TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes

Stimulation of the T-cell receptor (TCR) activates Ca2+ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca2+ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca2+ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca2+ currents as well as TCR-dependent Ca2+ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca2+ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.     

Cl-IB-MECA enhances TNF-α release in peritoneal macrophages stimulated with LPS

Adenosine receptor A3 (A3R) belongs to the Gi/Gq-coupled receptor family, that leads to the intracellular cAMP reduction and intracellular calcium increase, respectively. A3R is widely expressed and it can play a crucial role in many patho-physiological conditions, including inflammation. Here we investigate the effect of Cl-IB-MECA, A3R agonist, on the production of TNF-α. We found that Cl-IB-MECA enhances LPS-induced TNF-α release in peritoneal macrophages. This effect is reduced by MRS1191, A3R antagonist and by forskolin, activator of adenylyl cyclase. pIκBα increased in LPS+Cl-IB-MECA-treated macrophages, while total IκB kinase-β (IKKβ) reduced. Indeed, p65NF-κB nuclear translocation increased in cells treated with LPS+Cl-IB-MECA. Moreover, IMD 0354, IKKβ inhibitor, significantly abrogated the effect of Cl-IB-MECA on TNF-α release. Inhibition of protein kinase C (PKC) significantly reduced Cl-IB-MECA-induced TNF-α release in LPS-stimulated macrophages. Furthermore, LY-294002, PI3K inhibitor, reduced the TNF-α production enhanced by Cl-IB-MECA, although the phosphorylation status of Akt did not change in cells treated with LPS+Cl-IB-MECA than LPS alone. In summary, these data show that Cl-IB-MECA is able to enhance TNF-α production in LPS-treated macrophages in an NF-κB- dependent manner.     

HIV-2 Integrase Variation in Integrase Inhibitor-Na?ve Adults in Senegal,West Africa

Background

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.

Methods

We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.

Results

No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.

Conclusion

Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.     

No trade-off between learning ability and parasitoid resistance in Drosophila melanogaster

Learning ability and immunity to parasites are linked at the physiological level in several insect species. The aim of this work was to investigate the relationship between learning and immunity at an evolutionary level. We tested whether selection for improved learning ability in Drosophila melanogaster led to changes in parasitoid resistance as a correlated response. Similarly, we assayed whether selection for better parasitoid resistance led to a change in learning ability. There was no significant difference between selected and control lines in either case; the estimated confidence intervals for the differences indicate that a trade-off relationship is unlikely.     

Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8(+) T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20(+) B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8(+) T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.     

Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior.     

Oncologic Trogocytosis of an Original Stromal Cells Induces Chemoresistance of Ovarian Tumours

Background

The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.

Methodology/Principal Findings

We isolated an original type of stromal cells, referred to as “Hospicells” from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.

Conclusions/Significance

This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient''s tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.