Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc)

Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of K_D in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright © 2014 John Wiley & Sons, Ltd.     

Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Copper retention by a strain ofBacillus

Summary A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested.     

Iron metabolism genes in Antarctic notothenioids: A review

Iron is an essential element for metabolic processes intrinsic to life, but the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and ferritin, whereas the roles of others remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, and hepcidin. The present review aims at surveying the studies carried out on some of the most important genes involved in transport and storage of iron in Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absence of) hematocrit. This unique peculiarity among vertebrates makes this fish group a suitable system to studying the relationship between hemoglobin and iron metabolism and to understanding the adaptive changes occurred in Antarctic fish metabolism during their evolution to avoid the deleterious effects of iron overload in the absence of hemoglobin. The results summarised here indicate that the loss of hemoglobin in the most specialized group of Antarctic notothenioids, belonging to the Channychthyidae family, is accompanied by remodulation of the iron metabolism.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

Adrenergic origin of very low-frequency blood pressure oscillations in the unanesthetized rat

Spectral analysis of cardiovascular signals has been extensively used to investigate circulatory homeostatic mechanisms. However, the nature of very low-frequency (VLF) fluctuations remains unclear. Because we previously observed enhanced VLF fluctuations in blood pressure (BP) in the sympathectomized rat (a model characterized by markedly increased plasma epinephrine levels), the aims of our study were to assess whether the genesis of VLF fluctuations in BP depends on circulating catecholamines and to determine which adrenergic receptor(s) and which membrane ion channel(s) are involved. We used continuous intra-arterial BP recordings from unanesthetized unrestrained rats to compute the power of VLF fluctuations in BP in the intact condition, during acute ganglionic blockade with hexamethonium, and after restoration of BP levels by infusion (in addition to hexamethonium) of adrenergic agonists (epinephrine, norepinephrine, and clonidine) or nonadrenergic vasoconstrictors (vasopressin). Effects of infusion of specific adrenergic receptor blockers (propranolol, prazosin, and yohimbine) with hexamethonium and catecholamines and infusion of various membrane ion channel blockers on VLF fluctuations in BP were also evaluated. Our results are as follows. 1) Ganglionic blockade drastically reduced BP levels and VLF fluctuations. 2) All vasoconstrictors restored BP levels, but only adrenergic vasoconstrictors generated striking VLF fluctuations in BP. 3) Catecholamine-induced fluctuations were abolished by alpha2-, but not alpha1- or beta-, adrenergic receptor blockade and by Ba2+-sensitive K+ channel or L-type Ca2+ channel, but not by other ion channel, blockers. We conclude that, in the conscious, unrestrained ganglion-blocked rat, catecholamine infusion generates VLF fluctuations in BP through stimulation of alpha2-receptors and activation of Ba2+-sensitive K+ channels. These fluctuations may have (patho)physiological relevance under conditions of disrupted circulatory homeostasis.     

Wolbachia Reduces the Transmission Potential of Dengue-Infected Aedes aegypti

BackgroundDengue viruses (DENV) are the causative agents of dengue, the world’s most prevalent arthropod-borne disease with around 40% of the world’s population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia’s efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito’s ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites.Conclusions/SignificanceThe saliva-based traits reported here offer more disease-relevant measures of Wolbachia’s effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.     

Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids

Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a "pink-blooded" notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism.     

The likely impact of elevated [CO2], nitrogen deposition, increased temperature and management on carbon sequestration in temperate and boreal forest ecosystems: a literature review

Temperate and boreal forest ecosystems contain a large part of the carbon stored on land, in the form of both biomass and soil organic matter. Increasing atmospheric [CO2], increasing temperature, elevated nitrogen deposition and intensified management will change this C store. Well documented single-factor responses of net primary production are: higher photosynthetic rate (the main [CO2] response); increasing length of growing season (the main temperature response); and higher leaf-area index (the main N deposition and partly [CO2] response). Soil organic matter will increase with increasing litter input, although priming may decrease the soil C stock initially, but litter quality effects should be minimal (response to [CO2], N deposition, and temperature); will decrease because of increasing temperature; and will increase because of retardation of decomposition with N deposition, although the rate of decomposition of high-quality litter can be increased and that of low-quality litter decreased. Single-factor responses can be misleading because of interactions between factors, in particular those between N and other factors, and indirect effects such as increased N availability from temperature-induced decomposition. In the long term the strength of feedbacks, for example the increasing demand for N from increased growth, will dominate over short-term responses to single factors. However, management has considerable potential for controlling the C store.