A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin.

BACKGROUND: The activity of the renin-angiotensin system is usually evaluated as plasma renin activity (PRA, ngAI/ml per h) but the reproducibility of this enzymatic assay is notoriously scarce. We compared the inter and intralaboratory reproducibilities of PRA with those of a new automated chemiluminescent assay, which allows the direct quantification of immunoreactive renin [chemiluminescent immunoreactive renin (CLIR), microU/ml]. METHODS: Aliquots from six pool plasmas of patients with very low to very high PRA levels were measured in 12 centres with both the enzymatic and the direct assays. The same methods were applied to three control plasma preparations with known renin content. RESULTS: In pool plasmas, mean PRA values ranged from 0.14 +/- 0.08 to 18.9 +/- 4.1 ngAI/ml per h, whereas those of CLIR ranged from 4.2 +/- 1.7 to 436 +/- 47 microU/ml. In control plasmas, mean values of PRA and of CLIR were always within the expected range. Overall, there was a significant correlation between the two methods (r = 0.73, P < 0.01). Similar correlations were found in plasmas subdivided in those with low, intermediate and high PRA. However, the coefficients of variation among laboratories found for PRA were always higher than those of CLIR, ranging from 59.4 to 17.1% for PRA, and from 41.0 to 10.7% for CLIR (P < 0.01). Also, the mean intralaboratory variability was higher for PRA than for CLIR, being respectively, 8.5 and 4.5% (P < 0.01). CONCLUSION: The measurement of renin with the chemiluminescent method is a reliable alternative to PRA, having the advantage of a superior inter and intralaboratory reproducibility.

Lipoxygenase activities during development of root and nodule of soybean

The objective of this work was to evaluate root and nodule soybean lipoxygenases in Doko cultivar and in a near isogenic line lacking seed lipoxygenases, inoculated and uninoculated with Bradyrhizobium elkanii. The lipoxygenase activities from roots collected at 3, 5, 9, 13, 18 and 28 days post-inoculation and from nodules collected at 13, 18 and 28 days post-inoculation were measured. The pH-activity profiles from root and nodules suggested that the lipoxygenases pool expressed in these organs from Doko cultivar and triple-null near isogenic lines are similar. The root lipoxygenase activity of Doko and triple-null lines, inoculated and uninoculated, reduced over time. The highest lipoxygenase activity observed at the beginning of root formation suggests the involvement of this enzyme in growth and development of this organ. However, for nodules an expressive increase of lipoxygenase activity was noticed 28 days post-inoculation. Root and nodule showed, at least, two mobility groups for lipoxygenases in immunoblottings, with approximately 94 and 97 kDa.

Role of the naive and memory CD4+T-cell repertoire in transplantation

Purpose: The exact role of individual T cell-subsets in the development of rejection is not clearly defined. Given their distinct phenotypes, effector functions and trafficking patterns, naïve (CD45RBhiCD44lo) and memory (CD45RBloCD44hi) T cells may play distinct roles in anti-donor immunity after transplantation. Furthermore, only the CD4+CD45RBlo population contains CD4+CD25+ T cells, a subset with suppressive functions playing a major role in the maintenance of peripheral tolerance. The aim of this work was to study the contribution of these individual subsets in alloresponses via the direct and indirect pathways using a murine experimental model. Methods and materials: Purified naïve or memory CD4+ T cells were adoptively transferred into lymphopenic mice undergoing a skin allograft. Donor to recipient MHC combinations were chosen in order to study the direct and the indirect pathways of allorecognition separately. Graft survival and in vivo expansion, effector function and trafficking of the transferred T cells was assessed at different time points after transplantation. Results: We found that the cross-reactive CD4+CD45RBlo memory T-cell pool was heterogeneous and contained cells with regulatory potentials, both in the CD4+CD25+ and CD4+CD25-populations. CD4+ T cells capable of inducing strong primary alloreactive responses in vitro and rejection of a first allograft in vivo were mainly contained within the CD45RBhi naïve CD4+ T-cell compartment. CD4+CD45RBlo T cells proliferated less abundantly to allogeneic stimulation than their naïve counterparts both in vitro and in vivo, and allowed prolonged allograft survival even after the depletion of the CD4+CD25+ subset. Interestingly, CD4+CD25-CD45RBlo T cells were capable of prolonging allograft survival, mainly when the indirect pathway was the only mechanism of allorecognition. The indirect pathway response, which was shown to drive true chronic rejection and contribute to chronic allograft dysfunction, was predominantly mediated by naïve CD4+ T cells. Conclusion: This work provides new insights into the mechanisms that drive allograft rejection and should help develop new clinical immunosuppressive protocols. In particular, our results highlight the importance of selectively targeting individual T-cell subsets to prevent graft rejection but at the same time maintain immune protective responses to common pathogens.

