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Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate.

Insects are considered a suitable alternative feed for livestock production and their use is nowadays regulated in the European Union by the European Commission Regulation No. 893/2017. Insects have the ability to grow on a different spectrum of substrates, which could be naturally contaminated by mycotoxins. In the present work, the mycotoxin uptake and/or excretion in two different insect species, Alphitobius diaperinus (Lesser Mealworm, LM) and Hermetia illucens (Black Soldier Fly, BSF), grown on naturally contaminated substrates, was evaluated. Among all the substrates of growth tested, the Fusarium toxins deoxynivalenol (DON), fumonisin 1 and 2 (FB1 and FB2) and zearalenone (ZEN) were found in those based on wheat and/or corn. No mycotoxins were detected in BSF larvae, while quantifiable amount of DON and FB1 were found in LM larvae, although in lower concentration than those detected in the growing substrates and in the residual fractions. Mass balance calculations indicated that BSF and LM metabolized mycotoxins in forms not yet known, accumulating them in their body or excreting in the faeces. Further studies are required in this direction due to the future employment of insects as feedstuff.

Reaction of 2-benzyl-5-halopyridazin-3(2H)-ones (3) with Grignard reagents followed by quenching with electrophiles unexpectedly yielded 4,5-disubstituted pyridazin-3(2H)-ones instead of 5-substituted pyridazin-3(2H)-ones. These reactions represent the first examples of cine substitution in which the anionic σ(H)-adduct is quenched by electrophiles (other than a proton) before elimination takes place. Insight into the reaction mechanism led to the direct transformation of 2-benzylpyridazin-3(2H)-one (7) and 2-benzyl-6-chloropyridazin-3(2H)-one (9) into the corresponding C-4 alkyl and aryl derivatives (when Br(2) was used as the electrophile).

Because of its presence in many molecules of biological relevance, the conformational analysis of five-membered rings using (3)J(HH) scalar coupling data from NMR is a topic of considerable interest. Typically, conformational analysis involves the use of a well-established mathematical procedure, originally developed by de Leeuw et al., that fits two rigid conformations to the available experimental data. This so-called pseudorotation analysis approach is not without problems, however, as chemically unrealistic conformations are sometimes generated from the data. Here, we present our investigations in the use of time-averaged restrained molecular dynamics simulations as a generic tool to determine the conformations that agree with experimental (3)J(HH) scalar coupling data. For this purpose, a set of six ribose-based molecules has been used as model compounds. The influence of several modeling parameters is assessed and optimized values are proposed. The results obtained with the tar-MD approach are compared to those obtained from the two conformer fitting procedure. Interpretation of the latter is facilitated by the introduction of a fitting error analysis that allows mapping the solution space of the fitting procedure. The relative merits of both methods and the advantages that result from the use of a force field and a time-averaged restraint potential for the experimental data are discussed. When combined, both techniques allow an enhanced understanding of the molecules' conformational behavior and prevent possible overinterpretation. In view of the very reasonable computational burden of a tar-MD simulation for the systems investigated here, the approach should be generally applicable., (Copyright 2009 Wiley Periodicals, Inc.)

The first example of substitution reaction in the free alpha-position of N-confused calix[4]pyrroles is reported: azo-coupling with various arenediazonium salts. The obtained azocompounds were used for studies of their anion-binding properties by UV-Vis spectroscopy.

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor beta-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.

Interferon (IFN) alfa has been used widely for the treatment of chronic hepatitis C virus (HCV) infections but only a small number of patients treated have shown a sustained biochemical and virological response. Anti-envelope E1 and E2 antibody titers were assessed retrospectively before, during, and after treatment with IFN in order to evaluate their usefulness for the prediction and monitoring of therapy outcome in 115 patients infected chronically with HCV genotype 1b. At baseline, E2 induced more frequent and stronger immunogenic responses than E1, irrespective of patient response to therapy. E1 and E2 antibodies also tended to be higher in patients with a long-term or a transient response to IFN treatment than in patients who were absolute non-responders. In most patients, E1 and E2 antibody levels tended to be lower after treatment. This reduction was most pronounced and occurred most frequently in long-term responders to therapy. In this patient group, the reduction of E1 antibodies was more pronounced than that of E2 antibodies. In contrast to E2 antibodies, the decrease of E1 antibodies could already be observed at the end of therapy (week 24) and was significantly larger (p<0.05) than that observed in relapsers and non-responders. Thus, a sustained elevation of E1 antibodies seems to be associated with ongoing infection even when HCV RNA levels were undetectable in serum. Monitoring of E1 antibody titers may represent a useful additional marker to discriminate sustained responders from those who relapse in patients receiving interferon therapy., (Copyright 2000 Wiley-Liss, Inc.)

