Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

Antigen presentation to celiac lesion-derived T cells of a 33-mer gliadin peptide naturally formed by gastrointestinal digestion

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (alpha(2)-gliadin 56-88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.     

Hierarchical fine mapping of the cystic fibrosis modifier locus on 19q13 identifies an association with two elements near the genes CEACAM3 and CEACAM6

On 19q13, TGFB1 and the cystic fibrosis modifier 1 locus (CFM1) have been identified as modifiers of the course of the monogenic disease cystic fibrosis (CF). Recently, we have described a transmission disequilibrium at the microsatellite D19S197, localized between TGFB1 and CFM1. To map the corresponding molecular variants, we have selected informative SNP markers within a 600-kb area and compared two-marker-haplotype-distributions between phenotypically contrasting sib pair groups, intending to type only phylogenetically old markers by aiming for close-to-maximal polymorphism information content of the SNPs. Starting with a seed set of five SNPs that cover intermarker distances of up to 50 kb, we have iteratively added more SNPs to the map, until we could identify two genomic fragments of 3,289 and 2,052 bp for which pairs with contrasting phenotypes showed different haplotype distributions on the final 17-SNP-map (P _raw = 0.0002, P _corr17SNPs = 0.0106 and P _raw = 0.0008, P _corr17SNPs = 0.0469, respectively). Resequencing of these fragments of four unrelated individuals for each element showed that the mildly and severely affected pairs differ in seven SNPs and concordant pairs differ from discordant pairs in five SNPs. Annotation of these variants indicate that CEACAM6 and a regulatory element near the 3′ end of CEACAM3 are associated with CF disease severity and intrapair discordance, respectively. While our approach was only guided by the markers’ position, the involvement of genes from the CEACAM family in host defense and innate immunity designates these proteins as likely modifiers of the multi-organ disease cystic fibrosis which is known for its cytokine imbalance and pro-inflammatory phenotype.     

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.     

Create and assess protein networks through molecular characteristics of individual proteins

MOTIVATION: The study of biological systems, pathways and processes relies increasingly on analyses of networks. Most often, such analyses focus on network topology, thereby treating all proteins or genes as identical, featureless nodes. Integrating molecular data and insights about the qualities of individual proteins into the analysis may enhance our ability to decipher biological pathways and processes. RESULTS: Here, we introduce a novel platform for data integration that generates networks on the macro system-level, analyzes the molecular characteristics of each protein on the micro level, and then combines the two levels by using the molecular characteristics to assess networks. It also annotates the function and subcellular localization of each protein and displays the process on an image of a cell, rendering each protein in its respective cellular compartment. By thus visualizing the network in a cellular context we are able to analyze pathways and processes in a novel way. As an example, we use the system to analyze proteins implicated with Alzheimers disease and show how the integrated view corroborates previous observations and how it helps in the formulation of new hypotheses regarding the molecular underpinnings of the disease. AVAILABILITY: http://www.rostlab.org/services/pinat.     

Computational recognition of potassium channel sequences

MOTIVATION: Potassium channels are mainly known for their role in regulating and maintaining the membrane potential. Since this is one of the key mechanisms of signal transduction, malfunction of these potassium channels leads to a wide variety of severe diseases. Thus potassium channels are priority targets of research for new drugs, despite the fact that this protein family is highly variable and closely related to other channels, which makes it very difficult to identify new types of potassium channel sequences. RESULTS: Here we present a new method for identifying potassium channel sequences (PSM, Property Signature Method), which-in contrast to the known methods for protein classification-is directly based on physicochemical properties of amino acids rather than on the amino acids themselves. A signature for the pore region including the selectivity filter has been created, representing the most common physicochemical properties of known potassium channels. This string enables genome-wide screening for sequences with similar features despite a very low degree of amino acid similarity within a protein family.     