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Iowa Interstate Corridor Plan, 2013

The purpose of this Interstate Corridor Plan (plan) is to provide the Iowa Department of Transportation (Iowa DOT) with an initial screening and prioritization of interstate corridors/segments. This process evaluates the entire interstate system, independent of current financial constraints, using a select group of criteria weighted in terms of their relative significance. The resulting segments would then represent those areas that should be considered for further study (e.g., environmental, design, engineering), with the possibility of being considered for programming by the Iowa Transportation Commission. There was a dominant theme present in conversations with those department stakeholders who have a keen interest in the product of this planning effort. A statement that was often heard was that staff needed more information to help answer the question, “Where do we need to be looking to next, and when?” There was a strong desire to be able to use this plan to help populate that initial pool of candidate segments that would progress towards further study, as discussed below. It was this theme that framed the need for this plan and ultimately guided its development. Further study: As acknowledged at the beginning of this section, the product of this planning effort will be an initial screening and prioritization of interstate corridors/segments. While this initial screening will assist the Iowa DOT in identifying those areas that should be considered for further study, the plan will not identify specific projects or alternatives that could be directly considered as part of the programming process. Bridging the gap between this plan and the programming process are a variety of environmental, design, and engineering activities conducted by various Iowa DOT offices. It is these activities that will further refine the priority corridors/segments identified in this plan into candidate projects. In addition, should the evaluation process developed through this planning effort prove to be successful, it is possible that there will be additional applications, such as future primary system highway plans and statewide freight plans.

Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.

Late-onset cytomegalovirus (CMV) disease commonly occurs after discontinuation of antiviral prophylaxis. We determined the utility of testing CD8+ T-cell response against CMV as a predictor of late-onset CMV disease after a standard course of antiviral prophylaxis. Transplant patients at high-risk for CMV disease were enrolled. CD8+ T-cell-mediated immunity (CMI) was tested using the QuantiFERON-CMV assay at baseline, 1, 2 and 3 months posttransplant by measurement of interferon-gamma response to whole blood stimulation with a 21-peptide pool. The primary outcome was the ability of CMI testing to predict CMV disease in the first 6 months posttransplant. There were 108 evaluable patients (D+/R+ n = 39; D-/R+ n = 34; D+/R- n = 35) of whom 18 (16.7%) developed symptomatic CMV disease. At the end of prophylaxis, CMI was detectable in 38/108 (35.2%) patients (cutoff 0.1 IU/mL interferon-gamma). CMV disease occurred in 2/38 (5.3%) patients with a detectable interferon-gamma response versus 16/70 (22.9%) patients with a negative response; p = 0.038. In the subgroup of D+/R- patients, CMV disease occurred in 1/10 (10.0%) patients with a detectable interferon-gamma response (cutoff 0.1 IU/mL) versus 10/25 (40.0%) patients with a negative CMI, p = 0.12. Monitoring of CMI may be useful for predicting late-onset CMV disease.

Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

Proyecto de ejecución de zona lúdica de verano formada por piscinas, jacuzzi y edificio de servicios

