T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells.

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.

Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Aluminum hydroxy-interlayered minerals and chemical properties of a subtropical Brazilian Oxisol under no-tillage and conventional tillage

No tillage systems significantly influence the soil system, but knowledge about the effects on the mineralogy of tropical and subtropical soils is limited. This study evaluated the long-term effects (26 years) of no-tillage (NT) on aluminum hydroxy-interlayered minerals of a subtropical Oxisol in Southern Brazil (Guarapuava, PR), compared to the same soil under conventional tillage (CT). The clay fraction (< 2 µm) in soil samples of the surface horizons of a field experiment under both management systems was analyzed by X-ray diffraction (XRD) to identify and characterize Al hydroxy-interlayered minerals before and after treatment with sodium citrate to remove intra-layer material. Soil liquid (solution) and solid phases were also characterized. The contents of total organic C, exchangeable cations, P, and the values of extractable acidity and cation exchange capacity as well as electrical conductivity and levels of dissolved organic C, basic cations, aluminum, Si, and sulfur in the soil solution were higher in the NT soil. Under both soil management systems, more than 90 % of the total soluble Al was complexed with organic compounds, with similar Al activity. No significant changes were detected by 2:1 clay mineral XRD analyses in terms of extension or intercalation of Al-hydroxy-polymers in the no-tilled in comparison to the conventionally tilled soil. In both soil management systems, Al and Si activities in the soil solution indicated thermodynamic stability of 2:1 clay minerals with partially occupied by hydroxy-Al, suggesting deceleration in the intercalation process and a tendency of transforming clay minerals from extensive into partial intercalation.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Interpretation of plasma amino acids in the follow-up of patients: the impact of compartmentation

Results of plasma or urinary amino acids are used for suspicion, confirmation or exclusion of diagnosis, monitoring of treatment, prevention and prognosis in inborn errors of amino acid metabolism. The concentrations in plasma or whole blood do not necessarily reflect the relevant metabolite concentrations in organs such as the brain or in cell compartments; this is especially the case in disorders that are not solely expressed in liver and/or in those which also affect nonessential amino acids. Basic biochemical knowledge has added much to the understanding of zonation and compartmentation of expressed proteins and metabolites in organs, cells and cell organelles. In this paper, selected old and new biochemical findings in PKU, urea cycle disorders and nonketotic hyperglycinaemia are reviewed; the aim is to show that integrating the knowledge gained in the last decades on enzymes and transporters related to amino acid metabolism allows a more extensive interpretation of biochemical results obtained for diagnosis and follow-up of patients and may help to pose new questions and to avoid pitfalls. The analysis and interpretation of amino acid measurements in physiological fluids should not be restricted to a few amino acids but should encompass the whole quantitative profile and include other pathophysiological markers. This is important if the patient appears not to respond as expected to treatment and is needed when investigating new therapies. We suggest that amino acid imbalance in the relevant compartments caused by over-zealous or protocol-driven treatment that is not adjusted to the individual patient's needs may prolong catabolism and must be corrected

Functional role of RXRs and PPARgamma in mature adipocytes.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is abundantly expressed in adipocytes, and plays an important role in adipocyte differentiation and fat accretion. It is a heterodimeric partner of the retinoid X receptors alpha, beta and gamma, which are also expressed in the adipose tissue. As lethality of PPARgamma(-/-) and RXRalpha(-/-) mouse fetuses precluded the analysis of PPARgamma and RXRalpha functions in mature adipocytes, we generated RXRalpha(ad-/-) and PPARgamma(ad-/-) mice, in which RXRalpha and PPARgamma are selectively ablated in adult adipocytes, respectively. Even though the adiposity of RXRalpha(ad-/-) mice is similar to that of control mice when fed a regular diet, they are resistant to chemically and dietary-induced obesity. However, mature adipocytes lacking either both RXRalpha and RXRgamma or PPARgamma die, and are replaced by newly formed adipocytes. Thus, in adipocytes, RXRalpha is essential for lipogenesis, but RXRgamma can functionally replace RXRalpha for the adipocyte vital functions exerted by PPARgamma/RXR heterodimers.

