Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.     

Der Einfluss von Gebirge und See auf die 17-Ketosteroidausscheidung im Harn

Zusammenfassung Bei 11 Studenten aus Mainz wurde im September 1964 in Oberstdorf/Allgäu (843 m) und im September 1965 auf der Nordseeinsel Norderney täglich die Ausscheidung der 17-Ketosteroide untersucht, um den Adaptationsverlauf zu prüfen. Alle Versuchspersonen hatten eine erhöhte 17-KS-Ausscheidung,die an der Nordsee höher war als im Gebirge. Der Höhepunkt der Ausscheidung lag um den 10. Tag. Es bestanden individuelle Unterschiede in der 17-KS-Ausscheidung. Je niedriger diese vorher war, desto höher war die relative Steigerung im Gebirge und an der See.Während der Beobachtungsperiode waren wetterbedingte Abweichungen von den periodischen Schwankungen nachweisbar.

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The 17-ketosteroid excretion of 11 students from the University of Mainz was investigated during September 1964 in Oberstdorf (843 m) and in September 1965 on the North Sea island Norderney. All subjects had an increased 17-ketosteroid excretion which was higher at the sea than in the mountains.The maximum of the values was around the 10th day. There were individual differences; the lower the excretion at Mainz the higher was the relative increase at both locations. During the observation period deviations from the periodic changes were observed which were due to the effect of weather.

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Resume Afin d'examiner l'adaptation de 11 étudiants au milieu,on a analysé chaque jour leur excrétion de 17-kétostéroïde. Ces essais ont eu lieu en septembre 1964 à Oberstdorf/Allgäu (843 m d'altitude) et en septembre 1965 sur lïle de Norderney dans la Mer du Nord. On a pu déceler une augmentation de 17-kétostéroïde chez toutes les personnes examinées. Cette augmentation fut toutefois plus marquée au bord de la mer qu'en montagne. L'excrétion en fut maximum le 10ème jour.On a remarqué en outre des différences individuelle. Plus l'excrétion était faible au départ et plus l'augmentation relative était marquée ausi bien au bord de la mer qu'en montagne.Pendant la période des examens, on a pu prouver des divergences météorotropiques par rapport aux fluctuations périodiques.

     

Capillary blood perfusion during postischemic reperfusion in striated muscle.

Monitoring of nutritive blood flow in muscle is of particular importance to reconstructive surgeons, since ischemia/reperfusion in striated muscle is known to result in postischemic microvascular perfusion failure. Laser Doppler flowmetry has recently been introduced as an easy-to-use, noninvasive technique for continuous monitoring of microvascular tissue perfusion. Despite its popularity, there exists a great deal of controversy as to what actually generates the laser Doppler signal recorded from a given tissue. Intravital microscopy is a technique for direct visualization of the nutritional circulation in tissue. By using intravital microscopy, direct measurements of blood perfusion in individual segments of the nutritional microcirculation can be made. In 22 Syrian golden hamsters we performed laser Doppler flowmetry and intravital microscopy measurements in muscle tissue prior to and during reperfusion after 4 hours of tourniquet ischemia using the dorsal skinfold chamber model. Intravital microscopy (n = 10) revealed a heterogeneous capillary perfusion during the early reperfusion phase with a decrease (p less than 0.01) in functional capillary density to 49.4 +/- 17.0 percent of control. No recovery was observed after 24 hours of reperfusion. Laser Doppler flowmetry (n = 12) showed a parallel reduction of capillary red blood cell flux during the early perfusion phase to 43.9 +/- 22.6 percent of control values (p less than 0.01), and no recovery was observed after 24 hours of reperfusion. However, the laser Doppler flowmetry technique was not able to detect the capillary perfusion inhomogeneities shown by intravital microscopy. Postischemic reperfusion in striated muscle is characterized by a decrease in functional capillary density and a heterogeneous capillary perfusion. Laser Doppler flowmetry is a useful tool for monitoring microvascular tissue perfusion, although in striated muscle of the hamster it must be considered that accurate nutritional "capillary" flow readings can be grossly overestimated if larger vessels, such as arterioles and collecting venules, are contained in the measuring field of the laser Doppler probe.     

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.     

Pattern classification of kinematic and kinetic running data to distinguish gender,shod/barefoot and injury groups with feature ranking

The identification of differences between groups is often important in biomechanics. This paper presents group classification tasks using kinetic and kinematic data from a prospective running injury study. Groups composed of gender, of shod/barefoot running and of runners who developed patellofemoral pain syndrome (PFPS) during the study, and asymptotic runners were classified.

The features computed from the biomechanical data were deliberately chosen to be generic. Therefore, they were suited for different biomechanical measurements and classification tasks without adaptation to the input signals. Feature ranking was applied to reveal the relevance of each feature to the classification task.

Data from 80 runners were analysed for gender and shod/barefoot classification, while 12 runners were investigated in the injury classification task. Gender groups could be differentiated with 84.7%, shod/barefoot running with 98.3%, and PFPS with 100% classification rate. For the latter group, one single variable could be identified that alone allowed discrimination.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

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.     

Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.