Estudio transversal sobre la prevalencia de la Enfermedad Metabólica Ósea (EMO) y Nutrición Parenteral Domiciliaria (NPD) en España: datos del Grupo NADYA

Patients with intestinal failure who receive HPN are at high risk of developing MBD. The origin of this bone alteration is multifactorial and depends greatly on the underlying disease for which the nutritional support is required. Data on the prevalence of this disease in our environment is lacking, so NADYA-SEMPE group has sponsored this transversal study with the aim of knowing the actual MBD prevalence. MATERIAL AND METHODS: Retrospective data from 51 patients from 13 hospitals were collected. The questionnaire included demographic data as well as the most clinically relevant for MBD data. Laboratory data (calciuria, PTH, 25 -OH -vitamin D) and the results from the first and last bone densitometry were also registered. RESULTS: Bone mineral density had only been assessed by densitometry in 21 patients at the moment HPN was started. Bone quality is already altered before HPN in a significant percentage of cases (52%). After a mean follow up of 6 years, this percentage increases up to 81%. Due to retrospective nature of the study and the low number of subjects included it has not been possible to determine the role that HPN plays in MBD etiology. Only 35% of patients have vitamin D levels above the recommended limits and the majority of them is not on specific supplementation. CONCLUSIONS: HPN is associated with very high risk of MBD, therefore, management protocols that can lead to early detection of the problem as well as guiding for follow up and treatment of these patients are needed.

Genetic investigation of the functions of c-Myc and N-Myc in Hematopoietic stem cells

