Cannabinoids and Neuroprotection in Basal Ganglia Disorders

Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson’s disease and Huntington’s chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.     

Significance and limits in the use of predawn leaf water potential for tree irrigation

Research in estimating the water status of crops is increasingly based on plant responses to water stress. Several indicators can now be used to estimate this response, the most widely available of which is leaf water potential (Ψ_LWP) as measured with a pressure chamber. For many annual crops, the predawn leaf water potential (Ψ_PLWP), assumed to represent the mean soil water potential next to the roots, is closely correlated to the relative transpiration rate, RT. A similar correlation also holds for young fruit trees grown in containers. However, exceptions to this rule are common when soil water content is markedly heterogeneous. Two experimental conditions were chosen to assess the validity of this correlation for heterogeneous soil water content: 1) young walnut trees in split-root containers. The heterogeneity was created by two unequal compartments (20% and 80% of total volume), of which only the smaller was irrigated and kept at a moisture content higher than field capacity (permanent drainage). 2) adult walnut trees in an orchard. In this case, soil water heterogeneity was achieved by limiting the amount of localised irrigation (20% of the irrigated control) which was applied every evening. Values of sap flux and of minimum and predawn leaf water potentials with homogeneous and heterogeneous soil water content were compared for trees grown in the orchard and in containers. In spite of intense drought reflected by very low RT or stem water potential, Ψ_PLWP of trees under heterogeneous moisture conditions remained high (between -0.2 and -0.4 MPa) both in the orchard and in containers. These values were higher than those usually considered critical under homogeneous soil conditions. A semi-quantitative model, based on the application of Ohm's analogy to split-root conditions, is proposed to explain the apparently conflicting results in the literature on the relation between Ψ_PLWP and soil water potential. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bifidobacterium longum CECT 7347 modulates immune responses in a gliadin-induced enteropathy animal model

Coeliac disease (CD) is an autoimmune disorder triggered by gluten proteins (gliadin) that involves innate and adaptive immunity. In this study, we hypothesise that the administration of Bifidobacterium longum CECT 7347, previously selected for reducing gliadin immunotoxic effects in vitro, could exert protective effects in an animal model of gliadin-induced enteropathy. The effects of this bacterium were evaluated in newborn rats fed gliadin alone or sensitised with interferon (IFN)-γ and fed gliadin. Jejunal tissue sections were collected for histological, NFκB mRNA expression and cytokine production analyses. Leukocyte populations and T-cell subsets were analysed in peripheral blood samples. The possible translocation of the bacterium to different organs was determined by plate counting and the composition of the colonic microbiota was quantified by real-time PCR. Feeding gliadin alone reduced enterocyte height and peripheral CD4+ cells, but increased CD4+/Foxp3+ T and CD8+ cells, while the simultaneous administration of B. longum CECT 7347 exerted opposite effects. Animals sensitised with IFN-γ and fed gliadin showed high cellular infiltration, reduced villi width and enterocyte height. Sensitised animals also exhibited increased NFκB mRNA expression and TNF-α production in tissue sections. B. longum CECT 7347 administration increased NFκB expression and IL-10, but reduced TNF-α, production in the enteropathy model. In sensitised gliadin-fed animals, CD4+, CD4+/Foxp3+ and CD8+ T cells increased, whereas the administration of B. longum CECT 7347 reduced CD4+ and CD4+/Foxp3+ cell populations and increased CD8+ T cell populations. The bifidobacterial strain administered represented between 75-95% of the total bifidobacteria isolated from all treated groups, and translocation to organs was not detected. These findings indicate that B. longum attenuates the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in an animal model of gliadin-induced enteropathy.     

A simple mathematical model based on the cancer stem cell hypothesis suggests kinetic commonalities in solid tumor growth

Background

The Cancer Stem Cell (CSC) hypothesis has gained credibility within the cancer research community. According to this hypothesis, a small subpopulation of cells within cancerous tissues exhibits stem-cell-like characteristics and is responsible for the maintenance and proliferation of cancer.

Methodologies/Principal Findings

We present a simple compartmental pseudo-chemical mathematical model for tumor growth, based on the CSC hypothesis, and derived using a “chemical reaction” approach. We defined three cell subpopulations: CSCs, transit progenitor cells, and differentiated cells. Each event related to cell division, differentiation, or death is then modeled as a chemical reaction. The resulting set of ordinary differential equations was numerically integrated to describe the time evolution of each cell subpopulation and the overall tumor growth. The parameter space was explored to identify combinations of parameter values that produce biologically feasible and consistent scenarios.

