Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

Comprehensive Kinetics of Hydrolysis of Organotriethoxysilanes by 29Si NMR.

The kinetics of several representative hybrid precursors were studied via 29Si NMR: three alkyl precursors, methyltriethoxysilane, ethyltriethoxysilane, and propyltriethoxysilane; as well as two unsaturated radicals, vinyltriethoxysilane and phenyltriethoxysilane. The reaction rate is related to the chemical shift of 29Si in the NMR spectra, which gives information about the electronic density of the Si atoms and the inductive effects of substituents. The concentration of the precursors decreased exponentially with time, and the intermediate products of hydrolysis and the beginning of the condensation reactions showed curves characteristic of sequential reactions, with a similar distribution of the species as a function of the fractional conversion. For all of the precursors, condensation started when the most hydrolyzed species reached a maximum concentration of 0.30 M, when the precursor had run out. A prediction following the developed mathematical model fits the experimental results in line with a common pathway described by eight parameters.

Hsp90 activity is necessary to acquire a proper neuronal polarization.

Chaperones are critical for the folding and regulation of a wide array of cellular proteins. Heat Shock Proteins (Hsps) are the most representative group of chaperones. Hsp90 represents up to 1-2% of soluble protein. Although the Hsp90 role is being studied in neurodegenerative diseases, its role in neuronal differentiation remains mostly unknown. Since neuronal polarity mechanisms depend on local stability and degradation, we asked whether Hsp90 could be a regulator of axonal polarity and growth. Thus, we studied the role of Hsp90 activity in a well established model of cultured hippocampal neurons using an Hsp90 specific inhibitor, 17-AAG. Our present data shows that Hsp90 inhibition at different developmental stages disturbs neuronal polarity formation or axonal elongation. Hsp90 inhibition during the first 3h in culture promotes multiple axon morphology, while this inhibition after 3h slows down axonal elongation. Hsp90 inhibition was accompanied by decreased Akt and GSK3 expression, as well as, a reduced Akt activity. In parallel, we detected an alteration of kinesin-1 subcellular distribution. Moreover, these effects were seconded by changes in Hsp70/Hsc70 subcellular localization that seem to compensate the lack of Hsp90 activity. In conclusion, our data strongly suggests that Hsp90 activity is necessary to control the expression, activity or location of specific kinases and motor proteins during the axon specification and axon elongation processes. Even more, our data demonstrate the existence of a "time-window" for axon specification in this model of cultured neurons after which the inhibition of Hsp90 only affects axonal elongation mechanisms.

Oestradiol signalling through the Akt-mTORC1-S6K1.

The oestradiol plays an important role in normal brain development and exerts neuroprotective actions. Oestradiol is mainly produced in the ovary and in addition is locally synthesised in the brain. Most of the oestradiol functions have been associated with its capacity to directly bind and dimerize "classical oestrogen receptors" (ERs), alpha and beta. The ERs' actions have been classified as "genomic" and "non-genomic" depending on whether protein synthesis occurs through ER driven transcription or not. Indeed, recent evidence suggests that oestrogen may also act as a more general "trophic factor". Hence, we have studied the capacity of oestradiol to activate the PI3K/Akt pathway and its implication in axonal growth and neuronal morphogenesis. Our data show that when oestrogen receptors are blocked the axonal and dendritic lengths are reduced in mouse primary neurons. We found that Akt/Rheb/mTORC1 responds to ER activation in neurons and that some elements of this pathway are able to restore a normal neuronal morphology even in the presence of oestrogen receptor antagonist. All these data demonstrate a new mechanism regulated by oestradiol, at least in neuronal morphogenesis.

Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease.

