholesterol- Dependent Cytolysins

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three b-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs

Domain 2-Domain 3 interface features and Molecular simulations performed.

a<p>Pairs of residues with corresponding numbering are given in brackets.</p>b<p>The number of simulations where transitions of the Domain 2/TMH2 interface occurs is given in brackets. Except for PFO I MD simulations, only one simulation is presented in this work.</p