Résumé : c-Myc, le premier facteur de transcription de la famille Myc a été découvert il y a maintenant trente ans. Il reste à l'heure actuelle parmi les plus puissants proto-oncogènes connus. c-Myc est dérégulé dans plus de 50% des cancers, où il promeut la prolifération, la croissance cellulaire, et la néoangiogenèse. Myc peut aussi influencer de nombreuses autres fonctions de par sa capacité à activer ou à réprimer la transcription de nombreux gènes, et à agir globalement sur le génome à travers des modifications épigénétiques de la chromatine. La famille d'oncogènes Myc comprend, chez les mammifères, trois protéines structurellement proches: c-Myc, N-Myc et L-Myc. Ces protéines ont les mêmes proprietés biochimiques, exercent les mêmes fonctions mais sont le plus souvent exprimées de façon mutuellement exclusive. Myc a été récemment identifié comme un facteur clef dans la maintenance des cellules souches embryonnaires et adultes ainsi que dans la réacquisition des proprietés des cellules souches. Nous avons précédemment démontré que l'élimination de c-Myc provoque une accumulation de cellules souches hématopoïétiques (CSH) suite à un défaut de différenciation lié à la niche. Les CSH sont responsables de la production de tous les éléments cellulaires du sang pour toute la vie de l'individu et sont définies par leur capacité à s'auto-renouveler tout en produisant des précurseurs hématopoïétiques. Afin de mieux comprendre la fonction de Myc dans les CSH, nous avons choisi de combiner l'utilisation de modèles de souris génétiquement modifiées à une caractérisation systématique des schémas d'expression de c-Myc, N-Myc et L-Myc dans tout le système hématopoïétique. Nous avons ainsi découvert que les CSH les plus immatures expriment des quantités équivalentes de transcrits de c-myc et N-myc. Si les CSH déficientes en N-myc seulement ont une capacité d'auto-renouvellement à long-terme réduite, l'invalidation combinée des gènes c-myc et N-myc conduit à une pan-cytopénie suivie d'une mort rapide de l'animal, pour cause d'apoptose de tous les types cellulaires hématopoïétiques. En particulier, les CSH en cours d'auto-renouvelemment, mais pas les CSH quiescentes, accumulent du Granzyme B (GrB), une molécule fortement cytotoxique qui provoque une mort cellulaire rapide. Ces données ont ainsi mis au jour un nouveau mécanisme dont dépend la survie des CSH, à savoir la répression du GrB, une enzyme typiquement utilisée par le système immunitaire inné pour éliminer les tumeurs et les cellules infectées par des virus. Dans le but d'évaluer l'étendue de la redondance entre c-Myc et N-Myc dans les CSH, nous avons d'une part examiné des souris dans lesquelles les séquences codantes de c-myc sont remplacées par celles de N-myc (NCR) et d'autre part nous avons géneré une série allèlique de myc en éliminant de façon combinatoire un ou plusieurs allèles de c-myc et/ou de N-myc. Alors que l'analyse des souris NCR suggère que c-Myc et N-Myc sont qualitativement redondants, la série allélique indique que les efficiences avec lesquelles ces deux protéines influencent des procédés essentiels à la maintenance des CSH sont différentes. En conclusion, nos données génétiques montrent que l'activité générale de MYC, fournie par c-Myc et N-Myc, contrôle plusieurs aspects cruciaux de la fonction des CSH, notamment l'auto-renouvellement, la survie et la différenciation. Abstract : c-Myc, the first Myc transcription factor was discovered 30 years ago and is to date one of the most potent proto-oncogenes described. It is found to be misregulated in over 50% of all cancers, where it drives proliferation, cell growth and neo-angiogenesis. Myc can also influence a variety of other functions, owing to its ability to activate and repress transcription of many target genes and to globally regulate the genome via epigenetic modifications of the chromatin. The Myc family of oncogenes consists of three closely related proteins in mammals: c-Myc, N-Myc and L-Myc. These proteins share the same biochemical properties, exert mostly the same functions, but are most often expressed in mutually exclusive patterns. Myc is now emerging as a key factor in maintenance of embryonic and adult stem cells as well as in reacquisition of stem cell properties, including induced reprogramming. We previously showed that c-Myc deficiency can cause the accumulation of hematopoietic stem cells (HSCs) due to a niche dependent differentiation defect. HSCs are responsible for life-long replenishment of all blood cell types, and are defined by their ability to self-renew while concomitantly giving rise to more commited progenitors. To gain further insight into the function of Myc in HSCs, in this study we combine the use of genetically-modified mouse models with the systematic characterization of c-myc, N-myc and L-myc transcription patterns throughout the hematopoietic system. Interestingly, the most immature HSCs express not only c-myc, but also about equal amounts of N-myc transcripts. Although conditional deletion of N-myc alone in the bone marrow does not affect steady-state hematopoiesis, N-myc null HSCs show impaired long-term self-renewal capacity. Strikingly, combined deficiency of c-Myc and N-Myc results in pan-cytopenia and rapid lethality, due to the apoptosis of most hematopoietic cell types. In particular, self-renewing HSCs, but not quiescent HSCs or progenitor cell types rapidly up-regulate and accumulate the potent cytotoxic molecule GranzymeB (GrB), causing their rapid cell death. These data uncover a novel pathway on which HSC survival depends on, namely repression of GrB, a molecule typically used by the innate immune system to eliminate tumor and virus infected cells. To evaluate the extent of redundancy between c-Myc and N-Myc in HSCs, we examined mice in which c-myc coding sequences are replaced by that of N-myc (NCR) and also generated an allelic series of myc, by combinatorially deleting one or several c-myc and/or N-myc alleles. While the analysis of NCR mice suggests that c-Myc and N-Myc are qualitatively functionally redundant, our allelic series indicates that the efficiencies with which these two proteins affect crucial HSC maintenance processes are likely to be distinct. Collectively, our genetic data show that general "MYC" activity delivered by c-Myc and N-Myc controls crucial aspects of HSC function, including self-renewal, survival and niche dependent differentiation.

Gm(1,2,4,10,21) and Km(1) factors in vitreous humor.

The presence of Gm(1,2,4,10,21) and Km(1) factors in vitreous humor taken from human corpses was investigated. The results revealed a good agreement between the factors detected in this biological material and in blood. Their presence in vitreous humor is independent of the secretor type.