Growth promotion of pineapple 'vitória' by humic acids and burkholderia spp. during acclimatization

In vitro propagation of pineapple produces uniform and disease-free plantlets, but requires a long period of acclimatization before transplanting to the field. Quicker adaptation to the ex vitro environment and growth acceleration of pineapple plantlets are prerequisites for the production of a greater amount of vigorous, well-rooted planting material. The combination of humic acids and endophytic bacteria could be a useful technological approach to reduce the critical period of acclimatization. The aim of this study was to evaluate the initial performance of tissue-cultured pineapple variety Vitória in response to application of humic acids isolated from vermicompost and plant growth-promoting bacteria (Burkholderia spp.) during greenhouse acclimatization. The basal leaf axils were treated with humic acids while roots were immersed in bacterial medium. Humic acids and bacteria application improved shoot growth (14 and 102 %, respectively), compared with the control; the effect of the combined treatment was most pronounced (147 %). Likewise, humic acids increased root growth by 50 %, bacteria by 81 % and the combined treatment by 105 %. Inoculation was found to significantly increase the accumulation of N (115 %), P (112 %) and K (69 %) in pineapple leaves. Pineapple growth was influenced by inoculation with Burkholderia spp., and further improved in combination with humic acids, resulting in higher shoot and root biomass as well as nutrient contents (N 132 %, P 131 %, K 80 %) than in uninoculated plantlets. The stability and increased consistency of the host plant response to bacterization in the presence of humic substances indicate a promising biotechnological tool to improve growth and adaptation of pineapple plantlets to the ex vitro environment.

Anti-TNF-alpha therapy in patients with chronic non-infectious uveitis: the experience of Jules Gonin Eye Hospital.

BACKGROUND: The purpose of this study is to describe the experience of Jules Gonin Eye Hospital on the long-term outcome of anti-TNF-alpha therapy in chronic non-infectious uveitis. PATIENTS AND METHODS: We identified and followed those patients with chronic non-infectious uveitis who received systemic anti-TNF-alpha therapy. Anti-TNF-alpha therapy was administered when no response had been obtained with classical immunosuppressive therapies or in the presence of severe rheumatoid disease. RESULTS: Fifteen patients (28 eyes), 7 male and 8 female (mean age, 43 years; range: 7 to 70 years) were identified. Diagnoses included HLA-B27-associated anterior uveitis (n = 4), sarcoidosis (n = 2), juvenile idiopathic arthritis (n = 2), idiopathic retinal vasculitis with uveitis (n = 2), pars planitis (n = 2), Adamantiades-Behçet disease (n = 1), birdshot retinochoroidopathy (n = 1), and Crohn's disease (n = 1). Mean duration of ocular disease was 8 years (range: 1 to 29 years). Treatment with infliximab (n = 11), etanercept (n = 2), or adalimumab (n = 2) was initiated. One patient with etanercept was switched to infliximab due to lack of clinical response. Clinical and angiographic regression of uveitis was observed within the first two months of therapy in all patients, and was maintained throughout the entire follow-up period (mean 18 months; range: 3 - 72 months). Recurrence was observed in 3 patients, and resolved after adjustment of therapy. Adverse events were recorded in only one patient (arterial hypotension). CONCLUSIONS: In this series of patients with chronic non-infectious uveitis, anti-TNF-alpha therapy was effective and safe. Further clinical studies are needed to determine an adequate duration of therapy.

Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation.

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Molecular weight of hydroxyethyl starch : is there an effect on blood coagulation and pharmacokinetics ?

