Three-dimensional computer-assisted dissection of pancreatic lymphatic anatomy on human fetuses: a step toward automatic image alignment.

PURPOSE: Pancreatic cancer is the fourth cause of death by cancer worldwide. Lymph node (LN) involvement is known to be the main prognostic factor. However, lymphatic anatomy is complex and only partially characterized. The aim of the study was to study the pancreatic lymphatic system using computer-assisted anatomic dissection (CAAD) technique and also to update CAAD technique by automatizing slice alignment. METHODS: We dissected three human fetuses aged from 18 to 34 WA. 5-µm serial sections of duodeno-pancreas and spleen blocks were stained (hematoxylin-eosin, hematoxylin of Mayer and Masson trichrome), scanned, aligned and modeled in three dimensions. RESULTS: We observed a rich, diffuse but not systematized lymphatic network in the peri-pancreatic region. There was an equal distribution of LNs between the cephalic and body-tail portions. The lymphatic vascularization appeared in continuity from the celiac trunk to the distal ends of its hepatic and splenic arterial branches parallel to the nerve ramifications of the celiac plexus. We also observed a continuity between the drainage of the pancreatic head and the para-aortic region posteriorly. CONCLUSION: In view of the wealth of peri-pancreatic LNs, the number of LNs to harvest could be increased to improve nodal staging and prognostic evaluation. Pancreatic anatomy as described does not seem to be compatible with the sentinel LN procedure in pancreatic surgery. Finally, we are now able to offer an alternative to manual alignment with a semi-automated alignment.

Adherence in severe asthma.

Adherence in asthma is an important cause for concern. Although nearly 50% of asthma patients are considered poorly adherent to therapeutic advices, adherence is still difficult to assess, understand and improve despite major medical consequences. In this review, we revisited the literature of the last 10 years related to adherence in severe asthma. The concepts have changed and "compliance" is usually replaced by "adherence". Assessment of adherence is addressing ethical issues, but provides important insight into difficult-to-treat asthma. Different tools have been used but none is routinely recommended. Health-related outcomes (poor control, exacerbations, hospitalizations, lung function decline), which are clearly associated with severe asthma, are often worsened by non-adherence with consequences also on patient related outcomes (quality of life). The potential behaviour associated with non-adherence and all other related factors including easy-to-recognize psychological traits can help for patient's future management. Therapeutic educational interventions have been recognized with a scientifically proven efficiency even though evolution and improvements are needed. A multidisciplinary approach is required in severe asthma. Therapeutic adherence for a given patient is always a prerequisite to any other aspects when addressing severe asthma phenotypes. Severe asthma should be considered only in those who still experienced poor asthma outcomes despite optimal adherence. At a glance, poor adherence and severe asthma should be considered antinomic. Better understanding of the causes and customised management are potential future directions.

[Training simulation during the announcement of fetal malformation discovered on screening ultrasound: Results of a pilot study]. / Formation par simulation à l'annonce d'une malformation fœtale découverte en échographie de dépistage : résultats d'une étude pilote.

OBJECTIVES: Breaking bad news (BBN) to a pregnant woman with fetal abnormalities (FA) on ultrasound (US) examination is a challenge. Announcement technique influences patient reaction. Physicians receive little training in BBN. The simulation and using a BBN protocol as the English SPIKES protocol which guides the announcement consultation according to 6 steps (Setting Up, Perception, Invitation, Knowledge, Emotions and Empathy, Strategy and Summary) can be used for this teaching. The objective was to assess feasibility simulation scenarii of BBN for FA discovered during US and to evaluate the usefulness of SPIKES protocol in this situation. METHODS: Two scenarios have been created combining US simulator (US Mentor, Symbionix®) with simulated patient (SP). Scenarii objectives were to diagnose FA and break it to SP. Checklist derived from SPIKES was fulfilled by two investigators thanks to video recording, the SP and every participant (residents, physicians, fetal medicine specialists [FMS]). Participants filled out survey about the usefulness of this exercise too. RESULTS: Nine physicians (3 residents, 4 physicians, 2 FMS) produced 18 scenarii. Seventy-eight percent of physicians thought simulation was like real situation of BBN during US examination. Majority of participant (88%) found that this simulation training could help them to increase their ability to BBN and that it can be used to teach residents (89%) or physicians (100%). FMS had better SPIKES checklist than physicians (P<0,05). CONCLUSION: Simulation scenario of BBN for FA discovered during US is feasible by combining US simulator and SP. SPIKES protocol can be useful but a validated checklist should be created.