A MODEL OF CREATINE DEFICIENCY SYNDROMES IN 3D BRAIN CELL CULTURES BY KNOCKDOWN OF GAMT AND SLC6A8 GENES

La créatine joue un rôle essentiel dans le métabolisme cellulaire par sa conversion, par la creatine kinase, en phosphocreatine permettant la régénération de l'ATP. La synthèse de créatine, chez les mammifères, s'effectue par une réaction en deux étapes impliquant Γ arginine: glycine amidinotransférase (AGAT) et la guanidinoacétate méthyltransférase (GAMT). L'entrée de créatine dans les cellules s'effectue par son transporteur, SLC6A8. Les déficiences en créatine, dues au déficit en GAMT, AGAT ou SLC6A8, sont fréquentes et caractérisées par une absence ou une forte baisse de créatine dans le système nerveux central. Alors qu'il est connu que AGAT, GAMT et SLC6A8 sont exprimés par le cerveau, les conséquences des déficiences en créatine sur les cellules nerveuses sont peu comprises. Le but de ce travail était de développer de nouveaux modèles expérimentaux des déficiences en Cr dans des cultures 3D de cellules nerveuses de rat en agrégats au moyen de l'interférence à l'ARN appliquée aux gènes GAMT et SLC6A8. Des séquences interférentes (shRNAs) pour les gènes GAMT et SLC6A8 ont été transduites par des vecteurs viraux AAV (virus adéno-associés), dans les cellules nerveuses en agrégats. Nous avons ainsi démontré une baisse de l'expression de GAMT au niveau protéique (mesuré par western blot), et ARN messager (mesuré par qPCR) ainsi qu'une variation caractérisitique de créatine et guanidinoacétate (mesuré par spectrométrie de masse). Après avoir validé nos modèles, nous avons montré que les knockdown de GAMT ou SLC6A8 affectent le développement des astrocytes et des neurones ou des oligodendrocytes et des astrocytes, respectivement, ainsi qu'une augmentation de la mort cellulaire et des modifications dans le pattern d'activation des voies de signalisation impliquant caspase 3 et p38 MAPK, ayant un rôle dans le processus d'apoptose. - Creatine plays essential roles in energy metabolism by the interconversion, by creatine kinase, to its phosphorylated analogue, phosphocreatine, allowing the regeneration of ATP. Creatine is synthesized in mammals by a two step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Creatine is taken up by cells by a specific transporter, SLC6A8. Creatine deficiency syndromes, due to defects in GAMT, AGAT and SLC6A8, are among the most frequent inborn errors of metabolism, and are characterized by an absence or a severe decrease of creatine in central nervous system, which is the main tissue affected. While it is known that AGAT, GAMT and SLC6A8 are expressed in CNS, many questions remain on the specific effects of AGAT, GAMT and SLC6A8 deficiencies on brain cells. Our aim was to develop new experimental models of creatine deficiencies by knockdown of GAMT and SLC6A8 genes by RNAi in 3D organotypic rat brain cell cultures in aggregates. Specific shRNAs for the GAMT and SLC6A8 genes were transduced in brain cell aggregates by adeno-associated viruses (AAV). The AAV-transduced shRNAs were able to efficiently knockdown the expression of our genes of interest, as shown by a strong decrease of protein by western blotting, a decrease of mRNA by qPCR or characteristic variations of creatine and guanidinoacetate by tandem mass spectrometry. After having validated our experimental models, we have also shown that GAMT and SLC6A8 knockdown affected the development of astrocytes and neurons or oligodendrocytes and astrocytes, respectively. We also observed an increase of cell death and variations in activation pattern of caspase 3 and p38 MAPK pathways, involved in apoptosis, in our experimental model.

Steroid hormone pulsing drives cyclic gene expression.

Transcriptional cycling of activated glucocorticoid receptor (GR) and ultradian glucocorticoid secretion are well established processes. Ultradian hormone release is now shown to result in pulsatile gene transcription through dynamic exchange of GR with the target-gene promoter and GR cycling through the chaperone machinery.