Este trabajo final de carrera se basa en la redacción de un proyecto de ejecución de una zona lúdica situada en el interior del recinto de un camping, la cual estará formada por una piscina de grandes dimensiones formada por tres vasos de tipo desbordante, una piscina de tipo infantil, un jacuzzi y finalmente un edificio de servicios complementarios (bar, aseos y enfermería). La piscina grande estará formada por tres vasos donde el agua va deslizándose de uno a otro hasta ser recogida por un depósito de compensación desde donde se realizará la filtración y tratamiento antes de ser devuelta al piscina de nuevo. La instalación está también preparada para ser utilizado cada vaso como una piscina individual, recogiendo el agua por los rebosaderos laterales y conduciéndola al mismo vaso de compensación. El tratamiento del agua se realizará de forma física a través de la filtración mediante filtros de arena y de forma química analizando la cantidad de cloro residual y el nivel de PH y posteriormente añadiendo de forma automática las cantidades de cloro y corrector de PH adecuados mediante bombas dosificadoras de precisión. La piscina infantil es de pequeñas dimensiones y esta preparada para ser utilizada por niños de muy corta edad. Se diferencia de la piscina grande en el sistema de recirculación de agua (en este casos e utilizarán skimmers) y en el tratamiento posterior, ya que se utilizará el sistema de hidrólisis salina para su desinfección en lugar de añadir directamente cloro. Este sistema de tratamiento del agua no es tan agresivo como el tradicional, siendo cada vez mas utilizado en piscinas de mediano y pequeño tamaño. El jacuzzi es de grandes dimensiones, disponiendo de su propio sistema de tratamiento de agua, utilizando el sistema de hidrólisis salina como en el caso de la piscina infantil. El edificio de servicios dispone de zonas de bar, enfermería y aseos. Se ha descrito desde la estructura a las instalaciones, justificando las conclusiones a través de los cálculos correspondientes, bien de forma manual, bien utilizando diverso software especializado.

Human CD8 T-cell responses to Epstein-Barr virus and Cytomegalovirus in healthy donors and melanoma patients

