In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment.

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.

Ultrastructural Observations of the Vitelline Cells of Metamicrocotyla macracantha (Monogenea, Microcotylidae)

An electron microscopic study of the vitelline follicles of Metamicrocotyla macracantha (Alexander, 1954) Koratha,1955 showed that they are composed of cells in different stages of development. The immature cells have a large nucleus, nucleolus, cytoplasm with free ribosomes and few mitochondria. The developing vitelline cells present granules which are small in the early stages, increasing with maturity. The mature cells have an extensive granular endoplasmic reticulum and droplets of shell-protein; with maturation, clusters of shell protein and yolk bodies are formed and released in the ciliated vitelline ducts. Vitelline development is continuous and all of the cellular stages involved can be found in each follicle.

Clonal heterogeneity and chromosomal instability at disease presentation in high hyperdiploid acute lymphoblastic leukemia.

Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2-44.7%), compared with those in non-HeH ALL (3.2-6.4%), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.

Dosing regimen of gentamicin in neonates: evaluation of current guidelines based on a large population pharmacokinetic analysis

Objectives: Gentamicin is among the most commonly prescribed antibiotics in newborns, but large interindividual variability in exposure levels exists. Based on a population pharmacokinetic analysis of a cohort of unselected neonates, we aimed to validate current dosing recommendations from a recent reference guideline (Neofax®). Methods: From 3039 concentrations collected in 994 preterm (median gestational age 32.3 weeks, range 24.2-36.5) and 455 term newborns, treated at the University Hospital of Lausanne between 2006 and 2011, a population pharmacokinetic analysis was performed with NONMEM®. Model-based simulations were used to assess the ability of dosing regimens to bring concentrations into targets: trough ≤ 1mg/L and peak ~ 8mg/L. Results: A two-compartment model best characterized gentamicin pharmacokinetics. Model parameters are presented in the table. Body weight, gestational age and postnatal age positively influence clearance, which decreases under dopamine administration. Body weight and gestational age influence the distribution volume. Model based simulations confirm that preterm infants need doses superior to 4 mg/kg, and extended dosage intervals, up to 48 hours for very preterm newborns, whereas most term newborns would achieve adequate exposure under 4 mg/kg q. 24 h. More than 90% of neonates would achieve trough concentrations below 2 mg/L and peaks above 6 mg/L following most recent guidelines. Conclusions: Simulated gentamicin exposure demonstrates good accordance with recent dosing recommendations for target concentration achievement.

Application of a roughness-length representation to parameterize energy loss in 3-D numerical simulations of large rivers

Recent technological advances in remote sensing have enabled investigation of the morphodynamics and hydrodynamics of large rivers. However, measuring topography and flow in these very large rivers is time consuming and thus often constrains the spatial resolution and reach-length scales that can be monitored. Similar constraints exist for computational fluid dynamics (CFD) studies of large rivers, requiring maximization of mesh-or grid-cell dimensions and implying a reduction in the representation of bedform-roughness elements that are of the order of a model grid cell or less, even if they are represented in available topographic data. These ``subgrid'' elements must be parameterized, and this paper applies and considers the impact of roughness-length treatments that include the effect of bed roughness due to ``unmeasured'' topography. CFD predictions were found to be sensitive to the roughness-length specification. Model optimization was based on acoustic Doppler current profiler measurements and estimates of the water surface slope for a variety of roughness lengths. This proved difficult as the metrics used to assess optimal model performance diverged due to the effects of large bedforms that are not well parameterized in roughness-length treatments. However, the general spatial flow patterns are effectively predicted by the model. Changes in roughness length were shown to have a major impact upon flow routing at the channel scale. The results also indicate an absence of secondary flow circulation cells in the reached studied, and suggest simpler two-dimensional models may have great utility in the investigation of flow within large rivers. Citation: Sandbach, S. D. et al. (2012), Application of a roughness-length representation to parameterize energy loss in 3-D numerical simulations of large rivers, Water Resour. Res., 48, W12501, doi: 10.1029/2011WR011284.

Roles of canonical Wnt/TCF-1 signaling for hematopoiesis, lymphocyte development and function