Prevalence and Clinical Characteristics of HIV Type 1-Infected Patients Receiving Dialysis in Spain: Results of a Spanish Survey in 2006: GESIDA 48/05 Study

End-stage renal diseases (ESRD) are becoming more frequent in HIV-infected patients. In Europe there is little information about HIV-infected patients on dialysis. A cross-sectional multicenter survey in 328 Spanish dialysis units was conducted in 2006. Information from 14,876 patients in dialysis was obtained (81.6% of the Spanish dialysis population). Eighty-one were HIV infected (0.54%; 95% CI, 0.43-0.67), 60 were on hemodialysis, and 21 were on peritoneal dialysis. The mean (range) age was 45 (28-73) years. Seventy-two percent were men and 33% were former drug users. The mean (range) time of HIV infection was 11 (1-27) years and time on dialysis was 4.6 (0.4-25) years. ESRD was due to glomerulonephritis (36%) and diabetes (15%). HIV-associated nephropathy was not reported. Eighty-five percent were on HAART, 76.5% had a CD4 T cell count above 200 cells, and 73% had undetectable viral load. Thirty-nine percent of patients met criteria for inclusion on the renal transplant (RT) waiting list but only 12% were included. Sixty-one percent had HCV coinfection. HCV-coinfected patients had a longer history of HIV, more previous AIDS events, parenteral transmission as the most common risk factor for acquiring HIV infection, and less access to the RT waiting list (p < 0.05). The prevalence of HIV infection in Spanish dialysis units in 2006 was 0.54% HCV coinfection was very frequent (61%) and the percentage of patients included on the Spanish RT waiting list was low (12%).

Role of Notch signaling in the skin and cornea

Résumé : La voie de signalisation Notch est essentielle pour la différentiation de l'épiderme lors du développement embryonnaire de la peau. Il a été démontré que l'inactivation de Notch1 dans la peau de souris conduit à une hyperplasie de l'épiderme ainsi qu'à la formation subséquente de carcinomes basocellulaires ainsi que de plaques cornéennes. L'inactivation de Notch1 dans la cornée combinée à des lésions mécaniques démontre que les cellules progénitrices de la cornée se différentient en un épithélium hyperplasique et kératinisé comme la peau. Ce changement de destinée cellulaire conduit à une cécité cornéenne et implique des processus non-autonomes aux cellules épithéliales, caractérisés par la sécrétion de FGF-2 par l'épithélium Notch1-/- suivi d'une vascularisation et d'un remaniement du stroma sous-jacent. La déficience en vitamine A est connu comme cause de lésions cornéennes humaines (xérophtalmie sévère). En accord, nous avons trouvé que la signalisation Notch1 était liée au métabolisme de la vitamine A par la régulation de l'expression de CRBP1, nécessaire pour générer un pool de rétinol intracellulaire. La perte de Notch1 dans l'épiderme, l'autre récepteur de la famille présent dans la peau marine, ne conduit pas à un phénotype manifeste. Cependant, l'inactivation dans l'épiderme de Notch1 et Notch2 ensemble, ou de RBP-J, induit une dermatite atopique (DA) sévère chez les souris. De même, les patients souffrants de DA mais pas ceux souffrant de psoriasis ou de lichen plan, ont une réduction marquée de l'expression des récepteurs Notch dans la peau. La perte de Notch dans les keratinocytes conduit à une activation de la voie NF-κB, ce qui ensuite induit la production de TSLP, une cytokine profondément impliquée dans la pathogenèse de la DA. Nous démontrons génétiquement que TSLP est responsable de la DA ainsi que du développent d'un syndrome myéloprolifératif non-autonome aux cellules induit par le G-CSF. Cependant, ces souris avec une inactivation dans l'épiderme de Notch1 et Notch2 et aussi incapables de répondre au TSLP développent des tumeurs invasive sévères caractérisées par une haute activité de signalisation ß-catenin. TSLPR est identifié comme un potentiel suppresseur de tumeur non-autonome aux cellules tumorales; la transplantation de cellules hématopoïétiques TSLPR-/- dans des souris déficientes pour Notch est suffisant pour causer des tumeurs. Summary : The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. It has previously been demonstrated that Notch1 inactivation in marine skin results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors as well as corneal plaques. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1 deficient corneal progenitor cells differentiate into a hyperplasic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell non-autonomous processes, characterized by secretion of FGF-2 through Notch1-/- epithelium followed by vascularisation and remodelling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of CRBP1, required to generate a pool of intracellular retinol. Epidermal loss of Notch2, the other Notch receptor present in marine skin, doesn't lead to any overt phenotypes. However, postnatal epidermis-specific inactivation of both Notch1 and Notch2, or of RBP-J, induces the development of a severe form of atopic dermatitis (AD) in mice. Likewise, patients suffering from AD, but not psoriasis or lichen planas, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes leads to an activation of NF-κB signaling which in turn induces the production of Thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. We genetically demonstrate that TSLP is responsible for AD as well as the development of a cell non-autonomous G-CSF induced myeloproliferative disorder (MPD) in mice. However, these mice with conditional epidermal inactivation of Notch1 and Notch2 as well as incapable to respond to TSLP develop severe invasive tumors characterized by high ß-catenin signaling activity. TSLPR is identified as a potential cell non-autonomous tumor suppressor; transplantation of TSLPR-/- hematopoietic cells into epidermal Notch deficient mice is sufficient to cause tumors.

