Expansion and tissue infiltration of an allospecific CD4+CD25+CD45RO+IL-7Ralphahigh cell population in solid organ transplant recipients.

It has been recently shown (Seddiki, N., B. Santner-Nanan, J. Martinson, J. Zaunders, S. Sasson, A. Landay, M. Solomon, W. Selby, S.I. Alexander, R. Nanan, et al. 2006. J. Exp. Med. 203:1693-1700.) that the expression of interleukin (IL) 7 receptor (R) alpha discriminates between two distinct CD4 T cell populations, both characterized by the expression of CD25, i.e. CD4 regulatory T (T reg) cells and activated CD4 T cells. T reg cells express low levels of IL-7Ralpha, whereas activated CD4 T cells are characterized by the expression of IL-7Ralpha(high). We have investigated the distribution of these two CD4 T cell populations in 36 subjects after liver and kidney transplantation and in 45 healthy subjects. According to a previous study (Demirkiran, A., A. Kok, J. Kwekkeboom, H.J. Metselaar, H.W. Tilanus, and L.J. van der Laan. 2005. Transplant. Proc. 37:1194-1196.), we observed that the T reg CD25(+)CD45RO(+)IL-7Ralpha(low) cell population was reduced in transplant recipients (P < 0.00001). Interestingly, the CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population was significantly increased in stable transplant recipients compared with healthy subjects (P < 0.00001), and the expansion of this cell population was even greater in patients with documented humoral chronic rejection compared with stable transplant recipients (P < 0.0001). The expanded CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population contained allospecific CD4 T cells and secreted effector cytokines such as tumor necrosis factor alpha and interferon gamma, thus potentially contributing to the mechanisms of chronic rejection. More importantly, CD4(+)IL-7Ralpha(+)and CD25(+)IL-7Ralpha(+) cells were part of the T cell population infiltrating the allograft of patients with a documented diagnosis of chronic humoral rejection. These results indicate that the CD4(+)CD25(+)IL-7Ralpha(+) cell population may represent a valuable, sensitive, and specific marker to monitor allospecific CD4 T cell responses both in blood and in tissues after organ transplantation.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Antibiotic-induced modifications of the stiffness of bacterial membranes.

In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure.

Les biothérapies dans le lupus érythémateux disséminé et tes connectivites: nouvelles thérapeutiques [Treatment of connective tissue diseases with biological agents]

As B-cells are crucial for the production of antibodies and also in antigen presentation, they can play an important role in autoimmune connective tissue disease. B-cell surface antigens and receptors which are capable of activating B-cell function have been proposed as targets for therapy in these diseases. Anti-B cell treatments have been used recently in SLE and primary Sjogren's syndrome in a number of open studies, notably anti-CD20 (rituximab), with encouraging results. An anti-BAFF antibody (belimumab) has been tested in patients with SLE and also showed positive results in patients with increased levels of autoantibodies. In contrast, anti-TNF therapy in connective tissue disease and in RA can increase the levels of autoantibodies. Further studies are needed to define the place of these novel treatments in the management of autoimmune connective tissue diseases.

Major impact of body position on arterial stiffness indices derived from radial applanation tonometry in pregnant and non pregnant woman

