A icterícia por hiperbilirrubinemia indireta afeta mais de 60% dos recém-nascidos a termo. O tratamento, quando necessário, é realizado através da fototerapia. Não existem estudos na literatura avaliando os efeitos da fototerapia na função dos neutrófilos de recém-nascidos. O melhor entendimento da função dos neutrófilos nos recém-nascidos antes e após a fototerapia seria importante para avaliar as possíveis repercussões na expressão dos neutrófilos desencadeadas pelo tratamento fototerápico. O objetivo deste estudo foi avaliar e comparar a função dos neutrófilos, através da mensuração pela citometria de fluxo da expressão dos principais marcadores de superfície em recémnascidos ictéricos, antes e após 24 horas de fototerapia. Metodologia: Foram incluídos recém-nascidos com idade gestacional ≥ 35 semanas e peso de nascimento ≥ 2000g, que possuiam critérios da Academia Americana de Pediatria para tratamento fototerápico. Os critérios de exclusão foram: mal-formações congênitas, síndromes com alterações cromossômicas, erro inato do metabolismo, infecções do grupo STORCH, asfixia neonatal, sepse ou suspeita de sepse, exsanguineotransfusão, transfusão de hemocomponentes e uso de imunoglobulina. Foi realizada a avaliação de expressão da intensidade média de fluorescência (IMF) de CD10, CD11b, CD11c, CD15, CD16, CD18, CD62L, CD64 e CD66, antes do início e após 24 horas do início da fototerapia. Foram utilizados o teste T de Student para análise dos dados. Resultados: Foram incluídos 25 recém-nascidos no estudo, com idade mediana de 53 (27.5-75.5) horas de vida e bilirrubina média de 13.6±2.85 mg/dL. Não houve diferença estatística na expressão de CD11b, CD15, CD18, CD62L, CD64 e percentual de neutrófilos antes e após 24 horas de fototerapia. Ocorreu aumento da expressão de CD10 8 (p=0.038) e CD16 (p=0.017) e redução da expressão de CD11c (p=0.023) e CD66acde (p=0.004) após 24 horas de fototerapia. Conclusão: Os recém-nascidos submetidos ao tratamento fototerápico apresentaram aumento da expressão de CD10 e de CD16 e diminuição da expressão de CD11c e de CD66acde após 24 horas de exposição, que pode estar relacionado a um efeito antiinflamatório da fototerapia nos recém-nascidos expostos a este tratamento. / Jaundice due to indirect hyperbilirubinemia affects more than 60% of term neonates. The treatment when necessary is carried out using phototherapy. There are no studies in the literature evaluating the effect of phototherapy on the function of neonates' neutrophils. A better understanding of the function of neutrophils in neonates before and after phototherapy would be important in order to assess potential effects on the expression of neutrofils triggered by the phototherapy treatment. The aim of this study was to assess and compare the function of neutrophils by measuring the expression of the main surface markers in icteric neonates, using flow cytometry, before and after 24 hours of phototherapy. Methodology: Neonates at a gestational age ≥ 35 weeks and at a birth weight ≥ 2000g who met the criteria of the American Academy of Pediatrics for phototherapy were included. The exclusion criteria were: congenital malformations, syndromes with chromosomal alterations, inborn errors of metabolism, infections of the STORCH group, neonatal asphyxia, sepsis or suspicion of sepsis, exchange transfusion, transfusion of blood components, and use of immunoglobulin. The evaluation of the MFI expression of CD10, CD11b, CD11c, CD15, CD16, CD18, CD62L, CD64 and CD66 was performed before and 24 hours after the initiation of phototherapy. The chi-square and Student T tests were used for data analysis. Results: Twenty-five neonates were included in the study at the mean age of 53 (27.5- 75.5) hours of life and with a mean bilirubin level of 13.6±2.85 mg/dL. There was no statistical difference in the expression of CD11b, CD15, CD18, CD62L, CD64 and percentage of neutrophils before and after 24 hours of phototherapy. There was an increase in the expression of CD10 (p=0.038) and CD16 (p=0.017) and a reduction in 10 the expression of CD11c (p=0.023) and CD66acde (p=0.004) after 24 hours of phototherapy. Conclusion: The newborns submitted to phototherapy had increased expression of CD10 and CD16 and decreased expression of CD11c and CD66acde after 24 hours of exposure, which may be related to an anti-inflammatory effect of phototherapy on the neonates exposed to this treatment.

