Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Structure elucidation of eucommioside (2″-O-β-d-glucopyranosyl eucommiol) from Eucommia ulmoides

The isolation of eucommioside from Eucommia ulmoides is described. Chemical modifications and spectral evidence identify eucommioside as the 2″-O-β-d-glucopyranosyl derivative of eucommiol, a known cyclopentenoidtetrol previously isolated from the same plant.     

Retrovirus mediated gene transduction of human T-cell subsets

Purpose: Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukaemia. Graft versus host disease (GVHD) is a potentially life threatening complication of AlloBMT mediated by the T cells contained within the graft. In order to be able to control GVHD, the allogeneic T cells may be transduced with a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk). For this strategy to be successful, all subsets of T cells should be transduced to a similar extent. Also, the transduction protocol should not induce expression of unwanted homing receptors, nor should it lead to unwanted skewing of the T-cell receptor repertoire. We have studied the transduction efficiency of naïve and memory subsets of CD4+ and CD8+ T cells, and examined the transduced T-cell subsets for possible changes in T-cell receptor (TCR) repertoire and homing receptor expression. Methods: The cells were transduced using a Moloney murine retroviral vector carrying a conjugate of the genes encoding the truncated form of the cell surface marker, low affinity nerve growth factor receptor (LNGFR) and HSV-tk. Transduction efficiency and homing receptor expression were quantified by flow cytometry. TCR repertoire was determined by spectratyping. Results: We obtained a transduction efficiency of 30–50% of the cells, with no difference between the T-cell subsets. Cell surface receptors responsible for homing to skin, gastrointestinal tract or lymph nodes were practically absent at the end of 2 weeks in culture. The activation procedure seemed to favour the expansion of certain T-cell clones over polyclonal populations. However, there was no difference in the TCR repertoire between transduced and non-transduced cells. Conclusion: Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.     

Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.     

Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.     

Immunocompetence increases with larval body size in a phytophagous moth

Despite the obvious benefit of an immune system, its efficacy against pathogens and parasites may show great variation among individuals, populations and species. Understanding the causes of this variation is becoming a central theme in ecology. Many biotic and abiotic factors are known to influence immunocompetence (temperature, age, etc.). However, for a given age, size among individuals varies, probably as a result of accumulated resources. Thus, these variable resources could be allocated to immune defence and, consequently, body size may explain part of the variation in immune responsiveness. However, the influence of body size on immune defence is often overlooked. The present study investigates variations in haemocyte count and phenoloxidase activity in larvae of the phytophagous vine moth Eupoecilia ambiguella Hübner of the same age, although differing in body size. The measurements of immune function are made both when the insects are immunologically naïve and 24 h after a bacterial immune challenge. The base levels of these immune parameters do not covary with body size in naïve larvae. After the bacterial immune challenge, more haemocytes and phenoloxidase enzyme are mobilized, and the mobilization of these immune effectors is correlated positively with individual body size. Thus, larger larvae exhibit higher immunocompetence than smaller ones, suggesting that smaller larvae might be more vulnerable to infection. These results suggest that body size is probably an underestimated variable, which nevertheless modulates the insect immune system and should thus be considered as a covariate in insect immune system measurement. It is recommended therefore, that body size should be taken into account in ecological immunity studies with insects. © 2013 The Royal Entomological Society     

Plasmodium falciparum soluble extracts potentiate the suppressive function of polyclonal T regulatory cells through activation of TGFβ-mediated signals

Increased numbers of T regulatory cells (Tregs), key mediators of immune homeostasis, were reported in human and murine malaria and it is current opinion that these cells play a role in balancing protective immunity and pathogenesis during infection. However, the mechanisms governing their expansion during malaria infection are not completely defined. In this article we show that soluble extracts of Plasmodium falciparum (PfSEs), but not equivalent preparation of uninfected erythrocytes, induce the differentiation of polyclonally activated CD4(+) cells in Tregs endowed with strong suppressive activity. PfSEs activate latent TGFβ bound on the membrane of Treg cells, thus allowing the cytokine interaction with TGFβ receptor, and inducing Foxp3 gene expression and TGFβ production. The activation of membrane-bound latent TGFβ by PfSEs is significantly reduced by a broad-spectrum metalloproteinases inhibitor with Zn(++) -chelating activity, and completely inhibited by the combined action of such inhibitor and antibodies to a P. falciparum thrombospondin-related adhesive protein (PfTRAP). We conclude that Pf-Zn(++) -dependent proteinases and, to a lesser extent, PfTRAP molecules are involved in the activation of latent TGFβ bound on the membrane of activated Treg cells and suggest that, in malaria infection, this mechanism could contribute to the expansion of Tregs with different antigen specificity.     

Dietary zeolite supplementation reduces oxidative damage and plaque generation in the brain of an Alzheimer's disease mouse model

AimOxidative stress is considered one of the main events that lead to aging and neurodegeneration. Antioxidant treatments used to counteract oxidative damage have been associated with a wide variety of side effects or at the utmost to be ineffective. The aim of the present study was to investigate the antioxidant property of a natural mineral, the tribomechanically micronized zeolite (MZ).Main methodsCell death and oxidative stress were assessed in retinoic acid differentiated SH-SY5Y cells, a neuronal-like cell line, after a pro-oxidant stimulus. In vivo evaluation of antioxidant activity and amyloidogenic processing of beta amyloid have been evaluated in a transgenic model of aging related neurodegeneration, the APPswePS1dE9 transgenic mice (tg mice) after a five-month long period of water supplementation with MZ.Key findingsThe study showed that 24 h of cell pretreatment with MZ (1) protected the cells by radical oxygen species (ROS)-induced cell death and moreover (2) induced a reduction of the mitochondrial ROS production following a pro-oxidant stimulation. Looking for an antioxidant effect of MZ in vivo, we found (3) an increased activity of the endogenous antioxidant enzyme superoxide dismutase (SOD) in the hippocampus of tg mice and (4) a reduction in amyloid levels and plaque load in MZ treated tg mice compared to control tg mice.SignificanceOur results suggest MZ as a novel potential adjuvant in counteracting oxidative stress and plaque accumulation in the field of neurodegenerative diseases.