Pendant la grossesse, la pression artérielle reste stable malgré une nette augmentation du volume d'éjection systolique et du débit cardiaque. Cette stabilité vient d'un côté d'une vasodilatation périphérique entraînant une diminution des résistances périphériques et d'un autre côté d'une moindre rigidité des principales artères notamment l'aorte. En conséquence, l'amplitude des ondes de pouls est atténuée, de même que leur vitesse de propagation dans le sens tant antérogade que rétrograde (ondes réfléchies). Les ondes réfléchies tendent ainsi à atteindre l'aorte ascendante plus tard durant la systole, voire durant la diastole, ce qui peut contribuer à diminuer la pression puisée. La prééclampsie perturbe massivement ce processus d'adaptation. Il s'agit d'une maladie hypertensive de la grossesse engendrant une importante morbidité et mortalité néonatale et maternelle. Il est à remarquer que la diminution de la rigidité artérielle n'est pas observée chez les patientes atteintes avec pour conséquence une forte augmentation de la pression systolique centrale (aortique) par les ondes réfléchies. Ce fait a été établi grâce à l'existence de la tonométrie d'aplanation, une méthode permettant l'évaluation non invasive de l'onde de pouls centrale. Dans cette méthode, un senseur de pression piézo-électrique permet de capter l'onde de pouls périphérique, le plus souvent sur l'artère radiale. Par la suite, un algorithme validé permet d'en déduire la forme de l'onde de pouls centrale et de visualiser à quel moment du cycle cardiaque s'y ajoutent les ondes réfléchies. Plusieurs études font état d'une forte augmentation de la pression systolique centrale par les ondes réfléchies chez les patientes atteintes de prééclampsie, suggérant l'utilisation de cette méthode pour le diagnostic et le monitoring voire pour le dépistage de ces patientes. Pour atteindre ce but, il est nécessaire d'établir des normes en rapport notamment avec l'âge gestationnel. Dans la littérature, les données pertinentes actuellement disponibles sont variables, voire contradictoires. Par exemple, les ondes réfléchies proéminentes dans la partie diastolique de l'onde de pouls centrale disparaissaient chez des patientes enceintes au 3eme trimestre comparées à des contrôles non enceintes dans une étude lausannoise, alors que deux autres études présentent l'observation contraire. Autre exemple, certains auteurs décrivent une diminution progressive de l'augmentation systolique jusqu'à l'accouchement alors que d'autres rapportent un nadir aux environs du 6ème mois, suivi d'un retour à des valeurs plus élevées en fin de grossesse. Les mesures effectuées dans toutes ces études différaient dans leur exécution, les patientes étant notamment dans des postions corporelles différentes (couchées, semi-couchées, assises, en décubitus latéral). Or nous savons que le status hémodynamique est très sensible aux changements de position, particulièrement durant la grossesse où l'utérus gravide est susceptible d'avoir des interactions mécaniques avec les veines et possiblement les artères abdominales. Ces différences méthodologiques pourraient donc expliquer, au moins en partie, l'hétérogénéité des résultats concernant l'onde de pouls chez la femme enceinte, ce qui à notre connaissance n'a jamais été exploré. Nous avons mesuré l'onde de pouls dans les positions assise et couchée chez des femmes enceintes, au 3eme trimestre d'une grossesse non compliquée, et nous avons effectué une comparaison avec des données similaire obtenues chez des femmes non enceintes en bonne santé habituelle. Les résultats montrent que la position du corps a un impact majeur sur la forme de l'onde de pouls centrale. Comparée à la position assise, la position couchée se caractérise par une moindre augmentation systolique et, par contraste, une augmentation diastolique plus marquée. De manière inattendue, cet effet s'observe aussi bien en présence qu'en l'absence de grossesse, suggérant que la cause première n'en réside pas dans les interactions mécaniques de l'utérus gravide avec les vaisseaux sanguins abdominaux. Nos observations pourraient par contre être expliquées par l'influence de la position du corps, via un phénomène hydrostatique simple, sur la pression transmurale des artères éloignées du coeur, tout particulièrement celles des membres inférieurs et de l'étage abdominal. En position verticale, ces vaisseaux augmenteraient leur rigidité pour résister à la distension de leur paroi, ce qui y accroîtrait la vitesse de propagation des ondes de pression. En l'état, cette explication reste hypothétique. Mais quoi qu'il en soit, nos résultats expliquent certaines discordances entre les études conduites à ce jour pour caractériser l'influence de la grossesse physiologique sur la forme de l'onde de pouls central. De plus, ils indiquent que la position du corps doit être prise en compte lors de toute investigation utilisant la tonométrie d'applanation pour déterminer la rigidité des artères chez les jeunes femmes enceintes ou non. Il sera aussi nécessaire d'en tenir compte pour établir des normes en vue d'une utilisation de la tonométrie d'aplanation pour dépister ou suivre les patientes atteintes de prééclampsie. Il serait enfin intéressant d'évaluer si l'effet de la position sur la forme de l'onde de pouls central existe également dans l'autre sexe et chez des personnes plus âgées.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Tissue-adapted invasion strategies of the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae causes rice blast, the most serious foliar fungal disease of cultivated rice (Oryza sativa). During hemibiotrophic leaf infection, the pathogen simultaneously combines biotrophic and necrotrophic growth. Here, we provide cytological and molecular evidence that, in contrast to leaf tissue infection, the fungus adopts a uniquely biotrophic infection strategy in roots for a prolonged period and spreads without causing a loss of host cell viability. Consistent with a biotrophic lifestyle, intracellularly growing hyphae of M. oryzae are surrounded by a plant-derived membrane. Global, temporal gene expression analysis used to monitor rice responses to progressive root infection revealed a rapid but transient induction of basal defense-related gene transcripts, indicating perception of the pathogen by the rice root. Early defense gene induction was followed by suppression at the onset of intracellular fungal growth, consistent with the biotrophic nature of root invasion. By contrast, during foliar infection, the vast majority of these transcripts continued to accumulate or increased in abundance. Furthermore, induction of necrotrophy-associated genes during early tissue penetration, previously observed in infected leaves, was not seen in roots. Collectively, our results not only report a global characterization of transcriptional root responses to a biotrophic fungal pathogen but also provide initial evidence for tissue-adapted fungal infection strategies.

