Naturally Presented Peptides on Major Histocompatibility Complex I and II Molecules Eluted from Central Nervous System of Multiple Sclerosis Patients

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.T cells recognize antigen bound to MHC¹ molecules (1). CD4 as well as CD8 T cells have been shown to play a pathogenic role in various autoimmune diseases (2). Pathogenic T cells infiltrate the target organs and locally secrete proinflammatory cytokines and chemokines leading to tissue inflammation and possibly subsequent tissue destruction (3–5). Local presentation of autoantigens by MHC molecules in the target tissue of the autoimmune attack, i.e. the central nervous system (CNS) in multiple sclerosis (MS) or the pancreas in diabetes, is therefore a prerequisite for local immune amplification (6). MS is an inflammatory and neurodegenerative disease of the CNS leading to myelin and axonal loss (7). There are different disease courses, i.e. relapsing-remitting, secondary chronic progressive, and primary progressive disease. Potential autoantigens in MS include myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). It is thought that T cells enter the CNS from the systemic circulation and that they are subsequently reactivated in the CNS on MHC I and MHC II molecules expressed on local antigen-presenting cells (APC) (8).To date, naturally presented HLA-bound peptides from patients with MS thus far have not been isolated and identified. So far, only circumstantial evidence exists for the local presentation of autoantigens such as MBP on MHC molecules in CNS (9). The aim of this study consisted of the characterization of the MHC-bound peptide repertoire derived from brains of patients with MS. Cutting edge technology combining HPLC and tandem mass spectrometry has recently allowed us to define peptides presented on APC from bronchoalveolar lavage from lungs of sarcoidosis patients (10). Applying a similar method on autopsy material of MS patients, for the first time we demonstrated local presentation of previously known and potential novel autoantigens in MS.     

Parallel pattern of differentiation at a genomic island shared between clinal and mosaic hybrid zones in a complex of cryptic seahorse lineages

Diverging semi‐isolated lineages either meet in narrow clinal hybrid zones, or have a mosaic distribution associated with environmental variation. Intrinsic reproductive isolation is often emphasized in the former and local adaptation in the latter, although both reduce gene flow between groups. Rarely are these two patterns of spatial distribution reported in the same study system. Here, we report that the long‐snouted seahorse Hippocampus guttulatus is subdivided into discrete panmictic entities by both types of hybrid zones. Along the European Atlantic coasts, a northern and a southern lineage meet in the southwest of France where they coexist in sympatry—i.e., in the same geographical zone—with little hybridization. In the Mediterranean Sea, two lineages have a mosaic distribution, associated with lagoon‐like and marine habitats. A fifth lineage was identified in the Black Sea. Genetic homogeneity over large spatial scales contrasts with isolation maintained in sympatry or close parapatry at a fine scale. A high variation in locus‐specific introgression rates provides additional evidence that partial reproductive isolation must be maintaining the divergence. We find that fixed differences between lagoon and marine populations in the Mediterranean Sea belong to the most differentiated SNPs between the two Atlantic lineages, against the genome‐wide pattern of structure that mostly follow geography. These parallel outlier SNPs cluster on a single chromosome‐wide island of differentiation. Since Atlantic lineages do not map to lagoon‐sea habitat variation, genetic parallelism at the genomic island suggests a shared genetic barrier contributes to reproductive isolation in contrasting contexts–i.e., spatial versus ecological. We discuss how a genomic hotspot of parallel differentiation could have evolved and become associated both with space and with a patchy environment in a single study system.     

Comprehensive postmortem analyses of intestinal microbiota changes and bacterial translocation in human flora associated mice

Background

Postmortem microbiological examinations are performed in forensic and medical pathology for defining uncertain causes of deaths and for screening of deceased tissue donors. Interpretation of bacteriological data, however, is hampered by false-positive results due to agonal spread of microorganisms, postmortem bacterial translocation, and environmental contamination.

Methodology/Principal Findings

We performed a kinetic survey of naturally occurring postmortem gut flora changes in the small and large intestines of conventional and gnotobiotic mice associated with a human microbiota (hfa) applying cultural and molecular methods. Sacrificed mice were kept under ambient conditions for up to 72 hours postmortem. Intestinal microbiota changes were most pronounced in the ileal lumen where enterobacteria and enterococci increased by 3–5 orders of magnitude in conventional and hfa mice. Interestingly, comparable intestinal overgrowth was shown in acute and chronic intestinal inflammation in mice and men. In hfa mice, ileal overgrowth with enterococci and enterobacteria started 3 and 24 hours postmortem, respectively. Strikingly, intestinal bacteria translocated to extra-intestinal compartments such as mesenteric lymphnodes, spleen, liver, kidney, and cardiac blood as early as 5 min after death. Furthermore, intestinal tissue destruction was characterized by increased numbers of apoptotic cells and neutrophils within 3 hours postmortem, whereas counts of proliferative cells as well as T- and B-lymphocytes and regulatory T-cells decreased between 3 and 12 hours postmortem.