AIMS: Transmissible spongiform encephalopathies, also called prion diseases, are characterized by the cerebral accumulation of misfolded prion protein (PrP(SC) ) and subsequent neurodegeneration. However, despite considerable research effort, the molecular mechanisms underlying prion-induced neurodegeneration are poorly understood. Here, we explore the hypothesis that prions induce dysfunction of the PI3K/Akt/GSK-3 signalling pathway. METHODS: We employed two parallel approaches. Using cell cultures derived from mouse primary neurones and from a human neuronal cell line, we identified common elements that were modified by the neurotoxic fragment of PrP(106-126) . These studies were then complemented by comparative analyses in a mouse model of prion infection. RESULTS: The presence of a polymerized fragment of the prion protein (PrP(106-126) ) or of a prion strain altered PI3K-mediated signalling, as evidenced by Akt inhibition and GSK-3 activation. PI3K activation by the addition of insulin or the expression of a constitutively active Akt mutant restored normal levels of Akt and GSK-3 activity. These changes were correlated with a reduction in caspase activity and an increase in neuronal survival. Moreover, we found that activation of caspase 3, Erk and GSK-3 are common features of PrP(106-126) -mediated neurotoxicity in cellular systems and prion infection in the mouse cerebellum, while activation of caspase 12 and JNK was observed in cellular models. CONCLUSIONS: Our findings in cell culture and in vivo models of prion disease demonstrate marked alterations to the PI3K/Akt/GSK-3 pathway and suggest that two additional pathways contribute to PrP-induced neurotoxicity as responsible of JNK and caspase 12 activation.

WIP is a negative regulator of neuronal maturation and synaptic activity.

Wiskott-Aldrich syndrome protein (WASP) -interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP(-)(/-) hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP(-)(/-) neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity.

Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage.

Pig feces is an interesting biological sample to be implemented in metabolomics experiments by virtue of the information that can be deduced from the interaction between host and microbiome. However, pig fecal samples have received scant attention, especially in untargeted metabolomic studies. In this research, an analytical strategy was planned to maximize the identification coverage of metabolites found in pig fecal samples. For this purpose, two complementary platforms such as LC-QTOF MS/MS and GC-TOF/MS were used. Concerning sample preparation six extractant solvents with different polarity grade were tested to evaluate the extraction performance and, in the particular case of GC-MS, two derivatization protocols were compared. A total number of 303 compounds by combination of all the extractants and analytical platforms were tentatively identified. The main identified families were amino acids, fatty acids and derivatives, carbohydrates and carboxylic acids. For GC-TOF/MS analysis, the recommended extractant is methanol, while methoxymation was required in the derivatization protocol since this step allows detecting the α-keto acids, which are direct markers of the microbiome status. Concerning LC-QTOF MS/MS analysis, a dual extraction approach with methanol (MeOH) or MeOH/water and ethyl acetate is proposed to enhance the detection of polar and non-polar metabolites.

Pharmacological inhibition of GSK-3 is not strictly correlated with a decrease in tyrosine phosphorylation of residues 216/279.

Recent evidence suggests that intramolecular autophosphorylation is responsible for the tyrosine phosphorylation (pY) of residues 279 or 216 of glycogen synthase kinase-3 (GSK-3alpha or beta), an event that appears to play an important role in regulating this kinase. This provocative hypothesis was based on the capacity of certain nonselective GSK-3 inhibitors to alter both the activity of GSK-3 and its pY. Inhibitors of GSK-3 are not always capable of preventing this tyrosine phosphorylation, which may require an extended period of time. For example, although lithium chloride inhibits GSK-3 activity, this inhibition does not alter its pY content. Furthermore, even when GSK-3 activity is impaired, GSK-3 pY can still be modified by physiological or pharmacological agents. Taken together, these data indicate that GSK-3 kinase activity is not necessarily correlated with the extent of GSK-3 pY. We hypothesized that some as-yet-unidentified tyrosine kinases and phosphatases may also regulate this kinase.

Theoretical study on fulvic acid structure, conformation and aggregation. A molecular modelling approach.

The ubiquitous presence of humic substances (HS), combined with their ability to provide multiple sites for chemical reaction, makes them relevant to numerous biogeochemical processes such as mineral weathering, nutrient bioavailability, and contaminant transport. The reactivity of HS depends on their functional group chemistry and microstructure, which are in turn influenced by the composition of the surrounding media. In order to help towards an understanding of structure conformations and aggregation process of HS in soils and waters and to get a better knowledge of these kinds of materials, a fulvic acid (FA) has been modelled as a function of its ionic state under different conditions. Our proposed theoretical model based on the Temple-Northeastern-Birmingham (TNB) monomer fits well with experimental observations on the solubility (dipolar moment) and electronic and vibrational spectra of FAs. The presence of water molecules has a great stabilization effect on the electrostatic energy; this effect is greater as ionized rate increases. In vacuum, the non-ionized aggregated species are more stable than monomers because of the increase in their interaction due to H-bonding and non-bonding forces. When the molecules are ionized, no aggregation process takes place. In solution, the FA concentration is a critical factor for the aggregation. The system containing two FA molecules probably did not form aggregates because its equivalent concentration was too low. When the concentration was increased, the system gave rise to the formation of aggregates. The ionic state is another critical factor in the aggregation process. The ionized FA has a higher electric negative charge, which increases the energetic barriers and inhibits the approximation of FA caused by the Brownian movement.