Introdução: O acesso biliar ecoguiado é um método de drenagem alternativo à drenagem percutânea transhepática (DPTH) e à cirurgia em pacientes com obstrução biliar distal incurável que falharam drenagem por Colangiopancreatografia Endoscópica Retrógrada (CPRE). Nos casos em que a drenagem ecoguiada anterógrada transpapilar (ou transanastomótica) e o rendez-vous ecoguiado não podem ser realizados como primeira opção, a coledocoduodenostomia (CDT) e a hepaticogastrostomia (HPG) ainda podem ser realizadas em pacientes selecionados. Estas duas vias de drenagem não anatômicas criam uma fístula entra a via biliar e o estômago ou duodeno. Não há dados na literatura que determinem superioridade de uma ou outra técnica. Objetivo: Comparar o sucesso técnico, sucesso clínico e fatores associados entre as duas vias de drenagem em pacientes com obstrução da via biliar distal maligna incurável que não lograram sucesso na drenagem por CPRE ou rendez-vous ecoguiado. Métodos: Entre abril de 2010 e dezembro de 2013, 49 pacientes com obstrução biliar distal maligna incurável que falharam CPRE e rendez-vous ecoguiado foram randomizados para CDT ou HPG. Dados referentes ao sucesso técnico, sucesso clínico, tempo de procedimento, complicações, qualidade de vida e sobrevida foram coletados até três meses após o procedimento. Todos os procedimentos foram realizados em um centro terciário de endoscopia pelo mesmo endoscopista. Próteses biliares parcialmente recobertas (Boston Scientific, Wallflex, 10 mm, 8 cm ou 6 cm) foram utilizadas em todos os pacientes com sucesso técnico. Nos casos de HPG a punção ecoguiada foi intra-hepática no ducto hepático esquerdo. Nos casos de CDT a punção foi extra-hepática no segmento distal não obstruído do colédoco. Após a punção foi realizada colangiografia com introdução de um fio guia hidrofílico de 0,035 polegada. Dilatação com cateter e um dispositivo de needle knife foi realizada para permitir introdução do sistema de disparo da prótese biliar com 8,5 Fr. Resultados: Quarenta e nove procedimentos foram realizados (25 HPG e 24 CDT). Todos os pacientes tinham dilatação da via biliar intra e extra-hepática. A taxa de sucesso técnico foi de 96 % para HPG e de 91% para CDT (p = 0,609). A taxa de sucesso clínico foi de 91% para o grupo HPG e de 77% para o grupo CDT (p = 0,234). No grupo da HPG 5 pacientes (20%) tiveram complicações (3 sangramentos, 2 biliomas e uma bacteremia). No grupo da CDT 3 pacientes (12,5%) tiveram complicações (1 bilioma, 1 sangramento e 1 perfuração). Somente o caso da perfuração necessitou tratamento cirúrgico. As outras complicações foram tratadas clinicamente. O tempo de procedimento médio foi de 47,83 min para a HPG e de 48,88 min para a CDT (p = 0,843). Conclusão: O presente estudo não demonstrou diferença estatisticamente significante em relação ao sucesso técnico, sucesso clínico, complicações e tempo de procedimento entre os dois grupos estudados. Mais estudos são necessários para elucidar o papel de cada via de drenagem / Background: EUS-guided biliary access is an alternative for percutaneous access or surgery in patients with malignant unresectable distal biliary obstruction and failed ERCP. When rendezvous or anterograde transpapillary/transanastomotic intervention fails as primary drainage options, a Choledochoduodenostomy (CDT) or a Hepaticogastrostomy (HGT) can still be performed in selected patients. This procedure