Natural ligand motifs of H-2E molecules are allele specific and illustrate homology to HLA-DR molecules

Motifs of peptldes naturally associated with H-2Ek and Ed molecules were determined by (i) pool sequencing of natural ligand mixtures and (ii) sequencing of individual natural ligands followed by their alignment to the basic motif suggested by pool sequencing. The data reveal nine amino acid motifs with interaction sites at relative positions P1, P4, P6 and P9, with specificities that are identical at some but different at other anchor positions between Ed and Ek motifs, illustrating the different requirements for peptides to be presented by these two MHC molecules. The anchors with the most restricted specificity are P1 and P9. P1 is aliphatic for Ek and predominantly aromatic for Ed. P9 is positively charged for both molecules. P4 and P6 show a totally different amino acid preference between Ek and Ed ligand motifs. An alignment of Ed and Ek protein sequences to the recently reported HLA-DR1 pocket residues is in agreement with observed anchor residues in Ek and Ed motifs, thus confirming the predicted similarity of mouse class II E molecules with human DR molecules. Furthermore, this alignment was extended to the putative pockets of class II Eb and E* molecules, and allowed, together with sequence information of previously Identified natural ligands of Eb and E5 molecules, a prediction of their respective motifs. The information obtained by this study should be useful to identify putative class II E epltopes in proteins and to design peptides for blocking class II E molecule

Soluble Triggering Receptor Expressed on Myeloid Cells 1 Is Released in Patients with Stable Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is increasingly recognized as a systemic disease that is associated with increased serum levels of markers of systemic inflammation. The triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently identified activating receptor on neutrophils, monocytes, and macrophage subsets. TREM-1 expression is upregulated by microbial products such as the toll-like receptor ligand lipoteichoic acid of Gram-positive or lipopolysaccharides of Gram-negative bacteria. In the present study, sera from 12 COPD patients (GOLD stages I–IV, FEV1 51 ± 6%) and 10 healthy individuals were retrospectively analyzed for soluble TREM-1 (sTREM-1) using a newly developed ELISA. In healthy subjects, sTREM-1 levels were low (median 0.25 ng/mL, range 0–5.9 ng/mL). In contrast, levels of sTREM-1 in sera of COPD patients were significantly increased (median 11.68 ng/mL, range 6.2–41.9 ng/mL, P<.05). Furthermore, serum levels of sTREM-1 showed a significant negative correlation with lung function impairment. In summary, serum concentrations of sTREM-1 are increased in patients with COPD. Prospective studies are warranted to evaluate the relevance of sTREM-1 as a potential marker of the disease in patients with COPD

Biological and Pharmacokinetic Studies with β-Peptides

Interactions and cleavage reactions of β-amino acids and β-oligopeptides (up to nine residues, carrying the side chains of Ala, Val, Leu, Ile, Phe, Ser, Lys, and Hop) with biological systems, such as the most potent peptidases (pronase, proteinase K, 20S proteasome), microorganisms (Pseudomonas aeruginosa and Pseudomonas putida), and mammalian blood (intravenous application to rats) have been investigated and compared with ?-peptides. The results are: i) the three peptidases do not cleave β-peptides at all (within 24 h), and they are not inhibited by a β-peptide; ii) except for certain 3-aminobutanoic-acid (β-HAla) derivatives, neither free, nor N-acetyl-β-amino acids, nor β-peptides (offered as sole N and C source) lead to growth of the two bacteria tested; iii) two water-soluble β-heptapeptides (with Lys side chains) were shown to have elimination half-lives t1/2(β) of 3 and 10 h at 100- and 30-ng/ml levels, respectively, in the rodent blood – much larger than those of α-peptides. Thus, the preliminary results described here confirm the much greater stability of β-peptides, as compared to α-peptides, towards metabolization processes, but they also suggest that there may be interactions (by hitherto unknown mechanisms) between the worlds of α- and β-peptides

Regulatory T Cells and IL-10 Independently Counterregulate Cytotoxic T Lymphocyte Responses Induced by Transcutaneous Immunization

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T(reg)) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3(+) regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10(-/-)) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T(reg) in IL-10(-/-) mice and the use of B cell deficient JHT(-/-) mice, we can exclude T(reg) and B cells as source of IL-10 in the setting of TCI.We identify T(reg) and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T(reg) function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors

Dimerization of visinin-like protein 1 is regulated by oxidative stress and calcium and is a pathological hallmark of amyotrophic lateral sclerosis

AbstractRedox control of proteins that form disulfide bonds upon oxidative challenge is an emerging topic in the physiological and pathophysiological regulation of protein function. We have investigated the role of the neuronal calcium sensor protein visinin-like protein 1 (VILIP-1) as a novel redox sensor in a cellular system. We have found oxidative stress to trigger dimerization of VILIP-1 within a cellular environment and identified thioredoxin reductase as responsible for facilitating the remonomerization of the dimeric protein. Dimerization is modulated by calcium and not dependent on the myristoylation of VILIP-1. Furthermore, we show by site-directed mutagenesis that dimerization is exclusively mediated by Cys187. As a functional consequence, VILIP-1 dimerization modulates the sensitivity of cells to an oxidative challenge. We have investigated whether dimerization of VILIP-1 occurs in two different animal models of amyotrophic lateral sclerosis (ALS) and detected soluble VILIP-1 dimers to be significantly enriched in the spinal cord from phenotypic disease onset onwards. Moreover, VILIP-1 is part of the ALS-specific protein aggregates. We show for the first time that the C-terminus of VILIP-1, containing Cys187, might represent a novel redox-sensitive motif and that VILIP-1 dimerization and aggregation are hallmarks of ALS. This suggests that VILIP-1 dimers play a functional role in integrating the cytosolic calcium concentration and the oxidative status of the cell. Furthermore, a loss of VILIP-1 function owing to protein aggregation in ALS could be relevant in the pathophysiology of the disease

NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells

The phenotype of NFATc2−/− c3−/− (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4+ CD25+ T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR) and CD25. However, neither wild-type nor DKO CD4+ CD25+ regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4+ CD25− T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4+ CD25+ T reg cells but renders conventional CD4+ T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis