Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.     

AKT isoforms modulate Th1‐like Treg generation and function in human autoimmune disease

Foxp3⁺ regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNγ, together with the acquisition of a T helper 1 (Th1)‐like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1‐Tregs. Using a genome‐wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNγ secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1‐Treg generation ex vivo. Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1‐Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo‐isolated Tregs from untreated relapsing–remitting MS patients and that blockade of the pathway inhibits IFNγ secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.     

The quantitative analysis of the vascularization following two basic auditory canal skin incisions

Three groups of nine patients each were analyzed. The first two groups consisted of those that underwent tympanoplastic due to chronic inflammation of middle ear. Two different standard auditory canal skin incisions were applied, i.e. tympanomeatal flap (TMF) or vascular strip (VS). The third control group consisted of non-operated patients. All the operated patients were subjected to a quantitative analysis of the auditory canal revascularization by means of the Weibel stereological test method, i.e. the B 100 double network system. The density of capillaries, arterioles, venulolymphatic spaces and a total volume density of all vascular elements of the auditory canal skin were measured. The obtained results of vascularization were compared with those of the target control group. It was found out that there were no significant differences in vascularization of auditory canal skin between TMF and VS patients from one side and the control group on the other side.     

Learning Biomarkers of Pluripotent Stem Cells in Mouse

Pluripotent stem cells are able to self-renew, and to differentiate into all adult cell types. Many studies report data describing these cells, and characterize them in molecular terms. Machine learning yields classifiers that can accurately identify pluripotent stem cells, but there is a lack of studies yielding minimal sets of best biomarkers (genes/features). We assembled gene expression data of pluripotent stem cells and non-pluripotent cells from the mouse. After normalization and filtering, we applied machine learning, classifying samples into pluripotent and non-pluripotent with high cross-validated accuracy. Furthermore, to identify minimal sets of best biomarkers, we used three methods: information gain, random forests and a wrapper of genetic algorithm and support vector machine (GA/SVM). We demonstrate that the GA/SVM biomarkers work best in combination with each other; pathway and enrichment analyses show that they cover the widest variety of processes implicated in pluripotency. The GA/SVM wrapper yields best biomarkers, no matter which classification method is used. The consensus best biomarker based on the three methods is Tet1, implicated in pluripotency just recently. The best biomarker based on the GA/SVM wrapper approach alone is Fam134b, possibly a missing link between pluripotency and some standard surface markers of unknown function processed by the Golgi apparatus.     

3D QSAR study, synthesis, and in vitro evaluation of (+)-5-FBVM as potential PET radioligand for the vesicular acetylcholine transporter (VAChT)

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (～70-fold), only (+)-5-FBVM is selective for σ(1) receptors (～fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.     

Recent vegetation history of the North Adriatic grasslands: Expansion and decay of an anthropogenic habitat

A summary pollen diagram and old cartographic materials were used to reconstruct the recent vegetation history and trends of temporal dynamics of the North Adriatic Karst grasslands. Before the Roman period some evidence about grassland vegetation exists from Neolithic settlements, but deforestation, detectable on the landscape level because of pollen, started 2500-2000 years BP. A large-scale anthropogenically-driven process of clearance started in the Roman period as is reflected in the increasing pollen of grasses (Poaceae) and other herbaceous species (Apiaceae). The presence of pioneer trees (Quercus, Ostrya), juniper and grasses during 1000-400 years BP suggests that cycling processes of cultivation (strong grazing pressure), abandonment and re-cultivation were present. A 250-year-old map shows a peak of probable deforestation, where grasslands have 3.2 times larger surface area than on a recent land-use map in a 665.8 km² large pilot area. More than 60% of grasslands changed into forest since then. On a sub-plot area of 626 ha (1% of the pilot area), where habitat mappings were performed, only 12.8% were still grasslands without tall herb invasions or scrub encroachments. Another 27.1% were grasslands with early signs of reforestation. The traditionally open landscape of the North Adriatic Karst was able to recover to forest due to almost total abandonment and sufficient mesic climate conditions.     

Using the Simulated Patient Methodology to Assess Paracetamol-Related Counselling for Headache

Objectives

Firstly, to assess paracetamol-related counselling. Secondly, to evaluate the patient’s approach as a determinant of counselling and to test the acceptability of the simulated patient method in Slovenian pharmacies.

Methods

The simulated patient methodology was used in 17 community pharmacies. Three scenarios related to self-medication for headaches were developed and used in all participating pharmacies. Two scenarios were direct product requests: scenario 1: a patient with an uncomplicated short-term headache; scenario 2: a patient with a severe, long-duration headache who takes paracetamol for too long and concurrently drinks alcohol. Scenario 3 was a symptom-based request: a patient asking for medicine for a headache. Pharmacy visits were audio recorded and scored according to predetermined criteria arranged in two categories: counselling content and manner of counselling. The acceptability of the methodology used was evaluated by surveying the participating pharmacists.

Results

The symptom-based request was scored significantly better (a mean 2.17 out of a possible 4 points) than the direct product requests (means of 1.64 and 0.67 out of a possible 4 points for scenario 1 and 2, respectively). The most common information provided was dosage and adverse effects. Only the symptom-based request stimulated spontaneous counselling. No statistically significant differences in the duration of the consultation between the scenarios were found. There were also no significant differences in the quality of counselling between the Masters of Pharmacy and Pharmacy Technicians. The acceptability of the SP method was not as high as in other countries.

Conclusion

The assessment of paracetamol-related counselling demonstrates room for practice improvement.     

Intracranial aneurysm risk locus 5q23.2 is associated with elevated systolic blood pressure

Although genome-wide association studies (GWAS) have identified hundreds of complex trait loci, the pathomechanisms of most remain elusive. Studying the genetics of risk factors predisposing to disease is an attractive approach to identify targets for functional studies. Intracranial aneurysms (IA) are rupture-prone pouches at cerebral artery branching sites. IA is a complex disease for which GWAS have identified five loci with strong association and a further 14 loci with suggestive association. To decipher potential underlying disease mechanisms, we tested whether there are IA loci that convey their effect through elevating blood pressure (BP), a strong risk factor of IA. We performed a meta-analysis of four population-based Finnish cohorts (n(FIN) = 11 266) not selected for IA, to assess the association of previously identified IA candidate loci (n = 19) with BP. We defined systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure as quantitative outcome variables. The most significant result was further tested for association in the ICBP-GWAS cohort of 200 000 individuals. We found that the suggestive IA locus at 5q23.2 in PRDM6 was significantly associated with SBP in individuals of European descent (p(FIN) = 3.01E-05, p(ICBP-GWAS) = 0.0007, p(ALL) = 8.13E-07). The risk allele of IA was associated with higher SBP. PRDM6 encodes a protein predominantly expressed in vascular smooth muscle cells. Our study connects a complex disease (IA) locus with a common risk factor for the disease (SBP). We hypothesize that common variants in PRDM6 can contribute to altered vascular wall structure, hence increasing SBP and predisposing to IA. True positive associations often fail to reach genome-wide significance in GWAS. Our findings show that analysis of traditional risk factors as intermediate phenotypes is an effective tool for deciphering hidden heritability. Further, we demonstrate that common disease loci identified in a population isolate may bear wider significance.