Chemical Functionalization of Bioceramics To Enhance Endothelial Cells Adhesion for Tissue Engineering.

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

T cell affinity regulates asymmetric division, effector cell differentiation, and tissue pathology.

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

Comparative effects of oleoyl-estrone and a specific b3-adrenergic agonist (CL316, 243) on the expression of genes involved in energy metabolism of rat white adipose tissue

Background: The combination of oleoyl-estrone (OE) and a selective b3-adrenergic agonist (B3A; CL316,243) treatment in rats results in a profound and rapid wasting of body reserves (lipid). Methods: In the present study we investigated the effect of OE (oral gavage) and/or B3A (subcutaneous constant infusion) administration for 10 days to overweight male rats, compared with controls, on three distinct white adipose tissue (WAT) sites: subcutaneous inguinal, retroperitoneal and epididymal. Tissue weight, DNA (and, from these values cellularity), cAMP content and the expression of several key energy handling metabolism and control genes were analyzed and computed in relation to the whole site mass. Results: Both OE and B3A significantly decreased WAT mass, with no loss of DNA (cell numbers). OE decreased and B3A increased cAMP. Gene expression patterns were markedly different for OE and B3A. OE tended to decrease expression of most genes studied, with no changes (versus controls) of lipolytic but decrease of lipogenic enzyme genes. The effects of B3A were widely different, with a generalized increase in the expression of most genes, including the adrenergic receptors, and, especially the uncoupling protein UCP1. Discussion: OE and B3A, elicit widely different responses in WAT gene expression, end producing similar effects, such as shrinking of WAT, loss of fat, maintenance of cell numbers. OE acted essentially on the balance of lipolysislipogenesis and the blocking of the uptake of substrates; its decrease of synthesis favouring lipolysis. B3A induced a shotgun increase in the expression of most regulatory systems in the adipocyte, an effect that in the end favoured again the loss of lipid; this barely selective increase probably produces inefficiency, which coupled with the increase in UCP1 expression may help WAT to waste energy through thermogenesis. Conclusions: There were considerable differences in the responses of the three WAT sites. OE in general lowered gene expression and stealthily induced a substrate imbalance. B3A increasing the expression of most genes enhanced energy waste through inefficiency rather than through specific pathway activation. There was not a synergistic effect between OE and B3A in WAT, but their combined action increased WAT energy waste.

Insulin degradation by adipose tissue is increased in human obesity

White adipose tissue samples from obese and lean patients were used for the estimation ofinsulin protease and insulin:glutathione transhydrogenase using 1251-labeled insulin. There was no activity detected in the absence of reduced glutathione, which indicates that insulin is cleaved in human adipose "tissue through reduction of the disulfide bridge between the chains. O bese patients showed higher transhydrogenase activity (per U tissue protein wt, per U tissue wt, and in the total adipose tissue mass) than the lean group. There is a significant correlation between the activity per U tissue wt, and protein and total activity in the whole adipose tissue with respect to body mass index, with a higher activity in obese patients. The potential ofinsulin cleavage by adipose tissue in obese patients was a mean 5.6-fold higher than that in controla. The coexistence of high insulinemia and high cleavage capability implies that insulin secretion and turnover are increased in the o bese. Thus, white adipose tissue may be crucial in the control of energy availability through modulation ofinsulin cleavage.