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Current applications of cardiac magnetic resonance (CMR) imaging offer a wide spectrum of indications in the setting of acute cardiac care. In particular, CMR is helpful for the differential diagnosis of chest pain by detection of myocarditis and pericarditis. Also, takotsubo cardiomyopathy and acute aortic diseases can be evaluated by CMR and are important differential diagnoses in patients with acute chest pain. In patients with restricted windows for echocardiography, CMR is the method of choice to evaluate complications of acute myocardial infarction (AMI). In AMI, CMR allows for a unique characterization of myocardial damage by quantifying necrosis, microvascular obstruction, oedema (=area at risk), and haemorrhage. These capabilities will help us to understand better the pathophysiological events during infarction and will also allow to assess new treatment strategies in AMI. To what extent the information on tissue damage will guide patient management is not yet clear and further research in this field is warranted. In the near future, CMR will certainly become more routine in acute cardiac care units, as manufacturers are now focusing strongly on this aspect of user-friendliness. Finally, in the next decade or so, MRI of other nuclei such as fluorine and carbon might become a clinical reality, which would allow for metabolic and targeted molecular imaging with excellent sensitivity and specificity

Resultados materno-fetales de la gestación tras cirugía bariátrica.

BACKGROUND Obesity is the most frequent metabolic disease in the World, and is associated with several comorbidities. Bariatric procedures arise as a promising treatment when classical approach is ineffective. Half of the operated patients are reproductive-aged women and there is evidence that obesity is related to worse maternal and fetal outcomes. Because nutritional status is affected by bariatric surgery and is a vital component during pregnancy, the aim of our study is to asses the impact of bariatric surgery on pregnancy in these patients. MATERIAL AND METHODS We studied 10 women and 15 pregnancies following bariatric surgery between 2003 and 2009. The visits took place every three months by an obstetrician and an endocrinologist with experience in nutrition, recording clinical features and lab work. RESULTS We found iron deficiency in 80% of the pregnancies, vitamin D in 46,7%, vitamin A in 20%, vitamin E in 13,3% and vitamin B12 in 26,7%. There were no complications during pregnancy, except one case of gravidic hiperemesis. There were nine deliveries without malformations, three of them were small for gestational age newborns and one suffered aspiration pneumonia. There were three stillbirths and one preterm delivery with fetal death. CONCLUSIONS our results show fewer complications during pregnancy in these women than obese women and similar to general population.