Conclusions/Significance

We conclude that kinetics of ileal overgrowth with enterobacteria and enterococci in hfa mice can be used as an indicator for compromized intestinal functionality and for more precisely defining the time point of death under defined ambient conditions. The rapid translocation of intestinal bacteria starting within a few minutes after death will help to distinguish between relevant bacteria and secondary contaminants thus providing important informations for routine applications and future studies in applied microbiology, forensic pathology, and criminal medicine.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing

Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain‐containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein‐truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.     

Weitere Untersuchungen über die formative Wirkung des Lichtes und mechanischer Reize auf Pflanzen

Zusammenfassung der Ergebnisse Auf das Längenwachstum der Hypokotyle vonSinapis alba wirkt intermittierendes Licht stärker wachstumshemmend (etiolementsverhindernd) als (bei gleicher Reizsumme) kontinuierliches; die Hemmung wird weiterhin mit zunehmendem Zeitabstand zwischen den Teilreizen stärker. Hieraus kann geschlossen werden, daß jeder Teilreiz ein Refraktärstadium von mehreren Minuten Dauer induziert, das erst abgeklungen sein muß, wenn ein zweiter Reiz voll wirksam werden soll.Die formative Wirkung des Lichtes auf das Blatt ist anderer Natur; das zeigen auch ältere Beobachtungen über die unterschiedliche Abhängigkeit von der Lichtqualität.Auch durch mechanische Reize wird das Längenwachstum der Hypokotyle vonSinapis um so mehr gehemmt, je größer der Zeitabstand zwischen den Teilreizen der intermittierenden Reizung ist; auch hier machen sich also Refraktärstadien geltend.Diese Beobachtungen stützen die schon früher gezogene Schlußfolgerung, daß eine Komponente der formativen Wirkung des Lichtes in der Auslösung von Erregungsvorgängen im engeren Sinne besteht, wie sie ähnlich auch durch mechanische Reizung bedingt werden können.Die Beeinflussung der Gewebedifferenzierung in den Internodien vonVicia Faba durch mechanische Reize ist teilweise übereinstimmend mit der durch photische Reizung bedingten, so daß man für einen Teil dieser Beeinflussungen, namentlich für die Ausbildung von Wandverdickungen, vermuten darf, daß sie bei beiden Reizarten gemeinsam durch die Vermittlung jener Erregungsvorgänge entsteht.Mit 6 Textabbildungen.     

A hierarchical classification of freshwater mussel diversity in North America

Aim North America harbours the most diverse freshwater mussel fauna on Earth. This fauna has high endemism at the continental scale and within individual river systems. Previous faunal classifications for North America were based on intuitive, subjective assessments of species distributions, primarily the occurrence of endemic species, and do not portray continent‐wide patterns of faunal similarity. The aim of this study is to provide an analytical portrayal of patterns of mussel diversity in a hierarchical framework that informs the biogeographical history of the fauna. Location The study considered the mussel fauna of North America from the Rio Grande system northwards. Methods Patterns of mussel faunal similarity in 126 river systems or lake watersheds across North America were examined. The dataset was developed from the literature and consisted of recent species presence/absence (282 species) in each drainage unit; subspecies were not included. Patterns of mussel diversity were examined with hierarchical cluster analysis, based on a pairwise distance matrix between all drainage units. Results Cluster analysis revealed 17 faunal provinces within four major faunal regions: Mississippian, Atlantic, Eastern Gulf and Pacific. The Mississippian Region dominates the North American fauna with 11 provinces, including five not recognized by previous classifications: Mississippi Embayment, Upper Mississippi, Great Plains, Ohioan and Pontchartrain–Pearl–Pascagoula. Within the Eastern Gulf Region (containing three provinces), the Escambia–Choctawhatchee Province is distinctive from the Apalachicolan Province, under which it was previously subsumed. Patterns of diversity in the Atlantic Region (two provinces) and Pacific Region (one province) were similar to previous classifications. Main conclusions The classification proposed in this study largely corroborates earlier schemes based on the occurrence of endemic species but identifies additional heterogeneity that reflects unique assemblages of widely distributed species. The study proposes a hierarchical structure that illustrates relationships among these provinces. Although some provinces in the Mississippian Region have high endemism, all Mississippian provinces share a group of widely distributed species. The Atlantic and Eastern Gulf regions have distinctive, endemic faunas suggesting limited past connectivity with the Mississippian Region. The Pacific Region is the most distinct fauna in North America and bears close affinity to the Eurasian mussel fauna.