Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.     

Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida,Aspergillus and mucormycetes

Background aimsInvasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy.MethodsUsing the interferon-γ secretion assay, we isolated multi-specific human anti-fungal T cells after simultaneous stimulation with cellular extracts of Aspergillus fumigatus, Candida albicans and Rhizopus oryzae. Cells were phenotypically and functionally characterized by flow cytometry.ResultsOf a total of 1.1 × 10⁹ peripheral blood mononuclear cells, a median number of 5.2 × 10⁷ CD3⁺CD4⁺ T cells was generated within 12 days. This cell population consisted of activated memory T_H1 cells and reproducibly responded to a multitude of Aspergillus spp., Candida spp. and mucormycetes with interferon-γ production. On re-stimulation, the generated T cells proliferated and enhanced anti-fungal activity of phagocytes and showed reduced alloreactivity compared with the original cell fraction.ConclusionsOur rapid and simple method of simultaneously generating functionally active multi-specific T cells that recognize a wide variety of medically relevant fungi may form the basis for future clinical trials investigating adoptive immunotherapy in allogeneic hematopoietic stem cell transplantation recipients with invasive fungal infection.     

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.     

Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology

Physiological mechanisms of antennal sucrose perception in the honey bee were analysed using behavioural and electrophysiological methods. Following sucrose stimulation of the tip of a freely moving antenna, the latency of proboscis extension was 320–340 ms, 80–100 ms after the first activity in muscle M17 controlling this response. When bees were allowed to actively touch a sucrose droplet with one antenna, contacts with the solution were frequent with durations of 10–20 ms and average intervals between contacts of approximately 40 ms. High sucrose concentrations led to short and frequent contacts. The proboscis response and M17 activity were largely independent of stimulus duration and temporal pattern. Taste hairs of the antennal tip displayed spike responses to sucrose concentrations down to at least 0.1%. The first 25 ms of the response were suitable for discrimination of sucrose concentrations. This time interval corresponds to the duration of naturally occurring gustatory stimuli. Sucrose responses between different hairs on the same antenna showed a high degree of variability, ranging from less than five to over 40 spikes per 0.5 s for a stimulus of 0.1% sucrose. This variability of receptor responses extends the dynamic range of sucrose perception over a large range of concentrations.     

Refining the roots of the beewolf-Streptomyces symbiosis: antennal symbionts in the rare genus Philanthinus (Hymenoptera, Crabronidae)

Insects engage in symbiotic associations with a large diversity of beneficial microorganisms. While the majority of well-studied symbioses have a nutritional basis, several cases are known in which bacteria protect their host from pathogen infestation. Solitary wasps of the genera Philanthus and Trachypus (beewolves; Hymenoptera, Crabronidae) cultivate the actinomycete "Candidatus Streptomyces philanthi" in specialized antennal gland reservoirs. The symbionts are transferred to the larval cocoon, where they provide protection against pathogenic fungi by producing at least nine different antibiotics. Here we investigated the closest relatives of Philanthus and Trachypus, the rare genus Philanthinus, for the presence of antennal gland reservoirs and symbiotic streptomycetes. Molecular analyses identified "Ca. Streptomyces philanthi" in reservoirs of Philanthinus quattuordecimpunctatus. Phylogenies based on the 16S rRNA gene suggest that P. quattuordecimpunctatus may have acquired "Ca. Streptomyces philanthi" by horizontal transfer from other beewolf species. In histological sections and three-dimensional reconstructions, the antennal gland reservoirs were found to occupy six antennal segments (as opposed to only five in Philanthus and Trachypus) and to be structurally less complex than those of the evolutionarily more derived genera of beewolves. The presence of "Ca. Streptomyces philanthi" in antennal glands of Philanthinus indicates that the symbiosis between beewolves and Streptomyces bacteria is much older than previously thought. It probably evolved along the branch leading to the monophyletic tribe Philanthini, as it seems to be confined to the genera Philanthus, Trachypus, and Philanthinus, which together comprise 172 described species of solitary wasps.     

A ‘social’ gland in a solitary wasp? The postpharyngeal gland of female European beewolves (Hymenoptera,Crabronidae)

Exocrine glands play an important role in maintaining the integrity of colonies of social Hymenoptera. The postpharyngeal gland (PPG) of ants is crucial for the generation of a nest odour that enables nestmate recognition. The evolutionary history of this gland is unknown and it was thought to be restricted to ants. Here we describe an exocrine head gland in females of a solitary crabronid wasp, the European beewolf, Philanthus triangulum, that resembles the PPG of ants in many respects. The newly described gland has the same location and the same glove like shape as in ants, and it also has a monolayered epithelium with similar ultrastructure. Unlike in ants, the epithelium bears hairs that reach into the lumen of the gland. Although the PPG of beewolves serves a completely different function it is also associated to an allogrooming behaviour as in ants. Based on these morphological and behavioural similarities as well as similarities in the chemical composition of the content of the PPG of both taxa, we hypothesise that the PPGs of ants and beewolves have a common evolutionary origin. Thus, our results suggest that the PPG in ants might not have evolved in response to social requirements but might have already existed in solitary predecessors.     

Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.