Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.     

Chemokines and Glial Cells: A Complex Network in the Central Nervous System

Chemokines are small secreted proteins that are essential for the recruitment and activation of specific leukocyte subsets at sites of inflammation and for the development and homeostasis of lymphoid and nonlymphoid tissues. During the past decade, chemokines and their receptors have also emerged as key signaling molecules in neuroinflammatory processes and in the development and functioning of the central nervous system. Neurons and glial cells, including astrocytes, oligodendrocytes, and microglia, have been identified as cellular sources and/or targets of chemokines produced in the central nervous system in physiological and pathological conditions. In this article, we provide an update of chemokines and chemokine receptors expressed by glial cells focusing on their biological functions and implications in neurological diseases.     

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.     

Inhibition of HIV-1 Infection by Human α-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae

Background

α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology.

Methodology/Principal Findings

Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4⁺ T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity.

Conclusion/Significance

These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Formation and hydrolysis of triacylglycerol and sterols epoxides: role of unsaturated triacylglycerol peroxyl radicals

Epoxidation of unsaturated pure triacylglycerols (TAGs), cholesterol, and phytosterols was investigated using air and 18O2 oxidation experiments. Oxidized lipids were analyzed using both triple quadrupole mass spectrometry (MS), ion-trap MS in the direct infusion mode, and triple quadrupole MS in tandem with a liquid chromatograph (LC-MS/MS). Pure 1,2-distearoyl-3-oleoyl-glycerol (SSO) samples were heated in sealed vials under air or 18O2 atmosphere at 160 degrees C for 1 h. LC-MS/MS analysis of 18O-labeled oxidized TAGs revealed that hydroperoxides and epoxide TAGs are formed mainly during this first step. Then, oxidized TAGs were incubated under an inert atmosphere, separately with 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) at 160 degrees C for 90 min, and with cholesterol and stigmasterol at 100 degrees C for 10 min. Subsequent LC-MS/MS analysis revealed the occurrence of epoxidation products of PPO, cholesterol, and sitosterol. Therefore, we showed the epoxidation of unsaturated lipids proceeds readily in contact with hydroperoxide TAGs, in the absence of molecular oxygen. Dual oxidation experiments using both air and 18O2 allowed investigation of oxygen atom transfer during epoxidation of lipids. Moreover, the experimental oxidation design presented can be used to study fragmentation pathways, as illustrated for 5,6-epoxycholesterol (CE) on both triple quadrupole and ion-trap MS. We report for the first time the occurrence of 5,6;22,23-diepoxystigmasterol (StDE) and 5,6;22,23-diepoxybrassicasterol (BDE) in autoxidized vegetable oils. Additionally, acid-catalyzed hydrolysis of epoxidized lipids, with emphasis on phytosterol polyol formation, was investigated using a model gastric medium. For confirmation, almost all identified products were synthesized and characterized by MS.