Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.     

Inhibition of p38 MAPK suppresses inflammatory cytokine induction by etoposide, 5-fluorouracil, and doxorubicin without affecting tumoricidal activity

Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often experience debilitating fatigue similar to sickness behavior, a normal response to infection or tissue damage caused by the production of the inflammatory cytokines IL-1beta, TNF-alpha, and IL-6. The p38 mitogen activated protein kinase (p38 MAPK) plays a central role in the production of these cytokines and consequently the development of sickness behavior. Targeted inhibitors of p38 MAPK can reduce systemic inflammatory cytokine production and the development of sickness behavior. Several systemic cancer chemotherapy drugs have been shown to stimulate inflammatory cytokine production, yet whether this response is related to a common ability to activate p38 MAPK is not known and is the focus of this study. This understanding may present the possibility of using p38 MAPK inhibitors to reduce chemotherapy-induced inflammatory cytokine production and consequently treatment-related fatigue. One caveat of this approach is a potential reduction in chemotherapeutic efficacy as some believe that p38 MAPK activity is required for chemotherapy-induced cytotoxicity of tumor cells. The purpose of this study was to demonstrate proof of principal that p38 MAPK inhibition can block chemotherapy-induced inflammatory cytokine production without inhibiting drug-induced cytotoxicity using murine peritoneal macrophages and Lewis Lung Carcinoma (LLC1) cells as model cell systems. Using these cells we assessed the requirement of etoposide, doxorubicin, 5-fluorouracil, and docetaxel for p38 MAPK in inflammatory cytokine production and cytotoxicity. Study findings demonstrate that clinically relevant doses of etoposide, doxorubicin, and 5-FU activated p38 MAPK in both macrophages and LLC1 cells. In contrast, docetaxel failed to activate p38 MAPK in either cell type. Activation of p38 MAPK mediated the drug's effects on inflammatory cytokine production in macrophages but not LLC1 cytotoxicity and this was confirmed with inhibitor studies.     

Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

Background

The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcγR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcγR and the Fc domain of IgG depend on the IgG glycosylation state.

Methodology/Principal Findings

Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcγR and to an erythroleukemic cell line, K562, expressing FcγRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcγRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot.

Conclusions/Significance

We provide novel information about bacterial enzymatic modulation of the IgG/FcγR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.     

The Origin of the Vertebrate Eye

In his considerations of “organs of extreme perfection,” Charles Darwin described the evidence that would be necessary to support the evolutionary origin of the eye, namely, demonstration of the existence of “numerous gradations” from the most primitive eye to the most perfect one, where each such tiny change had provided a survival advantage (however slight) to the organism possessing the subtly altered form. In this paper, we discuss evidence indicating that the vertebrate eye did indeed evolve through numerous subtle changes. The great majority of the gradual transitions that did occur have not been preserved to the present time, either in the fossil record or in extant species; yet clear evidence of their occurrence remains. We discuss the remarkable “eye” of the hagfish, which has features intermediate between a simple light detector and an image-forming camera-like eye and which may represent a step in the evolution of our eye that can now be studied by modern methods. We also describe the important clues to the evolutionary origin of the vertebrate eye that can be found by studying the embryological development of our own eye, by examining the molecular genetic record preserved in our own genes and in the genes of other vertebrates, and through consideration of the imperfections (or evolutionary “scars”) in the construction of our eye. Taking these findings together, it is possible to discuss in some detail how the vertebrate eye evolved.     

C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.     

Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis.

In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.     

Secondary product formation in plant tissue cultures

The formation of secondary products in plant tissue culturesis reviewed. The conditions for the enhanced production of secondaryproducts, which include alkaloids, terpenoids, steroids andphenolics, can be regulated in a number of ways. For example,manipulation of secondary product formation is possible by varyingthe nutrient composition of the growth medium, light, temperatureand pH, and by the use of elicitors, permeabilisation and two-stagesystems. Molecular engineering and the use of biomass and large-scaleculture are described along with future prospects for the commercialproduction of secondary products from cell suspension cultures.     

The evolution of early vertebrate photoreceptors

Meeting the challenge of sampling an ancient aquatic landscape by the early vertebrates was crucial to their survival and would establish a retinal bauplan to be used by all subsequent vertebrate descendents. Image-forming eyes were under tremendous selection pressure and the ability to identify suitable prey and detect potential predators was thought to be one of the major drivers of speciation in the Early Cambrian. Based on the fossil record, we know that hagfishes, lampreys, holocephalans, elasmobranchs and lungfishes occupy critical stages in vertebrate evolution, having remained relatively unchanged over hundreds of millions of years. Now using extant representatives of these ‘living fossils’, we are able to piece together the evolution of vertebrate photoreception. While photoreception in hagfishes appears to be based on light detection and controlling circadian rhythms, rather than image formation, the photoreceptors of lampreys fall into five distinct classes and represent a critical stage in the dichotomy of rods and cones. At least four types of retinal cones sample the visual environment in lampreys mediating photopic (and potentially colour) vision, a sampling strategy retained by lungfishes, some modern teleosts, reptiles and birds. Trichromacy is retained in cartilaginous fishes (at least in batoids and holocephalans), where it is predicted that true scotopic (dim light) vision evolved in the common ancestor of all living gnathostomes. The capacity to discriminate colour and balance the tradeoff between resolution and sensitivity in the early vertebrates was an important driver of eye evolution, where many of the ocular features evolved were retained as vertebrates progressed on to land.