Summary The mechanisms regulating the protective immune T-cell responses generated against the persistent Epstein-Barr virus (EBV) and Cytomegaloviru_s (CNIV) remain poorly understood. We analyzed the dynamics of cellular differentiation and T-cell receptor (TCR) clonotype selection of EBV- and CMV-specific T-cells in healthy adults and melanoma patients. While these responses could be subdivided into four T lymphocyte populations, théir proportions varied between EBV and CMV specific responses. Phenotypic and TCR clonotypic analyses supported a linear model of differentiation from the early-differentiated (EM/CD28pos) subset to the late-differentiatdc (EMRA/CD28neg) subset. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, as some clonotypes were selected with differentiation, while others were not. Latedifferentiated CMV-specific clonotypes were mostly characterized by TCRs with lower dependency on CD8 co-receptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of four years. This work was extended to the study of EBV-specific CD8 T-cell responses in melanoma patients undergoing transient lymphodepletion, followed by adoptive cell transfer (ACT) and immune reconstitution for thè treatment of their tumors. Following treatment regimen, we first observed an increase in the proportion of virus-specific T-cells in 3 out of 5 patients, accompanied by a more differentiated phenotype (EMRA/CD28neg), compared to specific cells of healthy individuals. Yet, similarly to healthy donors, clonotype selection and composition of virus-specific T-cells varied along the pathway of cellular differentiation, with some clonotypes being selected with differentiation, while others were not. Intriguingly, no novel clonotypes emerged following transient immuno-suppression and homeostatic proliferation, finding which was subsequently explained by the absence of EBV reactivation. The distribution of each clonotype within early- and late-differentiated T-cell subsets in 4 out 5 patients was highly stable over time, with those clonotypes initially found before the start of treatment that were again present at specific differentiation stages after transient lymphodepletion and ACT. These findings uncover novel features of the highly sophisticated control of steady state protective T-cell immune responses against persistent herpesviruses in healthy adults. Furthermore they reveal the striking stability of these responses in terms of clonotype selection and composition with T-cell differentiation even in situations where the immune system has been. challenged. Résumé : Les mécanismes qui régulent les réponses immunitaires de type protectrices, générées contre les virus chroniquement persistants tels que l'Epstein-Barr (EBV) ou le Cytomegalo (CMV) restent largement inconnus. Nous avons analysé la différenciation des lymphocytes T spécifiques pour ces virus, ainsi que la composition des clonotypes T (par leur récepteur T) chez les donneurs sains. Les réponses immunes peuvent être classifiées en quatre souspopulations majeures de lymphocytes T, cependant, leur proportion varie entre les réponses spécifiques contre EBV ou CMV. Ces analyses soutiennent le modèle linéaire de différenciation, à partir de la population non différenciée (EM/CD28pos) vers la population plus différenciée (ENIIZA/CD28neg). De plus, nos données sur la composition clonale de ces cellules T spécifiques ont révélé des répertoires TCR restreints, pour la réponse anti-CMV, et relativement diversifiés contre EBV. Tous les clonotypes spécifiques de ces virus identifiés dans la sous-population différenciée EMRA/CD28neg, ont également été retrouvés dans la population de cellules "mémoires". Toutefois, de fortes différences ont été observées dans les schémas de domination de ces sous-populations, en effet, certains clonotypes étaient sélectionnés avec la différenciation, alors que d'autres ne l'étaient pas. Nous avons également démontré que ces clonotypes différenciés et spécifiques pour le CMV sont caractérisés par des TCRs à faible dépendance en regard de la coopération du corécepteur CD8. Néanmoins, tous les clonotypes affichent une avidité fonctionnelle similaire, suggérant un rôle compensatoire du CD8, dans le cas des clonotypes avec une faible avidité du TCR En définitive, la composition et la sélection des clonotypes spécifiques pour chaque virus et pour chaque sous-population suit un schéma de différenciation hautement conservé au cours du temps, avec la présence de ces mêmes clonotypes au même stade de différenciation sur une période de quatre ans. Ce travail a été étendu à l'étude des réponses T CD8+ spécifiques pour le virus EBV chez les patients atteints de mélanome et recevant dans le cadre du traitement de leurs tumeurs une lymphodéplétion transitoire, suivie d'un transfert adoptif de cellules et d'une reconstitution immunitaire. Au cours de cette thérapie, nous avons en premier lieu observé pour 3 des 5 patients une augmentation de la proportion de cellules T spécifiques pour le virus, accompagné d'un phénotype plus différencié (EMRA/CD28neg), et ceci comparativement à des cellules spécifiques d'individus sains. Pourtant, comme nous l'avons observé chez les donneurs sains, la sélection et la composition des clonotypes T spécifiques varient tout au long de la différenciation cellulaire, avec certains clonotypes sélectionnés et d'autres qui ne le sont pas. Étonnamment, aucun nouveau clonotype n'a émergé après l'immuno-suppression transitoire et la prolifération homéostatique. Cette observation trouve son explication par une absence de réactivation du virus EBV chez ces patients, et ce malgré leur traitement. De plus, la distribution de chaque clonotype parmi ces sous-populations non-différenciées et différenciées reste stable au cours du traitement. Ainsi, les mêmes clonotypes initialement identifiés avant le début du traitement sont présents aux mêmes stades de différenciation après la lymphodéplétion et la prolifération homéostatique. Ces résultats ont permis d'identifier de nouveaux mécanismes impliqués dans la régulation hautement «sophistiquée » des réponses immunitaires T contre les virus persistants EBV et CMV chez les donneurs sains. En particulier, ils révèlent la grande stabilité de ces réponses en termes de sélection et de composition des clonotypes avec la différenciation cellulaire, et ce dans les situations chroniques, ainsi que dans les situations dans lesquelles le système immunitaire a été profondément perturbé.

Fish passage assessment at the most downstream barrier of the Ebro River (NE Iberian Peninsula)

Fish passage at artificial barriers is necessary for the conservation of healthy fish stocks. The first barrier that migratory fish encounter when ascending the Ebro River is the Xerta Weir, where a pool-type fishway was constructed in 2008. From 2007 to 2010, boat electrofishing surveys were conducted in the Ebro River downstream of the Xerta Weir to assess the potential pool of species that could use the fishway. Nine native and 12 exotic species were captured, the latter comprising 62 % of the relative abundance and 70 % of the biomass. A combination of video recording, electrofishing and trapping was used to assess the effectiveness of the fishway in facilitating the passage of fish. Eight species were detected using the fishway, of which five were native (Liza ramada, Anguilla anguilla, Barbus graellsii, Gobio lozanoi and Salaria fluviatilis) and three exotic (Alburnus alburnus, Cyprinus carpio and Rutilus rutilus). Only L. ramada used the fishway in substantial numbers. The rate of fish passage was the highest from June to August and decreased afterwards. The effectiveness of the fishway might be lowered by areas of turbulence within the fishway and by distraction flows from a nearby hydropower station

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Volume-targeted and whole-heart coronary magnetic resonance angiography using an intravascular contrast agent.