Three Types of Tyrosine Hydroxylase-Positive CNS Neurons Distinguished by Dopa Decarboxylase and VMAT2 Co-Expression

Sumary 1. We investigate here for the first time in primate brain the combinatorial expression of the three major functionally relevant proteins for catecholaminergic neurotransmission tyrosine hydroxylase (TH), aromatic acid acid decarboxylase (AADC), and the brain-specific isoform of the vesicular monoamine transporter, VMAT2, using highly specific antibodies and immunofluorescence with confocal microscopy to visualize combinatorial expression of these proteins.2. In addition to classical TH, AADC, and VMAT2-copositive catecholaminergic neurons, two unique kinds of TH-positive neurons were identified based on co-expression of AADC and VMAT2.3. TH and AADC co-positive, but VMAT2-negative neurons, are termed “nonexocytotic catecholaminergic TH neurons.” These were found in striatum, olfactory bulb, cerebral cortex, area postrema, nucleus tractus solitarius, and in the dorsal motor nucleus of the vagus.4. TH-positive neurons expressing neither AADC nor VMAT2 are termed “dopaergic TH neurons.” We identified these neurons in supraoptic, paraventricular and periventricular hypothalamic nuclei, thalamic paraventicular nucleus, habenula, parabrachial nucleus, cerebral cortex and spinal cord. We were unable to identify any dopaergic (TH-positive, AADC-negative) neurons that expressed VMAT2, suggesting that regulatory mechanisms exist for shutting off VMAT2 expression in neurons that fail to biosynthesize its substrates.5. In several cases, the corresponding TH phenotypes were identified in the adult rat, suggesting that this rodent is an appropriate experimental model for further investigation of these TH-positive neuronal cell groups in the adult central nervous system. Thus, no examples of TH and VMAT2 co-positive neurons lacking AADC expression were found in rodent adult nervous system.6. In conclusion, the adult mammalian nervous system contains in addition to classical catecholaminergic neurons, cells that can synthesize dopamine, but cannot transport and store it in synaptic vesicles, and neurons that can synthesize only L-dopa and lack VMAT2 expression. The presence of these additional populations of TH-positive neurons in the adult primate CNS has implications for functional catecholamine neurotransmission, its derangement in disease and drug abuse, and its rescue by gene therapeutic maneuvers in neurodegenerative diseases such as Parkinson's disease.     

Functional characteristics of corticolous lichens in the understory of a tropical lowland rain forest

In tropical lowland forests, corticolous crustose green algal lichens are abundant and highly diverse. This may be related to adaptation to prevailing microenvironmental conditions including, for example, high precipitation and low light intensities. In the understory of a tropical lowland rain forest in French Guiana, we studied the morphology of crustose green algal lichens and measured gas exchange and chlorophyll a fluorescence. We found that (i) periods of thallus suprasaturation with water were reduced by the presence of water-repelling surface structures of mycobiont hyphae at the thallus surface and the medulla; (ii) photosynthesis was adapted to the low light intensities present in the understory; and (iii) photosynthesis was rapidly activated in fluctuating light. The combination of these three mechanisms enables corticolous lichens to implement specific morphological and physiological strategies, which may favour growth in the limiting understory habitat of tropical lowland rain forests.     

Decay-accelerating factor (CD55): a versatile acting molecule in human malignancies

The decay-accelerating factor (DAF, CD55) physiologically serves as an inhibitor of the complement system. Moreover, DAF is broadly expressed in malignant tumors. Here, DAF seems to dispose of several different functions reaching far beyond its immunological role, e.g., promotion of tumorigenesis, decrease of complement mediated tumor cell lysis, autocrine loops for cell rescue and evasion of apoptosis, neoangiogenesis, invasiveness, cell motility, and metastasis via oncogenic tyrosine kinase pathway activation, and specific seven-span transmembrane receptors (CD97) binding. Furthermore, DAF has already been included in diagnostic or therapeutic studies. Thereby, studies applying monoclonal anti-DAF antibodies and anti-DAF vaccination for a targeted therapy have been enrolled recently.