Eligibility for and outcome of treatment of latent tuberculosis infection in a cohort of HIV-infected people in Spain

BACKGROUND Previous studies have demonstrated the efficacy of treatment for latent tuberculosis infection (TLTBI) in persons infected with the human immunodeficiency virus, but few studies have investigated the operational aspects of implementing TLTBI in the co-infected population.The study objectives were to describe eligibility for TLTBI as well as treatment prescription, initiation and completion in an HIV-infected Spanish cohort and to investigate factors associated with treatment completion. METHODS Subjects were prospectively identified between 2000 and 2003 at ten HIV hospital-based clinics in Spain. Data were obtained from clinical records. Associations were measured using the odds ratio (OR) and its 95% confidence interval (95% CI). RESULTS A total of 1242 subjects were recruited and 846 (68.1%) were evaluated for TLTBI. Of these, 181 (21.4%) were eligible for TLTBI either because they were tuberculin skin test (TST) positive (121) or because their TST was negative/unknown but they were known contacts of a TB case or had impaired immunity (60). Of the patients eligible for TLTBI, 122 (67.4%) initiated TLTBI: 99 (81.1%) were treated with isoniazid for 6, 9 or 12 months; and 23 (18.9%) with short-course regimens including rifampin plus isoniazid and/or pyrazinamide. In total, 70 patients (57.4%) completed treatment, 39 (32.0%) defaulted, 7 (5.7%) interrupted treatment due to adverse effects, 2 developed TB, 2 died, and 2 moved away. Treatment completion was associated with having acquired HIV infection through heterosexual sex as compared to intravenous drug use (OR:4.6; 95% CI:1.4-14.7) and with having taken rifampin and pyrazinamide for 2 months as compared to isoniazid for 9 months (OR:8.3; 95% CI:2.7-24.9). CONCLUSIONS A minority of HIV-infected patients eligible for TLTBI actually starts and completes a course of treatment. Obstacles to successful implementation of this intervention need to be addressed.

Iron deficiency and anaemia in bariatric surgical patients: causes, diagnosis and proper management

Obesity-induced chronic inflammation leads to activation of the immune system that causes alterations of iron homeostasis including hypoferraemia, iron-restricted erythropoiesis, and finally mild-to-moderate anaemia. Thus, preoperative anaemia and iron deficiency are common among obese patients scheduled for bariatric surgery (BS). Assessment of patients should include a complete haematological and biochemical laboratory work-up, including measurement of iron stores, vitamin B12 and folate. In addition, gastrointestinal evaluation is recommended for most patients with iron-deficiency anaemia. On the other hand, BS is a long-lasting inflammatory stimulus in itself and entails a reduction of the gastric capacity and/or exclusion from the gastrointestinal tract which impair nutrients absorption, including dietary iron. Chronic gastrointestinal blood loss and iron-losingenteropathy may also contribute to iron deficiency after BS. Perioperative anaemia has been linked to increased postoperative morbidity and mortality and decreased quality of life after major surgery, whereas treatment of perioperative anaemia, and even haematinic deficiency without anaemia, has been shown to improve patient outcomes and quality of life. However, long-term follow-up data in regard to prevalence, severity, and causes of anaemia after BS are mostly absent. Iron supplements should be administered to patients after BS, but compliance with oral iron is no good. In addition, once iron deficiency has developed, it may prove refractory to oral treatment. In these situations, IV iron (which can circumvent the iron blockade at enterocytes and macrophages) has emerged as a safe and effective alternative for perioperative anaemia management. Monitoring should continue indefinitely even after the initial iron repletion and anaemia resolution, and maintenance IV iron treatment should be provided as required. New IV preparations, such ferric carboxymaltose, are safe, easy to use and up to 1000 mg can be given in a single session, thus providing an excellent tool to avoid or treat iron deficiency in this patient population.

Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency.

Starting from a cohort of 50 NADH-oxidoreductase (complex I) deficient patients, we carried out the systematic sequence analysis of all mitochondrially encoded complex I subunits (ND1 to ND6 and ND4L) in affected tissues. This approach yielded the unexpectedly high rate of 20% mutation identification in our series. Recurrent heteroplasmic mutations included two hitherto unreported (T10158C and T14487C) and three previously reported mutations (T10191C, T12706C and A13514G) in children with Leigh or Leigh-like encephalopathy. The recurrent mutations consistently involved T-->C transitions (p<10(-4)). This study supports the view that an efficient molecular screening should be based on an accurate identification of respiratory chain enzyme deficiency.