PURPOSE: To compare volume-targeted and whole-heart coronary magnetic resonance angiography (MRA) after the administration of an intravascular contrast agent. MATERIALS AND METHODS: Six healthy adult subjects underwent a navigator-gated and -corrected (NAV) free breathing volume-targeted cardiac-triggered inversion recovery (IR) 3D steady-state free precession (SSFP) coronary MRA sequence (t-CMRA) (spatial resolution = 1 x 1 x 3 mm(3)) and high spatial resolution IR 3D SSFP whole-heart coronary MRA (WH-CMRA) (spatial resolution = 1 x 1 x 2 mm(3)) after the administration of an intravascular contrast agent B-22956. Subjective and objective image quality parameters including maximal visible vessel length, vessel sharpness, and visibility of coronary side branches were evaluated for both t-CMRA and WH-CMRA. RESULTS: No significant differences (P = NS) in image quality were observed between contrast-enhanced t-CMRA and WH-CMRA. However, using an intravascular contrast agent, significantly longer vessel segments were measured on WH-CMRA vs. t-CMRA (right coronary artery [RCA] 13.5 +/- 0.7 cm vs. 12.5 +/- 0.2 cm; P < 0.05; and left circumflex coronary artery [LCX] 11.9 +/- 2.2 cm vs. 6.9 +/- 2.4 cm; P < 0.05). Significantly more side branches (13.3 +/- 1.2 vs. 8.7 +/- 1.2; P < 0.05) were visible for the left anterior descending coronary artery (LAD) on WH-CMRA vs. t-CMRA. Scanning time and navigator efficiency were similar for both techniques (t-CMRA: 6.05 min; 49% vs. WH-CMRA: 5.51 min; 54%, both P = NS). CONCLUSION: Both WH-CMRA and t-CMRA using SSFP are useful techniques for coronary MRA after the injection of an intravascular blood-pool agent. However, the vessel conspicuity for high spatial resolution WH-CMRA is not inferior to t-CMRA, while visible vessel length and the number of visible smaller-diameter vessels and side-branches are improved.

Forensic imaging: New challenges for radiographers : B-588

Purpose: Cross-sectional imaging techniques have pioneered forensic medicine. The involvement of a radiographer and formation of "forensic radiographers" allows an improvement of the quality of radiological examinations and facilitates the implementation of techniques, such as sample collections, and the performance of post-mortem angiography. Methods and Materials: During a period of three months, five radiographers with clinical experience have undergone a special training in order to learn procedures dedicated to forensic imaging. These procedures involved: I). acquisition of MDCT data, II). sample collection for toxicological or histological analyses by performing CT-guided biopsies and liquid sampling, III). post-mortem angiography and IV). post-processing of all data acquired. To perform the post-mortem angiography, radiographers were in charge of the preparation of the perfusion device and the investigated body. Therefore, cannulas were inserted into the femoral vessels and connected to the machine. For angiography, the radiographers had to synchronize the perfusion with the CT-acquisitions. Results: All five radiographers have acquired new skills to become "forensic radiographers". They were able to perform post-mortem MDCT, sample collection, post-mortem angiography and post-processing of the acquired data all by themselves. Most problems have been observed concerning the preparation of the body for post-mortem angiography. Conclusion: Our experience shows that radiographers are able to perform high quality examinations after a short period of training. Their collaboration is well accepted by the forensic team and regarding the increase of radiological exams in forensic department, it would be nonsense to exclude radiographers from the forensic-radiological team.

