Detailed investigations of proximal tubular function in Imerslund-Grasbeck syndrome

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B(12) receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B(12) binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis

Genomic expression and single-nucleotide polymorphism profiling discriminates chromophobe renal cell carcinoma and oncocytoma

<p>Abstract</p> <p>Background</p> <p>Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC.</p> <p>Methods</p> <p>Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors.</p> <p>Results</p> <p>A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.</p> <p>Conclusions</p> <p>Gene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.</p

Evaluation of diffusion weighted imaging in the context of multi-parametric MRI of the prostate in the assessment of suspected low volume prostatic carcinoma

Data from a multi-parameteric MRI study of patients with possible early-stage prostate cancer was assessed with a view to creating an efficient clinical protocol. Based on a correlation analysis suggesting that diffusion-weighted imaging (DWI) scores are more strongly correlated with overall PIRADS scores than other modalities such as dynamic contrast enhanced imaging or spectroscopy, we investigate the combination of T2-weighted imaging (T2w) and DWI as a potential diagnostic tool for prostate cancer detection, staging and guided biopsies. Quantification of the noise floor in the DWI images and careful fitting of the data suggests that the mono-exponential model provides a very good fit to the data and there is no evidence of non-Guassian diffusion for b-values up to 1000 s/mm2. This precludes the use of kurtosis or other non-Gaussian measures as a biomarker for prostate cancer in our case. However, the ADC scores for healthy and probably malignant regions are significantly lower for the latter in all 20 but one patient. The results suggest that a simplified mp-MRI protocol combining T2w and DWI may be a good compromise for a cost and time efficient, early-stage prostate cancer diagnostic programme, combining robust MR biomarkers for prostate cancer that can be reliably quantified and appear well-suited for general clinical practice

Le dopage en athlétisme

POITIERS-BU Médecine pharmacie (861942103) / SudocSudocFranceF

Expansion

L'expansion des cellules souches hématopoïétiques (CSH) représente une étape essentielle pour l'amélioration des greffes de moelle osseuse et le développement de nouveaux protocoles de thérapie cellulaire. L'utilisation de transferts de gènes codant des facteurs de transcription est une des voies pouvant mener à une expansion cellulaire efficace. Parmi ces facteurs, l'homéoprotéine HOXB4 est particulièrement intéressante car elle permet l'expansion très importante des CSH de souris, sans induire de leucémie même à long terme. Cependant, pour éviter tout effet délétère en rapport avec le transfert stable et la transcription constitutive du gène HOXB4 dans les cellules, nous avons utilisé la propriété des homéoprotéines à traverser passivement les membranes. Nous avons montré que les CSH et les progéniteurs hématopoïétiques immatures étaient nettement amplifiées, quand ces cellules étaient co-cultivées avec une lignée cellulaire stromale génétiquement modifiée pour secréter activement HOXB4. Le caractère pluripotent des cellules ainsi amplifiées était préservé. Nous avons testé, dans un modèle comparable, le rôle de HOXB4 sur l'expansion des progéniteurs lymphoïdes : nous avons montré que les progéniteurs immatures lympho-myéloïdes et pro-T/NK ainsi que les progéniteurs NK plus matures étaient clairement amplifiés. Nous avons aussi recherché une éventuelle activité synergique entre HOXB4 et d'autres homéoprotéines telles que HOXC4. Nous avons montré que HOXC4 était capable d'induire l'expansion des progéniteurs hématopoïétiques humain in vitro de façon comparable à HOXB4. La présence simultanée des deux homéoprotéines dans les co-cultures provoquait une expansion encore plus élevée qu'avec chacune d'entre elles. Notre méthode constitue donc une base pour le développement de nouvelles stratégies en thérapie cellulaire, par l'utilisation de cellules souches amplifiées in vitro mais non modifiées génétiquement

Enhanced Cytotoxic Activity of Ex Vivo-differentiated Human Natural Killer Cells in the Presence of HOXB4

International audienceWe have previously shown that human umbilical cord blood CD34 progenitor cells undergo in vitro differentiation into functional natural killer (NK) cells and that their coculture in the presence of HOXB4-transduced stromal MS-5 cells resulted in an increase in differentiated NK number. The present study was conducted to compare the stromal effect on NK lytic potential in the presence and absence of HOXB4. Our results provide evidence that HOXB4-transduced MS-5 cells as compared with transduced GFP (+) MS-5 cells induced highly differentiated cytotoxic NK cells. Importantly, this difference was not because of the expression of activating NK receptors but was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the upregulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy