An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies

The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.     

Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.     

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background

Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin''s lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.

Methodology/Principal Findings

The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin''s lymphomas with an indolent (EBV⁻ Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV⁺ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.

Conclusions/Significance

Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.     

Anandamide Suppresses Proliferation and Cytokine Release from Primary Human T-Lymphocytes Mainly via CB2 Receptors

Background

Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB₁R and CB₂R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB₁ and CB₂ receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.

Methodology/Significance

In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-α and INF-γ from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB₂R, since it could be mimicked by the CB₂R selective agonist JWH-015, and could be blocked by the specific CB₂R antagonist SR144528. Instead the selective CB₁R agonist ACEA, or the selective CB₁R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17.

Conclusions/Significance

Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB₂R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.     

Noah's nectar: The proteome content of a glass of red wine

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of proteins present in red wines. Surprisingly, although it is stated that red wines are in general fined with egg albumin, for all Italian wines investigated (in the areas around Chiari and Verona as well as in the Chianti area) we find that the only fining agent used is bovine casein, just like in white wines. Although the typical levels of casein found range between 45 to 85 μg/L, in one case as little as 3.8 μg/L of casein could be detected, an extremely high level of sensitivity, close to our lower detection limit of 1 μg/L reported for white wines. As a result of such treatments, very small amounts of residual proteins in red wines could be identified: essentially no residual grape proteins (except for thaumatin), but only traces of proteins from Saccharomyces cerevisiae and a few proteins from plant pathogens and fungi (e.g., Botryotinia fuckeliana, Sclerotinia sclerotiorum, Aspergillus aculeatus). Contrary to what has been found in white wines, the best capture efficiency with CPLLs has occurred at pH 7.2 and pH 9.3, with minimal capture at pH 3.3. The fact that such very low levels of fining agents can still be detected in treated red wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.     

Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography-mass spectrometry (GC-MS) with selective ion monitoring (SIM)

Phytosterols (beta-sitosterol, cholestanol and campesterol) and cholesterol precursors (desmosterol and lathosterol), have been suggested as important biochemical markers of intestinal cholesterol absorption and liver biosynthesis, respectively, and as useful clinical parameters in the study of hypercholesterolemia, beta-sitosterolemia, atherosclerosis and cardiovascular disease, including pharmacological response to hypolipidemic agents. We developed an optimised analytical method for the simultaneous analysis of cholestanol, desmosterol, lathosterol, campesterol and beta-sitosterol in plasma using capillary gas chromatography coupled to mass spectrometry (GC-MS) with multiple selected ion monitoring (SIM). This method is based on the alkaline hydrolysis of sterol esters, extraction of free sterols and derivatization. The recovery of all sterols was in the range 76-101%. Within-day relative standard deviations (R.S.Ds.) and the between-day R.S.Ds. of cholestanol, desmosterol, lathosterol, campesterol and beta-sitosterol were less than 8%, and their plasma levels in 161 normal subjects were (mean+/-S.D.) 4.73+/-2.57, 2.37+/-1.04, 6.23+/-3.14, 3.67+/-1.95 and 5.92+/-3.62 micromol/l, respectively.