Résumé Fondement : le développement de solutions d'hydroxy-éthyl-amidons (HEAS) avec peu d'impact sur la coagulation sanguine, mais un effet supérieur sur la volémie, par comparaison aux HEAS couramment utilisés, est d'un grand intérêt clinique. Nous posons l'hypothèse que des solutions de haut poids moléculaire et de bas degré de substitution possèdent ces caractéristiques. Méthode : trente porcs ont été perfusés avec trois HEAS différents (20 ml/kg) de même degré de substitution (0.42) mais de poids moléculaire différent (130, 500 et 900 kDa). Une série de prélèvements sanguins ont été effectués sur 24 heures, sur lesquels des analyses de coagulation sanguine étaient effectuées par thromboélastographie et dosages plasmatiques. De plus, la concentration plasmatique ainsi que le poids moléculaire in vivo ont été déterminés, ainsi que des paramètres de pharmacocinétiques, ceci en se basant sur un modèle bi-compartimental. Résultats : les analyses de thromboélastographie et les tests de coagulation plasmatique n'ont pas démontré d'altération plus marquée de la coagulation sanguine après l'utilisation des solutions des HAES 500 et HAES 900, par comparaison avec celle de HAES 130. Par contre, les HAES 500 et HAES 900 ont présenté une plus grande aire sous la courbe (area under the curve), dans la relation concentration en fonction du temps [1542 (142) g min litre-1, p<0.001, 1701 (321) g min litre-1, p<0.001] par rapport au HAES 130 [1156 (223) g min litre-1]. La demi-vie alpha (t ½α) était plus longue pour les HAES 500 [53.8 (8.6) min, p<0.01] et HAES 900 [57.1 (12.3) min, p<0.01 ]que pour le HAES 130 [39.9 (10.7) min]. La demi-vie beta (t½β) était par contre similaire pour les trois types de HAES [de 332 (100) à 381 (63) min]. Conclusions : pour les HAES de bas degré de substitution, le poids moléculaire n'est pas un facteur clé en ce qui concerne l'altération de la coagulation. La persistance intravasculaire initialement plus longue des HAES de haut poids moléculaire et bas degré de substitution pourrait résulter dans un plus long effet volémique de ces substances. Abstract Background: The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties. Methods: Thirty pigs were infused with three different HES solutions (20 ml kg-1) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph® analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model. Results: Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre-1, P<0.001, 1701 (321) g min litre-1, P<0.001] than HES 130 [I 156 (223) g min litre-1] and alpha half life (t ½α) was longer for HES 500 [53.8 (8.6) min, P<0.01 ] and HES 900 [57. I (I 2.3) min, P<0.01 ] than for HES 130 [39.9 (I 0.7) min]. Beta half life (t½β), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]. Conclusions. In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight lowsubstituted HES might result in a longer lasting volume effect.

Alpha-band suppression in the visual word form area as a functional bottleneck to consciousness.

The current state of empirical investigations refers to consciousness as an all-or-none phenomenon. However, a recent theoretical account opens up this perspective by proposing a partial level (between nil and full) of conscious perception. In the well-studied case of single-word reading, short-lived exposure can trigger incomplete word-form recognition wherein letters fall short of forming a whole word in one's conscious perception thereby hindering word-meaning access and report. Hence, the processing from incomplete to complete word-form recognition straightforwardly mirrors a transition from partial to full-blown consciousness. We therefore hypothesized that this putative functional bottleneck to consciousness (i.e. the perceptual boundary between partial and full conscious perception) would emerge at a major key hub region for word-form recognition during reading, namely the left occipito-temporal junction. We applied a real-time staircase procedure and titrated subjective reports at the threshold between partial (letters) and full (whole word) conscious perception. This experimental approach allowed us to collect trials with identical physical stimulation, yet reflecting distinct perceptual experience levels. Oscillatory brain activity was monitored with magnetoencephalography and revealed that the transition from partial-to-full word-form perception was accompanied by alpha-band (7-11 Hz) power suppression in the posterior left occipito-temporal cortex. This modulation of rhythmic activity extended anteriorly towards the visual word form area (VWFA), a region whose selectivity for word-forms in perception is highly debated. The current findings provide electrophysiological evidence for a functional bottleneck to consciousness thereby empirically instantiating a recently proposed partial perspective on consciousness. Moreover, the findings provide an entirely new outlook on the functioning of the VWFA as a late bottleneck to full-blown conscious word-form perception.