Summary : The canonical Wnt signaling pathway plays key roles in the maintenance of self-renewing tissues, like the gut or the skin. In contrast, the role of this pathway in hematopoiesis remains poorly defined. Wnt ligands transmit signals through ß-catenin which activates gene transcription upon its association with Lymphoid Cell Enhancer/T Cell Factor (LEF/TCF). Currently, v-catenin is the only alternative factor known to transduce canonical Wnt signals. The ß-/γ-catenin bindiná domain in TCF-1 is required to partly rescue thymopoiesis and NK cell development in TCF-1-deficient mice. However, T cell development and hematopoiesis w-as normal in mice deficient of ß-catenin, or of γ-catenin. Surprisingly we found that hematopoiesis and thymopoiesis was also normal in the combined absence of ß- and γ-catenin. Reporter assays showed that double-deficient lymphocytes were still able to transduce canonical wnt signals. These data provided evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of ß- and γ-catenin. There exist numerous TCF-1 isoforrns including those that harbor the N-terminal ß-/y-catenin binding domain or that contains a C-terminal CRARF domain whose role in vivo has not been previously tested. We found that the CRARF domain influences lymphocyte development in conjunction with the N-treminal ß-/γ-catenin binding. The presence of the two domains directs thymocytes to the CD8+ T cell lineage whereas NK cell development is abolished. Roles of the canonical Wnt/TCF-1 pathway for lymphocyte function have not been defined. We demonstrate that TCF-1 deficient CDBT T cells mount a normal primary response to viral infection but these T cells fail to expand upon restimulation. The failure of CD8+ T cells to respond to IL-2 during primary infection seems to account for this phenotype. Thus, TCF-1 is essential for programming functional CD8+ T cell memory. Collectively, these data provide significant new insights into the role of Wnt/TCF-1 pathway for lymphocyte development and function and suggest a novel mechanism of Wnt signal transuction in hematopoietic cells. Résumé : La voie de signalisation canonique Wnt joue un rôle prépondérant dans le renouvellement de tissus, comme l'intestin ou la peau. Son rôle dans l'hématopoïèse est quant à lui mal défini. Le ligand Wnt transmet le signal via la ß-catenin qui active la transcription de gènes cibles quand il est associé avec Lymphoid Cell Enhancer,~T Cell Factor (LEF/TCF). Actuellement, la γ-catenin est le seul autre facteur connu pouvant se substituer à la fonction de la ß-catenin. Un variant de TCF-1 contenant le domaine liant ß-/,~-catenin est capable de restaurer le développement des lymphocytes T et NK en l'absence de TCF-1. Cependant la thymopoïèse et l'hématopoïèse sont normales dans les souris déficientes pour la ß-catenin ou la γ-catenin. De façon surprenante, nous avons trouvé que l'hématopoïèse et le développement des lymphocytes sont normaux lors de l'absence combinée de ß-/γ-catenin. De plus, la transduction des signaux de la voie de signalisation Wnt est maintenue dans des lymphocytes déficients pour ß-/γ-catenin. Ces résultats démontrent que les cellules hématopoïétiques peuvent transmettre les signaux de la voie canonique Wnt lors de l'absence combinée de la ß et la γ -catenin. Il existe de nombreuses isofonnes de TCF-1, y compris certaines qui comprennent un domaine qui lie ß-/γ-catenin du côté N-terminus ou qui contiennent un domaine CRARF du côté C-terminus. Nous montrons ici que le domaine CRARF influence le développement des lymphocytes en conjonction avec le domaine liant ß-/γ-catenin. La présence des deux domaines dirige les thymocytes vers la lignée de cellules T CD8, alors que le développement des cellules NK est aboli. Au-delà de sa fonction sur le développement des lymphocytes, le rôle de la soie de signalisation canonique Wnt/TCF-1 lors d'une infection n'a pas été défini. Nous avons montré que les cellules T CD8, déficientes pour TCF-1, développent une réponse primaire normale à une infection virale, mais qu'elles ne s'accumulent pas après restimulation. L'incapacité des cellules TCD8 à répondre à l'IL-2 durant la réponse primaire peut expliquer ce phénotype. Ainsi; TCF-1 est essentiel pour la programmation de cellules T CD8 mémoires fonctionnelles. L'ensemble de ces résultats fournit de nouveaux aperçus du rôle de la voie de signalisation Wnt/TCF-1 pour le développement et la fonction des lymphocytes et suggèrent un nouveau mécanisme de transduction du signal Wnt dans les cellules hématopoïétiques.

No Role for Cerebrospinal Fluid Myelin Basic Protein Levels in Patients Treated for Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Central nervous system prophylaxis of childhood acute lymphoblastic leukemia has dropped rates of relapses but has been associated with neurotoxicity and imaging abnormalities. Predictors of neurotoxicity are lacking, because of inconsistency between clinical symptoms and imaging. Some have suggested that cerebrospinal fluid myelin basic protein (MBP) levels to be of potential interest. A retrospective analysis of MBP levels in correlation with clinical and radiologic data is presented. MATERIALS AND METHODS: MBP levels obtained at the time of intrathecals, charts, and neuroradiology reports were retrospectively analyzed. Academic achievement data were obtained from phone contacts with patients and families. RESULTS: We retrieved 1248 dosages of MBP in 83 patients, 381 neurologic examinations in 34 patients and 69 neuroradiologic investigations in 27 patients. Fifty-two patients had abnormal MBP levels. Radiologic anomalies were present in 47% of those investigated, 14% of them having school difficulties. Proportions of patients with school difficulties in the groups with abnormal MBP levels but no radiologic anomalies or with no radiologic investigations were 0% and 3%, respectively, which was lower than in the group of patients with normal MBP levels (100%, 22%, and 5%, respectively). DISCUSSION: Notwithstanding the retrospective character of our study, we conclude that there is limited usefulness of systematic dosage of MBP as indicator of treatment-induced neurotoxicity in acute lymphoblastic leukemia patients.