Recent Observations of the Distribution and Status of Bluntnose Darters and Crystal Darters in Iowa

The distribution and status of Iowa's fishes were last extensively described in Iowa Fish and Fishing (Harlan et al. 1987). Since then, numerous fish collections have been made in Iowa's interior and bordering rivers and streams. Excluding non-native species, there have been three documented accounts of new fish species distributional records in Iowa since 1987. In this paper, I describe new collections of Crystal Darter (Crystallaria asprella) and Bluntnose Darter (Etheostoma chlorosomum) from the Mississippi River. The first documented specimen of C. asprella in Iowa was collected in Pool 11 of the Upper Mississippi River (UMR) in 1995. One specimen of E. chlorosomum was collected in Pool 13 of the UMR in 1998, and another was collected in 1999. The bluntnose darter had not been collected since 1975 and was generally thought to be extirpated in Iowa.

Signalling pathways in skin homeostasis

SUMMARY The results presented here contribute to a better understanding of the crucial molecular relationships and signalling cues exchanged by several fundamental cell types (epidermal keratinocytes, dermal fibroblasts, immune and endothelial cells) of the skin. Importantly we provide evidence to directly implicate Wnt/ß-catenin signalling as a putative player in different cell types (keratinocytes and neutrophils) in mediation of the cutaneous inflammatory response (Fart A). Finally we highlight the importance of several molecules, specifically expressed in the hair follicle stem cell niche to the morphogenesis and homeostasis of the hair follicle (Part B). PART A Currently the body of work pertaining to Wnt signalling and immune cells largely focuses on Wnt signalling in the development of these cells. The data presented here suggests a novel mechanism in which Wnt signalling appears to modulate immune cell recruitment to the skin. Keratinocytes are major contributors to early inflammatory responses by the release of chemokines which recruit immune cells. The resultant inflammatory response is a dynamic process of sequentially infiltrating immune cells governed by a network of growth factors, chemokines and cytokines. In wild type mice the response is typified by a rapid and substantial infiltration of neutrophils followed at later time points by macrophages and Tcells. The expression of the canonical Wnt pathway activating ligand, Wnt3a, is able to induce a strong neutrophil infiltration in the dermis. This response originates in keratinocytes, as it is abrogated upon keratinocyte-specific ablation of ß-catenin. Notably, this suggests that the crucial cross talk between these resident cells and recruited immune cells is, in part, mediated by Wnt signalling. In corroboration of this role of Wnt-mediated recruitment of neutrophils, expression of the Wnt inhibitory ligand sFRPI during acute inflammation results in a dramatic 'dampening' of immune cell infiltration in particular of neutrophil chemoattraction. Importantly, an intrinsic Wnt signalling pathway is essential for neutrophil chemoattraction in response to inflammatory stimuli. There is a marked reduction of neutrophil infiltration in mice grafted with a ß-catenin deficient bone marrow upon TPA induced cutaneous inflammation. Additionally, neutrophils lacking Wnt/ß-catenin fail to respond to IFNγ, an early inflammatory cue, in vitro. In combination, these data indicate a potent function of Wnt signalling in immune cell recruitment and the modulation of the inflammatory response. PART B Tissue specific stem cells form the cellular base on which tissue homeostasis and repair of adult tissue relies. The maintenance of this stem cell pool is highly dependent on the immediate environment or niche. We have identified three genes, the fibroblast growth factor receptor 1 (FGFR1), serpin protease inhibitor (serpin F1) and the haematopoietic cell phosphatase (Hcph) to be specifically expressed in a small population of stromal cells which are in close contact to bulge stem cells. These specialized stromal cells might represent an essential mesenchymal component of the skin stem cell niche and may regulate stem cell proliferation and differentiation. Multiple FGFR1 isoforms are generated through alternate transcript splicing and are able to interact with both FGFs and cell adhesion molecules. Two predominant forms of the receptor are FGFR1-α and FGFR1-ß. Expression of a dominant negative form of the alpha isoform prevents hair follicle morphogenesis altogether. Given that