[Absent or hypoplastic thymus: A marker for 22q11.2 microdeletion syndrome in case of polyhydramnios]. / La dysgénésie thymique : marqueur de microdélétion 22q11.2 dans le bilan d'un hydramnios.

OBJECTIVES: In prenatal diagnosis of 22q11.2 microdeletion syndrome, without cardiac malformation or multiple associated congenital anomalies, we study the presence of polyhydramnios and its association with thymic dysgenesis. MATERIALS AND METHODS: This was a multicenter retrospective observational study. It was performed in two multidisciplinary centers for prenatal diagnosis in the south of France between January 1, 2010 and June 30, 2013. Inclusion criteria were prenatal diagnosis of 22q11.2 deletion syndrome. We excluded from the study any fetus with cardiac malformation or multiple associated congenital anomalies. RESULTS: During the inclusion period, eleven antenatal diagnoses of 22q11.2 microdeletion syndrome have been made. Six cases were excluded: 5 fetuses with cardiac malformation and one with multiple associated congenital anomalies. Therefore, five cases of isolated polyhydramnios were included. All 5 fetuses had a thymic dysgenesis: 3 had a thymic agenesis and 1 thymic hypoplasia diagnosed by sonography and 1 had a thymic agenesis diagnosed by retrospective reading of fetal MRI. CONCLUSION: When faced with a polyhydramnios, the presence of a thymic dysgenesis should be search for by ultrasound screening and would alert to the possibility of a 22q11.2 microdeletion syndrome. The confirmation of this is diagnosis by amniocentesis would enable improved antenatal support for parents and would enable early implementation of the multidisciplinary neonatal care that is required to avoid serious complications of this syndrome.

Assignment of the human tyrosine hydroxylase gene to chromosome 11.

Using a panel of human-mouse somatic cell hydrids and a cDNA probe for human tyrosine hydroxylase, we have assigned the structural gene for tyrosine hydroxylase to chromosome 11 by Southern blotting techniques.

Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor.

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).

Molecular genetics of catecholamines as an approach to the biochemistry of manic-depression.

Manic depressive illness has been clearly established to exhibit a strong genetic component and is therefore amenable to linkage analysis using random DNA markers. In view of the catecholamine hypothesis of this disorder, the gene encoding tyrosine hydroxylase (TH) the limiting enzyme in catecholamines is a good candidate to investigate. This gene has been localized to chromosome 11 in close linkage with Harvey-ras-1. The various transcriptional and post-transcriptional mechanisms that modulate short and long-term TH activity are discussed. Human tyrosine hydroxylase is coded by at least three distinct mRNAs derived from a single gene. This variation has clear functional consequences and could represent a novel mode of regulating catecholamines levels in normal and pathological neurons.

[Asthmatic emergencies: a psychosocial approach to behaviour of asthmatics]. / L'asthme aux urgences: une approche psychosociale des conduites des malades.

Emergency department admissions for acute asthma often show that patients have failed to manage their disease well. Such admissions may reflect poor asthma control, as defined in the literature, often due to a lack of education and medical follow-up. If patient-centred education has been recognised as effective, what are the best locations and the best moments in the patient's disease history to provide this education? The French Guidelines (HAS) on therapeutic education suggested that education should take place in the emergency department. Moreover, the emergency department is a unique healthcare situation and opportunity for therapeutic education. In the emergency department, a better analysis and understanding of an asthmatic's health orientated behaviour by a psychosocial interview may improve the patient's decision making and lead to an appropriate education programme.

Expression of human dopamine beta-hydroxylase in mammalian cells infected by recombinant vaccinia virus. Mechanisms for membrane attachment.