Treatment of large bone defects with the Ilizarov technique.

Between 1985 and 1990 we treated 11 large segmental bone defects (average 6.7 cm) in ten patients with the Ilizarov technique. Open fractures, type III according to Gustilo, represented the largest group (8 of 11 cases). The average delay before the Ilizarov technique was initiated was 8.9 months. The external fixator was usually maintained for 1 year. Bone regeneration was obtained in every case. Consolidation was not fulfilled with this technique in three cases. The complications observed were one refracture, four leg-length discrepancies (average 1.5 cm), and five axial deformities exceeding 5 degrees. No pin-track infection was observed. In our limited series of four type IIIC open fractures treated by the Ilizarov technique, no patients required amputation. The Ilizarov technique is particularly useful in the treatment of large bone defects, without major complications, especially if there is an adequate initial debridement.

A modular approach for integrative analysis of large-scale gene-expression and drug-response data

High-throughput technologies are now used to generate more than one type of data from the same biological samples. To properly integrate such data, we propose using co-modules, which describe coherent patterns across paired data sets, and conceive several modular methods for their identification. We first test these methods using in silico data, demonstrating that the integrative scheme of our Ping-Pong Algorithm uncovers drug-gene associations more accurately when considering noisy or complex data. Second, we provide an extensive comparative study using the gene-expression and drug-response data from the NCI-60 cell lines. Using information from the DrugBank and the Connectivity Map databases we show that the Ping-Pong Algorithm predicts drug-gene associations significantly better than other methods. Co-modules provide insights into possible mechanisms of action for a wide range of drugs and suggest new targets for therapy

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

The tumor suppressor p16Ink4a regulates T lymphocyte survival.

In contrast to other cell cycle inhibitors, the tumor suppressor p16Ink4a is not detectable or expressed at very low levels in embryonic and adult mouse tissues, and therefore it has often been considered as a specialized checkpoint protein that does not participate in the control of normal cell cycle progression. However, Ink4a-/- mice possess increased thymus size and cellularity, thus suggesting the involvement of p16(Ink4a) in the control of thymocyte proliferation. In this study, we found increased numbers of CD8 and CD4 T lymphocytes in thymus and spleen from Ink4a-/- mice. Unexpectedly, this was not related to an increase in T-cell division rates, which were similar in lymphoid organs of Ink4a-/- and wild-type mice. In contrast, T-cell apoptosis rates were significantly decreased in thymus and spleen from Ink4a-/- mice. Moreover, whereas p16Ink4a-deficient and wild-type T cells were equally sensitive to Fas or TCR-mediated apoptosis, the former were clearly more resistant to apoptosis induced by oxidative stress or gamma irradiation. Our results indicate that p16Ink4a function is associated with T-cell apoptosis, and subsequently contributes to the control of T-cell population size in lymphoid organs.

Quantification, self-renewal, and genetic tracing of FL1+ tumor-initiating cells in a large cohort of human gliomas.

Evidence has emerged that the initiation and growth of gliomas is sustained by a subpopulation of cancer-initiating cells (CICs). Because of the difficulty of using markers to tag CICs in gliomas, we have previously exploited more robust phenotypic characteristics, including a specific morphology and intrincic autofluorescence, to identify and isolate a subpopulation of glioma CICs, called FL1(+). The objective of this study was to further validate our method in a large cohort of human glioma and a mouse model of glioma. Seventy-four human gliomas of all grades and the GFAP-V(12)HA-ras B8 mouse model were analyzed for in vitro self-renewal capacity and their content of FL1(+). Nonneoplastic brain tissue and embryonic mouse brain were used as control. Genetic traceability along passages was assessed with microsatellite analysis. We found that FL1(+) cells from low-grade gliomas and from control nonneoplasic brain tissue show a lower level of autofluorescence and undergo a restricted number of cell divisions before dying in culture. In contrast, we found that FL1(+) cells derived from many but not all high-grade gliomas acquire high levels of autofluorescence and can be propagated in long-term cultures. Moreover, FL1(+) cells show a remarkable traceability over time in vitro and in vivo. Our results show that FL1(+) cells can be found in all specimens of a large cohort of human gliomas of different grades and in a model of genetically induced mouse glioma as well as nonneoplastic brain. However, their self-renewal capacity is variable and seems to be dependent on the tumor grade.