FGFR1-ß signals principally through the FGF ligands, this data indicates that FGF signalling is dispensable for follicle morphogenesis. Moreover the loss of follicular morphogenesis upon suggests a requirement for signalling via cell adhesion molecule association with the receptor as FGFR1 α has a greater affinity for these molecules. The expression of the second candidate niche gene serpin f1, lead to the complete ablation of hair follicle morphogenesis. The serpin f1 product, pigment-epithelial derived factor (PEDF) has potent anti-angiogenic effects. Immunohistochemical analysis using CD31, a endothelial cell marker, revealed that although these cells are present, they have are disorganised and do not form vessels. Interestingly, endothelial cells have been found to contribute to the neuronal stem cell niche and our results suggest a similar mechanism in the skin. SHP1, the Hcph gene product, is a phosphatase which acts in the haematopoetic system. Motheaten mice carrying spontaneous mutations in the Hcph gene have patchy alopecia in their skin and severe defects in their haematopoietic system. However the haematopoietic rescue of the mouse does not result in normal follicular homeostasis. Additionally, ablation of Hcph in either the dermal or keratinocyte compartments of the skin produces hair follicles with abberant morphologies. This data indicates that although SHP1 is not essential for hair follicle morphogenesis it is required in both epidermal and dermal compartments to maintain follicular morphology. RÉSUMÉ PARTIE A Jusqu'à présent, les travaux dédiés à l'étude de la voie de signalisation Wnt dans le système immunitaire se sont essentiellement concentrés sur son rôle dans le développement des cellules immunitaires. Les données présentées ici suggèrent fortement et de manière nouvelle, l'existence d'un mécanisme par lequel la voie de signalisation Wnt/ß-caténine module le recrutement de cellules immunitaires dans un tissu périphérique, la peau, et ainsi la réponse inflammatoire cutanée. La réponse inflammatoire cutanée est un processus dynamique d'infiltration séquentielle de diverses cellules immunitaires, orchestré par un réseau de facteurs de croissance, chémokines et cytokines. Les kératinocytes sont des contributeurs majeurs à la réponse inflammatoire précoce par la libération de chémokines qui permettent ensuite de recruter les cellules immunitaires. Dans des souris sauvages, la réponse est d'abord caractérisée par une infiltration rapide et substantielle de neutrophiles, suivie par celle des macrophages et des lymphocytes T. L'expression d'un ligand activateur de le voie canonique de signalisation Wnt (après injection infra-dermique de fibroblastes sur-exprimant Wnt-3a) induit une infiltration dermique très marquée de neutrophiles. De plus, la réponse est éliminée en l'absence de ß-caténine spécifiquement dans les kératinocytes, indiquant que ces cellules sont à l'origine de la réponse. De manière remarquable, ceci suggère qu'une signalisation cruciale entre ces cellules résidentes de la peau et les cellules immunitaires recrutées est, au moins en partie, médiée par la voie Wnt. Corroborant ce rôle de la voie Wnt/ß-caténine dans le recrutement des neutrophiles, l'expression d'un ligand inhibiteur de la voie (sFRP1) résulte au cours d'une inflammation aigüe en une réduction spectaculaire de l'infiltration des cellules immunitaires en général, et des neutrophiles en particulier. De manière importante, la voie de signalisation Wnt est intrinsèquement requise pour la chémoattraction des neutrophiles en réponse à un stimulus inflammatoire. En effet, suite à une inflammation cutanée induite par un ester de phorbol (TPA), une réduction notable de l'infiltration des neutrophiles est observée dans des souris préalablement greffées avec de la moelle osseuse constituée de cellules déficientes en ß-caténine. De plus, in vitro, les neutrophiles sans ß-caténine ne répondent pas à une stimulation par l'interféron γ, qui est pourtant un signal inflammatoire établi in vivo. En conclusion, nos données indiquent que la voie de signalisation Wnt/ß-caténine joue une fonction active dans le recrutement des cellules immunitaires vers un organe périphérique, la peau, ainsi que dans la modulation, à plusieurs niveaux, de la réponse inflammatoire cutanée. PARTIE B Les cellules souches tissu-spécifiques forment la base cellulaire sur laquelle repose l'homéostase et la réparation tissulaires chez l'adulte. La maintenance de ce réservoir de cellules souches est hautement dépendante de leur environnement cellulaire