Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Glutamatergic input-output properties of thalamic astrocytes

Astrocytes in the somatosensory ventrobasal (VB) thalamus of rats respond to glutamatergic synaptic input with metabotropic glutamate receptor (mGluR) mediated intracellular calcium ([Ca²?](i)) elevations. Astrocytes in the VB thalamus also release the gliotransmitter (GT) glutamate in a Ca²?-dependent manner. The tripartite synapse hypothesis posits that astrocytic [Ca²?](i) elevations resulting from synaptic input releases gliotransmitters that then feedback to modify the synapse. Understanding the dynamics of this process and the conditions under which it occurs are therefore important steps in elucidating the potential roles and impact of GT release in particular brain activities. In this study, we investigated the relationship between VB thalamus afferent synaptic input and astrocytic glutamate release by recording N-methyl-D-aspartate (NMDA) receptor-mediated slow inward currents (SICs) elicited in neighboring neurons. We found that Lemniscal or cortical afferent stimulation, which can elicit astrocytic [Ca²?](i) elevations, do not typically result in the generation of SICs in thalamocortical (TC) neurons. Rather, we find that the spontaneous emergence of SICs is largely resistant to acute afferent input. The frequency of SICs, however, is correlated to long-lasting afferent activity. In contrast to short-term stimulus-evoked GT release effects reported in other brain areas, astrocytes in the VB thalamus do not express a straightforward input-output relationship for SIC generation but exhibit integrative characteristics.

Moderate relationships between NAA and cognitive ability in healthy adults:implications for cognitive spectroscopy

Background: Proton Magnetic Resonance Spectroscopy (H-MRS) is a non-invasive imaging technique that enables quantification of neurochemistry in vivo and thereby facilitates investigation of the biochemical underpinnings of human cognitive variability. Studies in the field of cognitive spectroscopy have commonly focused on relationships between measures of N-acetyl aspartate (NAA), a surrogate marker of neuronal health and function, and broad measures of cognitive performance, such as IQ. Methodology/Principal Findings: In this study, we used H-MRS to interrogate single-voxels in occipitoparietal and frontal cortex, in parallel with assessments of psychometric intelligence, in a sample of 40 healthy adult participants. We found correlations between NAA and IQ that were within the range reported in previous studies. However, the magnitude of these effects was significantly modulated by the stringency of data screening and the extent to which outlying values contributed to statistical analyses. Conclusions/Significance: H-MRS offers a sensitive tool for assessing neurochemistry non-invasively, yet the relationships between brain metabolites and broad aspects of human behavior such as IQ are subtle. We highlight the need to develop an increasingly rigorous analytical and interpretive framework for collecting and reporting data obtained from cognitive spectroscopy studies of this kind. © 2014 Patel, Blyth, Griffiths, Kelly and Talcott.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (Review)

The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but suprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral factors that influence disease progression will help provide an evidence based approach were realistic expectations regarding patient prognosis can be ascertained.

BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Safety and efficacy of ledipasvir-sofosbuvir in black patients with hepatitis C virus infection: A retrospective analysis of phase 3 data.

UNLABELLED: Black patients chronically infected with genotype 1 hepatitis C virus (HCV) have historically had lower rates of response to interferon-based treatment than patients of other races. In the phase 3 ION program, the single-tablet regimen of the NS5A inhibitor ledipasvir and NS5B nucleotide polymerase inhibitor sofosbuvir was shown to be safe and highly effective in the general population. The aim of this study was to evaluate the safety and efficacy of ledipasvir/sofosbuvir in black patients using data from the three open-label ION clinical trials, which evaluated the safety and efficacy of 8, 12, and 24 weeks of ledipasvir/sofosbuvir with or without ribavirin for the treatment of treatment-naïve and treatment-experienced patients with genotype 1 HCV, including those with compensated cirrhosis. The primary endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). For our analysis, rates of SVR12, treatment-emergent adverse events, and graded laboratory abnormalities were analyzed in black versus non-black patients. Of the 1949 patients evaluated, 308 (16%) were black. On average, black patients were older, had higher body mass index, were more likely to be IL28B non-CC, and had a lower serum alanine aminotransferase at baseline than non-black patients. Overall, 95% of black and 97% of non-black patients achieved SVR12. The rate of relapse was 3% in black patients as compared with 2% in non-black patients. The most common adverse events included fatigue, headache, nausea, and insomnia. The majority of adverse events occurred more frequently in the ribavirin-containing arms of the studies. No differences were observed in overall safety by race. CONCLUSION: A once-daily dosage of ledipasvir/sofosbuvir was similarly effective in black and non-black patients with genotype 1 HCV infection. The addition of ribavirin did not appear to increase SVR12 but was associated with higher rates of adverse events.

