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Background: Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. Results: We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. Conclusions: Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment. [ABSTRACT FROM AUTHOR]

In rice, plants exposure to low water temperature ( T w ) treatment during reproductive stage severely affects the yield-related traits, diminishes physiological and metabolic processes leading to low yield. In higher plants, the glutamate metabolism playes central roles in the amino acid metabolism, plant defense and productivity. In order to understand how low T w treatment during reproductive stage affects the yield and relative physiological parameters of glutamate metabolism, the rice were subjected to 17 °C low T w for varying length of time (three, six, nine, twelve, and fifteen days) at reproductive stage (microsporogenesis). The indexes of yield, yield-related traits, amino acid content, enzyme activities of glutamate metabolism, and the relationships among them were investigated, respectively. The results revealed that the changes degree of yield, yield-related traits and glutamate metabolism varies with rice varieties and duration of exposure. Multiple stepwise regression identified glutamate (Glu) content and glutamate dehydrogenase (GDH) activity as important traits that influenced the grain yield and spikelet sterility, respectively. By adjusting the glutamate metabolism level of leaves in rice, we can thus improve rice cold tolerance, and reduce the effect of low T w (17 °C) treatment on grain yield and spikelet sterility during reproductive stage. [ABSTRACT FROM AUTHOR]

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members., Results: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses., Conclusions: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

Objective: To determine the functions of in vitro primed Natural Killer (NK) cells in Human Immunodeficiency Virus (HIV-1) infection and the role of IL-2, IL-12 and IL-15 in enhancing the NK survival and activity in terms of viral suppression and of purging of HIV provirus., Methods: Peripheral Blood Mononuclear Cells (PBMCs) and CD4+ T lymphocytes cells obtained from eight healthy donors were infected in vitro with HIV-1 and p24 was measured with and without IL-2, IL-12 and IL-15. We studied the effect of NK pulsed in vitro with IL-2, IL-12 and IL-15 on HIV replication by measurement of p-24 and DNA-provirus load when added into the culture of PBMCs and CD4+ T lymphocytes cells infected in vitro. We evaluated the effect of NK cells pulsed with IL-2, IL-12 and IL-15 on HIV replication and DNA-load into the culture of CD4+ T lymphocytes cells and PBMCs by trans-well chamber., Results: We found high levels of p24 in the supernatants of PBMCs and CD4+ T lymphocytes cells cultured with IL-2, IL-12, and IL-15. We observed a significant reduction of p24 in the culture both of infected PBMCs and CD4+ T lymphocytes cells in which was added NK pulsed with IL-15. We did not obtain the some results with NK pulsed with IL-2 and IL-12. We observed a power effect of NK pulsed with IL-15 on HIV-DNA. The trans-well chamber experiments showed that the effect of NK is both direct and both mediated by realizing of soluble factors., Conclusions: This study highlights some important effects of IL 15 on NK in HIV patients anyway our results are preliminary and descriptive and others studies will be needed to provide rationale for immune therapies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: There has been an ever growing interest in the search for new anti-tumor compounds that do not interact with MDR1-Pgp and MRP1 drug transporters and so circumvent the effect of these proteins conferring multidrug resistance (MDR) and poor prognosis in AML patients. We have investigated the cytotoxic activity of the strong glutathione S-transferase (GST) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on AML (HL60) cell lines., Methods: Functional drug efflux studies and cell proliferation assays were performed on both sensitive and MDR AML (HL60) cells after incubation with NBDHEX. Moreover, the mode of cell death (apoptosis vs. necrosis) as well as the correlation between NBDHEX susceptibility and GST activity or Bcl-2 expression was investigated., Results: NBDHEX is not a substrate of either MDR1-Pgp or MRP1 efflux pumps; in fact, it is not only cytotoxic toward the parental HL60 cell line, but also overcomes the MDR phenotype of its HL60/DNR and HL60/ADR variants., Conclusions: The data herein reported show that NBDHEX mediates efficient killing of both MDR1-Pgp and MRP1 over-expressing AML cells. Therefore, this drug can potentially be used as an effective agent for treating MDR in AML patients.