Dopamine beta-hydroxylase (DBH) is found in neurosecretory vesicles in both membrane-bound and soluble forms. We expressed various human DBH cDNAs in two mammalian cell lines, using the vaccinia virus expression system. The expression of a full-length DBH cDNA (DBH-f) reproduced the native DBH electrophoretic pattern and led to the synthesis of an active enzyme composed of two subunits of 77 and 73 kDa. In contrast, a truncated cDNA lacking the first ATG (DBH-t) generated a single band of 73 kDa. Analysis of mutated recombinant clones demonstrates that the two polypeptides do not result from the use of an alternative translation initiator codon. These results, combined with deglycosylation experiments, allow us to attribute the double band pattern to an optional cleavage of the signal peptide. When the NH2-terminal extremity is shortened, cleavage becomes obligatory, underlining the role of the first 14 amino acids in the regulation of the cleavage of the signal peptide. Subcellular analysis of recombinant DBH-t and DBH-f proteins indicates that DBH is anchored to the membrane by two distinct mechanisms; one of them is due to the non-removal of the signal peptide, whereas the second one is independent of the presence of the signal sequence. Moreover, quantification of the fractionation experiments suggests that the two modes of membrane attachment are additive.

Cloning of quail tyrosine hydroxylase: amino acid homology with other hydroxylases discloses functional domains.

A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C-terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.

Time course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine.

A single injection of reserpine causes a long lasting enhancement of the activity of tyrosine hydroxylase (TH), the enzyme catalyzing the rate-limiting step in the biosynthesis of catecholamines. A sensitive method has been developed to assay both TH mRNA level and enzyme activity in tissue from a single rat. The time course of the induction was analysed in adrenals, locus coeruleus and substantia nigra. In both locus coeruleus and adrenals reserpine caused respectively 4.2- and 4.5-fold increase of TH mRNA which was maximal 2 days after drug injection. This increase is about twice that of the enzyme activity. No change was observed in substantia nigra. The effect lasted longer in locus coeruleus than in adrenal. In the latter, TH mRNA had almost returned to initial values at day 4 whereas at this time it is 3-fold higher in locus coeruleus and still significant at day 18. This result suggests that induction of TH results from an enhanced transcription of the TH gene. The time course difference between locus coeruleus and adrenals is most likely to result from a difference in the stability of TH mRNA in the two structures.

[Histochemical localization of messenger RNA coding for tyrosine hydroxylase: in situ hybridization with a single-stranded cDNA probe]. / Localisation histochimique des ARN messagers codant pour la tyrosine hydroxylase: hybridation in situ à l'aide d'une sonde ADNc monocaténaire.

The expression of tyrosine-hydroxylase (TH) gene was analysed in tissue sections of bovine adrenal glands, by in situ hybridization using a single-stranded cDNA probe. Tissue fixation and hybridization conditions were found that led to a specific and sensitive detection of TH.

Complete coding sequence of rat tyrosine hydroxylase mRNA.

Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed.

Localization of the human tyrosine hydroxylase gene to 11p15: gene duplication and evolution of metabolic pathways.

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH) are consecutive enzymes in the metabolic pathway leading to the production of catecholamine neurotransmitters. A comparison of recently available sequence data of these enzymes in the rat indicates about 70% homology in the 3' coding regions. We have localized TH by in situ hybridization to human chromosome region 11p15. Consideration of this assignment and that of PAH to chromosome 12, together with the known distribution of other pairs of related genes on these two chromosomes, provides convincing evidence of their ancestral relationship and suggests a role for gene duplication in the diversification of metabolic pathways in the vertebrate ancestors of mammals.

Amphiphilic and nonamphiphilic forms of bovine and human dopamine beta-hydroxylase.

We show that human and bovine dopamine beta-hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholinesterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II----Da4I----Dna4. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide, and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.

Predicted amino acid sequence of bovine tyrosine hydroxylase and its similarity to tyrosine hydroxylases from other species.

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.

Retinoic acid induces cholinergic differentiation of cultured newborn rat sympathetic neurons.

Many studies provide evidence that retinoic acid (RA), an endogenous derivative of vitamin A, plays a role in the development of the nervous system. We now report that RA controls the neurotransmitter phenotype of post-mitotic rat sympathetic neurons in cell culture. RA added to the culture medium increased the specific activity of choline acetyltransferase (ChAT) and the level of acetylcholine (ACh). Concomitantly, RA reduced the specific activities of two catecholamine synthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and the level of norepinephrine (NE). After a 2 week treatment with 5 microM RA, ChAT was increased by 5-10 fold, whereas TH and DBH were decreased by 10-15 fold and 2-3 fold, respectively, as compared to sympathetic neurons grown in the absence of RA. The modulation of the activity of the three enzymes was dose-dependent and followed a similar time course. The decrease of TH expression was demonstrated to be due to a decreased number of TH molecules.