Conclusions/Significance

Certain kinetic relationships apparently must be satisfied to sustain solid tumor growth and to maintain an approximate constant fraction of CSCs in the tumor lower than 0.01 (as experimentally observed): (a) the rate of symmetrical and asymmetrical CSC renewal must be in the same order of magnitude; (b) the intrinsic rate of renewal and differentiation of progenitor cells must be half an order of magnitude higher than the corresponding intrinsic rates for cancer stem cells; (c) the rates of apoptosis of the CSC, transit amplifying progenitor (P) cells, and terminally differentiated (D) cells must be progressively higher by approximately one order of magnitude. Simulation results were consistent with reports that have suggested that encouraging CSC differentiation could be an effective therapeutic strategy for fighting cancer in addition to selective killing or inhibition of symmetric division of CSCs.     

The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.The genetic drift of seasonal influenza viruses and the occasional emergence of pandemic strains represent a continuing and serious burden on human health. Pandemic influenza viruses arise at irregular intervals, can infect up to 50% or more of the population, and vary in disease severity. Most notably, the H1N1 Spanish influenza pandemic of 1918 killed an estimated 20 to 50 million people worldwide, and the 1957 H2N2 Asian flu and 1968 H3N2 Hong Kong flu pandemics killed between 0.5 and 1 million people in the United States alone (30). The ongoing danger of influenza was recently emphasized by the emergence of the novel H1N1 pandemic virus from Mexico in April of 2009. The urgent need to speed up vaccine production was highlighted by this outbreak because over 340,000 confirmed cases and 4,100 deaths had occurred worldwide during the 6 months that were necessary to produce a vaccine using current procedures (39).As the major surface antigen of influenza A viruses, the hemagglutinin (HA) envelope glycoprotein is the primary source of natural immunity and the key target in vaccination. However, changes in the antigenic sites of the HA protein due to antigenic drift result in lost or diminished immunity acquired from previous infection or vaccination (35). This necessitates the production of new vaccines against seasonal influenza viruses each year. The HA protein also plays a central role in the emergence of human pandemic influenza viruses. There are 16 known antigenic subtypes of HA proteins in influenza A viruses (H1 through H16), and a pandemic occurs when an influenza virus that has an HA protein to which most of the population lacks immunity acquires the ability to be efficiently transmitted from person to person.The HA protein has multiple roles in the virus life cycle, notably receptor binding and membrane fusion. The protein is synthesized as a single precursor protein, HA₀, that trimerizes and becomes glycosylated in the endoplasmic reticulum as it traffics to the cell surface (33). The HA protein contains multiple disulfide bonds and is cleaved into a mature form consisting of two subunits, HA₁ and HA₂ (9, 18). HA₂ and the N- and C-terminal portions of HA₁ form a membrane-proximal stalk that mediates membrane fusion during viral entry (40). A receptor-binding domain (HA-RBD) forms the distal head of the molecule and is inserted into the HA₁ subunit. During virus entry, the HA-RBD engages sialic acid-containing receptors on the surface of the host cell, and the virion is subsequently internalized by endocytosis (33). Structurally and functionally, the HA-RBD is a member of the lectin superfamily, and the specificity of the binding pocket contributes to the host range of influenza viruses. For example, α(2,6)-containing sialosides are typically preferred by the HA protein from human viruses and α(2,3) sialosides by the HA proteins from avian viruses (13, 28). Upon triggering by the low-pH environment of endosomes, the HA protein undergoes an irreversible conformational change (6, 40) during which the intact HA-RBDs dissociate from the stalk of the trimer (3, 14, 19, 21). This observation, together with the manner in which the lectin-like domain is inserted as a folded module into the full-length HA protein, led us to hypothesize that the HA-RBD is able to adopt its native structure in isolation. Proper folding of the isolated HA-RBD into its native immunogenic structure has important therapeutic implications because the domain contains all of the known HA antigenic epitopes responsible for antibody recognition (5), and producing a protein-based influenza vaccine composed of isolated HA-RBD would dramatically speed up vaccine development during the early stages of a pandemic.In a recently published report, a construct of the 2009 pandemic H1N1 HA protein that encompasses the HA-RBD, designated HA_63-286-RBD, was expressed in Escherichia coli as inclusion bodies, refolded and purified, and used as a vaccine to produce immunity in ferrets (2). In this report, we show that this construct behaves as a stable, structured protein in solution, can be readily crystallized, and indeed adopts a structure that is virtually indistinguishable from that in the H1N1 HA protein ectodomain (41).