Next to the "normal" calix[4]pyrrole 1, the N-confused calix[4]pyrrole 2 is formed in substantial amounts (up to 22 % yield) as side product in the acid-catalyzed condensation reaction of ketones and pyrrole. In some cases, doubly N-confused calix[4]pyrroles are also formed.

Severe combined immunodeficient (SCID) mice accept human xenografts and can act as a model for human immune functions. Murine natural killer cells (NK), however, represent an important barrier for the reconstitution of SCID mice with human peripheral blood leukocytes (Hu-PBL). We investigated the effect on Hu-PBL survival of pretreatment with TM-beta1, a rat monoclonal antibody for the mouse IL-2 receptor beta chain. TM-beta1 greatly improved the survival of Hu-PBL. Human lymphocytes, predominantly T cells, survived in the peritoneum and infiltrated spleen and lungs already 1 week after engraftment and liver and thymus from 2 weeks on. Secondary humoral responses were evaluated with Hu-PBL from a donor immune to hepatitis-B surface Ag (HBsAg) and tetanus toxoid (TT). TM-beta1 pretreatment enhanced the recall Ig response to HBsAg and did not affect the baseline anti-TT Ig production. In conclusion, TM-beta1 pretreatment of SCID mice significantly improves the survival and functionality of the Hu-PBL graft.

Cotreatment or pretreatment of several human myeloid cell lines (KG1, HL60, U937, THP1) with the differentiation inducer DMSO was found to potentiate the antiproliferative and cytotoxic effects of TNF. In addition, TNF-resistant monocytic cell lines could be sensitized to TNF cytotoxicity by DMSO treatment. Other highly polar molecules, known to be potent differentiation inducers, showed similar effects to those of DMSO. The potentiating effect of DMSO was related neither to an up-regulation of TNF receptor expression nor to an alteration in the rate of TNF internalization and degradation. We present evidence that the TNF activities are p55 TNF receptor-mediated and are not due to insertion of TNF into lipid bilayers, an effect that could be susceptible to DMSO, as this component has been described to modify cell membrane characteristics. DMSO-induced potentiation of TNF cytostasis/cytotoxicity was restricted to myeloid leukemia cell lines. In non-myeloid cells such as fibrosarcomas, myosarcomas, thymomas, or carcinomas, DMSO was found either not to alter or to inhibit TNF-induced cell death. The latter results are in good agreement with data reported by others who suggested that DMSO could act as a scavenger of TNF-induced toxic radical formation. The potential correlation in myeloid cells between DMSO-induced changes in the cells' differentiation status and DMSO-enhanced TNF-susceptibility is discussed.

Treatment of the human myelomonocytic U937 and THP1 cell lines for 24 h with 180 mM of the differentiation inducer DMSO, resulted in priming these cells to subsequent LPS-induced cytolysis. The observed cytotoxicity was LPS dose-dependent and characterized by a prolonged lag phase with detectable effects only appearing after 8 h. LPS-induced apoptotic cell death in DMSO-pretreated U937 cells as indicated by the appearance of 200 basepair DNA fragments upon agarose gel electrophoresis of total cellular DNA. Furthermore, DMSO pretreatment potentiated the cells' capacity to produce cytokines, especially TNF, upon LPS stimulation. This endogenously present TNF was metabolized by the cells. These observations suggested that the LPS-induced cytostasis/cytotoxicity was mediated through TNF. Indeed, medium conditioned by LPS-stimulated U937-DMSO cells was found to exert a cytotoxic effect on U937-DMSO cells that was completely neutralized by anti-human TNF antiserum. Such TNF-like activities were not only present in the supernatant but also at the level of the cell membrane of LPS-stimulated U937-DMSO cells. Apart from TNF, other exogenously applied recombinant cytokines (IL1, IL6, IFN gamma, GM-CSF) were not cytotoxic to U937-DMSO cells. Thus, DMSO-pretreated myelomonocytic cells become sensitive to LPS-induced cytotoxicity, which is, at least in part, mediated through endogenous TNF.