Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Fibrinogen alpha chain O-glycopeptides as possible markers of urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infection leading to substantial morbidity and considerable health care expenditures across all ages. Here we present an exploratory UPLC-MS study of human urine in the context of febrile, complicated urinary tract infection aimed to reveal and identify possible markers of a host response on infection. A UPLC-MS based workflow, taking advantage of Ultra High Resolution (UHR) Qq-ToF-MS, and multivariate data handling were applied to a carefully selected group of 39 subjects with culture-confirmed febrile Escherichia coli UTI. Using a combination of unsupervised and supervised multivariate modeling we have pinpointed a number of peptides specific for UTI. An unequivocal structural identification of these peptides, as O-glycosylated fragments of the human fibrinogen alpha 1 chain, required MS2 and MS3 experiments on two different MS platforms: ESI-UHR-Qq-ToF and ESI-ion trap, a blast search and, finally, confirmation was achieved by matching experimental tandem mass spectra with those of custom synthesized candidate-peptides.In conclusion, exploiting non-targeted UPLC-MS based approach for the investigation of UTI related changes in urine, we have identified and structurally characterized unique O-glycopeptides, which are, to our knowledge, the first demonstration of O-glycosylation of human fibrinogen alpha 1-chain.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.     

L-Lysine alpha-oxidase: physicochemical and biological properties

This review summarizes data on the properties of L-lysine -oxidase, an enzyme that belongs to the group of oxidases of L-amino acids. This enzyme acts virtually only on L-lysine with a rather low K _m yielding -keto--aminocaproic acid. The decrease in the level of the essential amino acid L-lysine and the formation of hydrogen peroxide during the reaction possibly provide the basis for the unique properties of L-lysine -oxidase: cytotoxic, antitumor, antimetastatic, antiinvasive, antibacterial, and antiviral activities, as well as an immunomodulating effect. Native L-lysine -oxidase and its immobilized forms are promising tools for determination of concentration of L-lysine in various biological materials.     

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.     

Antigen-specific,MHC-unrestricted T cells

The published record suggests that in the majority of cases the antigen is recognized by the T cell receptor (TCR) as a complex of a foreign antigen and amino acid residues contributed by the major histocompatibility complex (MHC) antigens, and the antigen-specific, MHC-restricted effector function is an unambiguous result of this process. Alternatively, the T cell receptor may recognize a particular conformational form of the antigen which is dictated by the allelic differences in the MHC, resulting also in MHC-restricted recognition. When, however, a T cell which phenotypically fulfills all the requirements necessary to perform antigen specific, MHC-restricted function, shows a lack of MHC restriction, there are two possible explanations: 1) In addition to the MHC-restricted, antigen-specific T cell receptor the cell expresses, or has newly acquired the expression of another, MHC-unrestricted (NK-like) receptor, or 2) The specific antigen recognized by the T cell receptor, is able to bind to the receptor and activate the T cell without being presented by the MHC molecule. While the first possibility has been extensively described in the literature as well as other articles in this issue, the second possibility has not been dealt with to the same extent and is the primary focus of this review.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.