immédiat, encore appelé «niche des cellules souches». Dans la peau, ces cellules stromales spécialisées représentent un compartiment mésenchymateux essentiel de la niche des cellules souches en régulant leurs prolifération et différentiation. Nous avons identifié trois gènes, le «récepteur 1 àux facteurs de croissance des fibroblastes » (Fgfr1 ), l' «inhibiteur de protéase à sérine » (serpinf1 ou pedf) et la « phosphatase des cellules hématopoiétiques » (Hcph ou Ptpn6), comme spécifiquement exprimés par une petite population de cellules stromales qui sont étroitement associées aux cellules souches de la peau (localisées au niveau du bombement du follicule pileux). Pour analyser leur fonction dans ce contexte, nous avons utilisé un test de reconstitution complète de peau murine en combinaison à des. transductions géniques basées sur l'utilisation de lentivirus. Ce test repose sur le mélange de deux compartiments cellulaires, épidermique (kératinocytes) et dermique (fibroblastes), greffés sur une zone ouverte de peau du dos d'une souris pour ensemble reconstituer la peau. Des isoformes multiples de FGFR1 sont générées par épissage alternatif de transcrits et sont capables d'interagir à la fois avec les FGFs (facteurs de croissance des fibroblastes) et les molécules d'adhésion cellulaires. Les deux formes prédominantes du récepteur, FGFR1-α et FGFR1-ß, ne différent que par le «domaine ressemblant aux immunoglobulines 1 » (immunoglobulin-like 1 domain), absent de FGFR1-ß. De plus, FGFR1-ß a une affinité plus grande pour les FGFs et plus faible pour les molécules d'adhésion cellulaires telles que la Ncadhérine (connue pour activer FGFR). La sur-expression de l'une ou l'autre des formes n'empêche pas la morphogenèse folliculaire mais conduit à la formation de follicules aberrants. Toutefois, une différence phénotypique majeure est observée lorsqu'une forme «Dominant-Négatif » (DN) est exprimée dans le compartiment dermique. La sur-expression de FGFR1-ß DN conduit en effet à la formation de follicules petits et tronqués, avec des gaines épithéliales et un bulbe élargis ainsi qu'une petite papille dermique. Par contre, l'expression de FGFR1-α DN abolit complètement la morphogenèse folliculaire. Etant donné que la signalisation par FGFR1-ß est principalement dépendante des ligands FGFs, ces données indiquent que la signalisation par ceux-cì est non-nécessaire à la morphogenèse folliculaire. De plus, l'abolition du processus par la sur-expression de FGFR1-a DN suggëre une signalisation nécessaire entre le récepteur FGFR1 et une ou des molécules d'adhésion cellulaire. L'expression de notre second candidat comme gène spécifique de la niche des cellules souches de la peau, serpinf1, prévient la morphogenèse folliculaire. Seules de petites structures ressemblant à des cystes sont observées après reconstitution de la peau. De plus, dans ces transplants, aucune cellule CD34-positive (marqueur des cellules souches) n'est retrouvée associé à ces cystes. Le produit du gène serpin f1, le «facteur dérivé d'épithélium pigmentaire » (PEDF) est un puissant facteur anti-angiogénique. Nous avons donc analysé la vascularisation des transplants par immunohistochirnies utilisant CD31, un marqueur des cellules endothéliales. Nos résultats révèlent que les cellules endothéliales sont bien présentes, mais de manière désorganisée et ne formant pas de vaisseaux. De manière intéressante, les cellules endothéliales contribuent activement à la niche des cellules souches neuronales, et nos résultats suggèrent donc l'existence possible d'un mécanisme similaire dans la peau. SHP1, le produit du gène Hcph, est une phosphatase quì agit dans le système hématopoiétique. Les souris « motheaten »qui portent des mutations spontanées du gène ont une alopécie inégale au niveau de la peau et de sévères troubles du système hématopoiétique. Pour s'assurer que le phénotype observé au niveau de la peau n'est pas une conséquence d'un défaut du système hématopoiétique, nous avons transplanté des souris Hcph -/- avec de la moelle osseuse sauvage afin de restaurer la fonction de SHP 1 dans le système hématopoiétique. Toutefois, le défaut de morphologie folliculaire est maintenu. De plus, l'ablation d'Hcph dans le compartiment dermique ou épidermique d'essais de reconstitution de peau conduit à la production de follicules pileux avec des morphologies aberrantes. Ces données indiquent que SHP1 n'est pas essentiel à la morphogenèse folliculaire mais est toutefois requis à la fois dans les compartiments épidermiques et dermiques pour la maintenance de la forme du follicule.