Withdrawal of Antidementia Drugs in Older People: Who, When and How?

The evidence base to guide withdrawal of antidementia medications in older people with dementia is limited; while some randomised controlled studies have considered discontinuation of cholinesterase inhibitors, no such studies examining discontinuation of the N-Methyl-D-aspartate receptor antagonist memantine have been conducted to date. The purpose of this opinion article was to summarise the existing evidence on withdrawal of cholinesterase inhibitors and memantine, to highlight the key considerations for clinicians when making these prescribing decisions and to offer guidance as to when and how treatment might be discontinued. Until the evidence-base is enhanced by the findings of large scale randomised controlled discontinuation trials of ChEIs and memantine which use multiple, clinically relevant cognitive, functional and behavioural outcome measures, clinicians’ prescribing decisions involve balancing the risks of discontinuation with side-effects and costs of continued treatment. Such decisions must be highly individualised and patient-centred.

Insight into the mechanism of galactokinase: role of a critical glutamate residue and helix/coil transitions

Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics (MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted that two regions (residues 174-179 and 231-240) had different dynamics as a consequence. Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg- 228. These three residues were identified as important in catalysis in previous computational studies on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in activity with little change in stability. When Arg-228 was changed to methionine, the enzyme’s interaction with both ATP and galactose was affected. This variant was significantly less stable than the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies demonstrate the importance of a negative charge at position 174 and highlight the critical role of the dynamics in to key regions of the protein. We postulate that these regions may be critical for mediating the enzyme’s structure and function. 

Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model

TORRES, F ; FILHO, M.S. ; ANTUNES, C. ; KALININE, E. ; ANTONIOLLI, E. ; PORTELA, Luis Valmor ; SOUZA, Diogo Onofre ; TORT, A. B. L. . Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model. Experimental Neurology , v. 221, p. 296-306, 2010

Modifications neurométaboliques et microstructurales à la suite d'une commotion cérébrale chez les athlètes féminines.

L’utilisation de méthodes d’investigation cérébrale avancées a permis de mettre en évidence la présence d’altérations à court et à long terme à la suite d’une commotion cérébrale. Plus spécifiquement, des altérations affectant l’intégrité de la matière blanche et le métabolisme cellulaire ont récemment été révélées par l’utilisation de l’imagerie du tenseur de diffusion (DTI) et la spectroscopie par résonance magnétique (SRM), respectivement. Ces atteintes cérébrales ont été observées chez des athlètes masculins quelques jours après la blessure à la tête et demeuraient détectables lorsque les athlètes étaient à nouveau évalués six mois post-commotion. En revanche, aucune étude n’a évalué les effets neurométaboliques et microstructuraux dans la phase aigüe et chronique d’une commotion cérébrale chez les athlètes féminines, malgré le fait qu’elles présentent une susceptibilité accrue de subir ce type de blessure, ainsi qu’un nombre plus élevé de symptômes post-commotionnels et un temps de réhabilitation plus long. Ainsi, les études composant le présent ouvrage visent globalement à établir le profil d’atteintes microstructurales et neurométaboliques chez des athlètes féminines par l’utilisation du DTI et de la SRM. La première étude visait à évaluer les changements neurométaboliques au sein du corps calleux chez des joueurs et joueuses de hockey au cours d’une saison universitaire. Les athlètes ayant subi une commotion cérébrale pendant la saison ont été évalués 72 heures, 2 semaines et 2 mois après la blessure à la tête en plus des évaluations pré et post-saison. Les résultats démontrent une absence de différences entre les athlètes ayant subi une commotion cérébrale et les athlètes qui n’en ont pas subie. De plus, aucune différence entre les données pré et post-saison a été observée chez les athlètes masculins alors qu’une diminution du taux de N-acetyl aspartate (NAA) n’a été mise en évidence chez les athlètes féminines, suggérant ainsi un impact des coups d’intensité sous-clinique à la tête. La deuxième étude, qui utilisait le DTI et la SRM, a révélé des atteintes chez des athlètes féminines commotionnées asymptomatiques en moyenne 18 mois post-commotion. Plus spécifiquement, la SRM a révélé une diminution du taux de myo-inositol (mI) au sein de l’hippocampe et du cortex moteur primaire (M1) alors que le DTI a mis en évidence une augmentation de la diffusivité moyenne (DM) dans plusieurs faisceaux de matière blanche. De iii plus, une approche par région d’intérêt a mis en évidence une diminution de la fraction d’anisotropie (FA) dans la partie du corps calleux projetant vers l’aire motrice primaire. Le troisième article évaluait des athlètes ayant subi une commotion cérébrale dans les jours suivant la blessure à la tête (7-10 jours) ainsi que six mois post-commotion avec la SRM. Dans la phase aigüe, des altérations neuropsychologiques combinées à un nombre significativement plus élevé de symptômes post-commotionnels et dépressifs ont été trouvés chez les athlètes féminines commotionnées, qui se résorbaient en phase chronique. En revanche, aucune différence sur le plan neurométabolique n’a été mise en évidence entre les deux groupes dans la phase aigüe. Dans la phase chronique, les athlètes commotionnées démontraient des altérations neurométaboliques au sein du cortex préfrontal dorsolatéral (CPDL) et M1, marquées par une augmentation du taux de glutamate/glutamine (Glx). De plus, une diminution du taux de NAA entre les deux temps de mesure était présente chez les athlètes contrôles. Finalement, le quatrième article documentait les atteintes microstructurales au sein de la voie corticospinale et du corps calleux six mois suivant une commotion cérébrale. Les analyses n’ont démontré aucune différence au sein de la voie corticospinale alors que des différences ont été relevées par segmentation du corps calleux selon les projections des fibres calleuses. En effet, les athlètes commotionnées présentaient une diminution de la DM et de la diffusivité radiale (DR) au sein de la région projetant vers le cortex préfrontal, un volume moindre des fibres de matière blanche dans la région projetant vers l’aire prémotrice et l’aire motrice supplémentaire, ainsi qu’une diminution de la diffusivité axiale (DA) dans la région projetant vers l’aire pariétale et temporale. En somme, les études incluses dans le présent ouvrage ont permis d’approfondir les connaissances sur les effets métaboliques et microstructuraux des commotions cérébrales et démontrent des effets délétères persistants chez des athlètes féminines. Ces données vont de pair avec la littérature scientifique qui suggère que les commotions cérébrales n’entraînent pas seulement des symptômes temporaires.

Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model

TORRES, F ; FILHO, M.S. ; ANTUNES, C. ; KALININE, E. ; ANTONIOLLI, E. ; PORTELA, Luis Valmor ; SOUZA, Diogo Onofre ; TORT, A. B. L. . Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model. Experimental Neurology , v. 221, p. 296-306, 2010

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1 (SOCS1)

Abstract: Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFN-γ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFN-γ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFN-γ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in H SC activation. Liver fibrosis was induced in Socs1[superscript -/-]Ifng[superscript -/-] mice with dimethylnitrosamine or carbon tetrachloride. Ifng[superscript -/-] and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1[superscript -/-]Ifng[superscript -/-] mice showed elevated serum ALT levels and increased liver fibrosis com-pared to mice Ifng[superscript -/-]. The latter group showed higher alanine aminotransferase (ALT) levels and fibrosis than C57BL/6 controls. The livers of Socs1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. Socs1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from Socs1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of Socs1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFN-γ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.

Crassaminicella profunda gen. nov., sp nov., an anaerobic marine bacterium isolated from deep-sea sediments

A novel, anaerobic, chemo-organotrophic bacterium, designated strain Ra1766HT, was isolated from sediments of the Guaymas basin (Gulf of California, Mexico) taken from a depth of 2002 m. Cells were thin, motile, Gram-stain-positive, flexible rods forming terminal endospores. Strain Ra1766H(T) grew at temperatures of 25-45 degrees C (optimum 30 degrees C), pH 6.7-8.1 (optimum 7.5) and in a salinity of 5-60 g l(-1) NaCl (optimum 30 g l(-1)). It was an obligate heterotrophic bacterium fermenting carbohydrates (glucose and mannose) and organic acids (pyruvate and succinate). Casamino acids and amino acids (glutamate, aspartate and glycine) were also fermented. The main end products from glucose fermentation were acetate, butyrate, ethanol, H-2 and CO2. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, nitrate, nitrite and Fe(III) were not used as terminal electron acceptors. The predominant cellular fatty acids were C-14 : 0, C-16:1 omega 7, C-16:1 omega 7 DMA and C-16:0. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipids. The G +C content of the genomic DNA was 33.7 molo/o. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Ra1766H(T) was affiliated to cluster XI of the order Clostridia les, phylum Firmicutes. The closest phylogenetic relative of Ra1766H(T) was Geosporobacter subterraneus (94.2% 16S rRNA gene sequence similarity). On the basis of phylogenetic inference and phenotypic properties, strain Ra1766H(T) (=DSM 27501(T)=JCM 19377(T)) is proposed to be the type strain of a novel species of a novel genus, named Crassaminicella pro funda.