Retention of cobalt on a humin derived from brown coal.

In this work, the retention of cobalt on a humin (HU) derived from a brown coal is studied. Through a systematic and coordinated investigation of the behavior of the metal ions in solution (speciation diagrams as a function of pH) and their adsorption and precipitation processes with reactive functional groups of the solid (sorption isotherms), the interactions of different Co(II) species with HU are probed. To further confirm the nature of these interactions, the complementary spectroscopic techniques of FTIR, Raman microspectroscopy, UV-visible absorption and XRD are employed. Molecular modeling techniques are used to gain information about the stability of different Co(II) species as a function of pH, as well as the stability of Co(II) species complexed with benzoic acid, a common surface component of humic substances. It is found that the selectivity that humin has for different Co(II) species, as well as the amount of Co(II) that it can retain, are both highly dependent on pH. Through Raman microspectroscopy measurements, the presence and location of Co(OH)(2) precipitates on the surface of HU is confirmed.

Amyloid precursor protein proteoglycan is increased after brain damage.

The beta-amyloid peptide (Abeta or A4) is produced by proteolytic cleavage from amyloid precursor protein (APP). The progressive cerebral deposition of this peptide is one of the most important features of Alzheimer's disease. From the study of normal and transfected cells, two APP processing pathways have been proposed as physiological alternatives. One of these can produce Abeta or amyloidogenic peptides, whereas the second does not. However, it is not completely clear how APPs are post-translationally modified, proteolytically processed and metabolized in the brain. We report here that APPs also exist as proteoglycan, chondroitin-sulfate (ChS). We have identified in normal rat brain a complex pool of 8 to 130 kDa ChS-core proteins. The main portion of these proteoglycan (PGs) APPs contains complete amyloidogenic sequence, suggesting a novel proteolytic processing of APP from the amino-terminal to the transmembrane region. This population appears augmented after brain damage. These findings may have significant implications in understanding the initial deposition and kinetics of amyloid aggregation in a pathological situation like Alzheimer's disease.

Effect of pH on the aggregation of a gray humic acid in colloidal and solid states.

Gray humic acids have a marked colloidal character, a large number of surface functional groups, and are subject to aggregation phenomena. They are able to complex soluble pollutants, and initiate flocculation processes as a function of environmental conditions. The aim of this work is to study the aggregation of a gray humic acid, which is stable in colloidal dispersion, by means of photon correlation spectroscopy, and molecular modeling. The effect of this aggregation in the solid state is also studied by means of N2 (to 77 K) and CO2 (to 273 K) adsorption isotherms, as well as FT-IR absorption. The variation of the colloid's zeta potential and size, with pH, reflects the ionization of the carboxylic and phenolic acidic groups, and a linear dependence of size on zeta potential. The decrease in the size of the colloids seems to be more affected by the ionization of the phenolic acid groups, than by that of the carboxylic acid groups, which is likely because in the case of the ionized carboxylic groups the humic colloids are still capable of generating H-bonds. In the solid state, aggregation effects are illustrated by a decrease in surface area, and a disappearance of certain micropores, with increasing pH. These features are likely due to an inhibition of aggregation in the colloidal state as a consequence of the increase in charge that results from ionization of the acidic groups, and also to an increased hindrance to H-bond formation, due to the loss of protons during the above-mentioned ionization process.

Molecular cloning, expression pattern and chromosomal mapping of pig CD9 antigen.

CD9 is a member of the transmembrane-4 superfamily of surface molecules that seems to have a relevant role in cell migration and adhesion, as well as malignant progression. This work describes the isolation of the cDNA coding for the porcine CD9 molecule. Pig CD9 cDNA was isolated from a smooth muscle cDNA library and contains a 678-bp open reading frame with its predicted polypeptide sequence of 226 amino acids. The deduced amino acid sequence conserves the main characteristics of TM4 proteins, including the presence of four transmembrane domains. Like their homologous molecules from other species, pig CD9 has two extracellular regions of a different size with the minor loop bearing two possible glycosylation sites. The pig CD9 gene was localized to chromosome 5q25 by using a somatic cell hybrid panel. Analysis of CD9 expression in different porcine cells and tissues demonstrated that CD9 mRNA is ubiquitously expressed.

Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N.

The neuropeptides neurotensin and neuromedin N (from 10(-12) M to 10(-9) M) have been shown in this study to stimulate significantly in vitro several steps of the phagocytic process: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and production of superoxide anion measured by nitroblue tetrazolium reduction in resting peritoneal macrophages from BALB/c mice. A dose-response relationship was observed, with a maximal stimulation of the phagocytic process at 10(-11) M. The two neuropeptides induced no change of intracellular cyclic AMP in murine macrophages. Moreover, adherence and chemotaxis decreased significantly in the presence of EGTA (1 mM), a chelator of extracellular Ca2+, or ryanodine (0.5 mM), a blocker of a Ca(2+)-gated channel from the endoplasmic reticulum, in both controls and samples with the addition of neurotensin or neuromedin N. These results suggest that there is no relation between the cAMP messenger system and the phagocytic process stimulation in murine peritoneal macrophages by neurotensin or neuromedin N. In addition, the results observed with EGTA and ryanodine could indicate that these two neuropeptides produce their effects through an increase of intracellular Ca2+ concentration.

Molecular cloning and characterization of the pig homologue to human CD29, the integrin beta1 subunit.

BACKGROUND: CD29 is the beta1 subunit, a member of the integrin gene superfamily that function as receptor for cell adhesion molecules of the extracellular matrix. Porcine integrin beta1 subunit is involved in rejection of pig-to-human tissue xenografts as target of the natural antibodies present in the human serum. Moreover since CD29, as part of the beta1 integrins very late antigen 4 (VLA-4) and VLA-6, is involved in homing and differentiation of haematopoietic progenitor cells, its characterization in pig is critical to study the interaction of porcine adhesion molecules with human ligands in the induction of donor-specific tolerance toward porcine antigens, a process extremely desirable to prevent rejection of xenogeneic organs. METHODS: The porcine CD29 cDNA has been isolated from a cDNA library and its structure determined. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of CD29 in different tissues. RESULTS: The nucleotide sequence of the porcine cDNA includes an open reading frame encoding a polypeptide of 798 amino acids. Expression analysis showed that porcine CD29 is expressed in all lymphoid tissues tested and, in lower amounts, in nonlymphoid tissues. Pig CD29 deduced amino acid sequence displays extensive conservation compared with CD29 sequences from other species and a common structural feature with all the other CD29 molecules analyzed in mammals, including the 12 potential N-glycosilation sites. Punctual changes between human and swine CD29 molecule into the ligand binding domain, and/or into the regulatory domain, suggest potential differences between human and porcine CD29 relative to the human CD29 ligands. CONCLUSIONS: Cloning of the swine CD29 gene offers a new tool for an alternative protocol of removing xenoreactive antibodies in the recipient. In addition, the determination of the differences between human and swine CD29 will help to understand the adhesion molecule-ligand interactions and their function across the swine-human barrier in xenotransplantation.

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors.

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.

A new epitope on sheep CD45R molecule detected by a monoclonal antibody.

This paper describes the production and characterization of a monoclonal antibody (mAb), Co-46D5, which recognizes a new epitope on the isoform of the homologous sheep leukocyte common antigen (LCA) or CD45. This nmAb was submitted to the 3rd workshop on ruminant leukocyte antigens and was assigned to a cluster reactive with B- and T-cells subsets. Co-46D recognizes a 220 kDa molecule on peripheral blood mononuclear cells (PBMC) and spleen cells but not on thymocytes. Flow cytometry (FCM) analysis shows that Co-46D5 reacted with 30% of PBMC and 50% of spleen cells and more than 95% of cells freshly isolated from lymphoid follicles of the ileal Peyer's patches (IPP) of young lambs. By immunohistochemistry, the antigen was detected mainly on B-cell areas of lymph nodes and spleen. It was also found on a subpopulation of medullar thymocytes. Based on these results, we assume that Co-46D5 recognizes a new epitope on the largest isoform of the sheep CD45 receptor, probably on the homologous to the human CD45RA isoform.