creates a new \" \" y I w one route or the other should be recommended. Aim: To compare technical and clinical success and possible associated factors between the two different drainage routes CDT and HGT in patients with distal unresectable malignant biliary obstruction that failed standard ERCP and EUS-guided rendez vouz (RV) maneuver. Methods: Between April/2010 and December/2013 49 consecutive jaundiced patients with distal unresectable malignant biliary obstruction that failed previous ERCP and EUS-guided RV maneuver were elected randomly to undergo either EUS-guided CDT or HGT. Data including indications, clinical and technical success, procedural times and complications with a three-month follow-up were prospectively collected in a database. All procedures were performed in a tertiary center by the same endoscopist. A partially covered SEMS (Boston Scientific, Wallflex, 10 mm, 8 cm or 6 cm) was used in all technically successful procedures. After puncture of left hepatic duct in case of HGT or the distal unobstructed segment of common bile duct in case of CDT a cholangiogram was obtained followed by advancement of a 0,035-inch guide wire into the biliary system. Bougies and wire-guided needle-knife were used to perform track dilation to allow passage of an 8.5 Fr stent delivery system. Results: Forty-nine cases (25 HGT and 24 CDT) were performed. All patients had intra and extra hepatic biliary dilation. Technical success rate was 96 % for HGT and 91% for CDT (p = 0.609). Clinical success rate was 91% for HPG and 77% for CDT (p = 0.234). In the HGT group five patients (20%) had complications (3 bleeding, 2 bilomas and 1 bacteremia). In the CDT group 3 patients (12.5%) had complications (1 biloma, 1 bleeding and 1 perforation). Only the perforation patient required surgery. All other complications were managed clinically. The median procedural time was 47.83 min for HGT and 48.88 min for CDT (p = 0.843). Conclusion: No significant difference was found in regards to technical or clinical success, complications and procedure time between the two drainage routes. More studies are needed to clarify situations in which the CDT or the HGT should be advocated

Η ηπατική εγκεφαλοπάθεια είναι ένα σύνθετο νευροψυχιατρικό σύνδρομο το οποίο εκδηλώνεται κυρίως σε συνθήκες ηπατικής κίρρωσης. Διάφορες παθολογικές και τοξικές καταστάσεις μπορεί να επηρεάσουν την ηπατική λειτουργία σε τέτοια βαθμό ώστε να προκληθεί ηπατική εγκεφαλοπάθεια. Το οξειδωτικό στρές έχει ενεπλακεί σε διάφορες μελέτες στην παθογένεση της ηπατικής εγκεφαλοπάθειας. Επιπλέον, η ανάπτυξη αυξημένου οξειδωτικού στρές υπό την επίδραση αποφρακτικού ικτέρου έχει μετρηθεί σε διάφορα όργανα πειραματοζώων, συμπεριλαμβανομένου και του εγκεφάλου. Ο σκοπός της παρούσας μελέτης ήταν να ανιχνεύσει της αλλαγές στις αποφρακτικές συνδέσεις των τριχοειδικών αγγείων του εγκεφάλου που συσχετίζονται με την οκκλουδίνη. Για να το επιτύχουμε αυτό χρησιμοποιήσαμε ένα μοντέλο απολίνωσης του κοινού χοληδόχου πόρου (BDL) σε πειραματόζωα (αρουραίους). Στο πείραμα 1 η έκφραση της οκκλουδίνης εκτιμήθηκε μέσω της μεθόδου ποσοτικοποίησης κατά Westernblot. Σε αυτό το πείραμα χρησιμοποιήθηκαν πέντε (BDL) και πέντε ψευδώς χειρουργηθέντα πειραματόζωα (sham). Τα πειραματόζωα θανατώθηκαν δέκα ημέρες μετα το χειρουργείο. Στην κατά Westernblot ποσοτικοποίηση παρατηρήθηκε μεγάλη μείωση της ποσότητας της οκκλουδίνης στα BDL πειραματόζωα σε σχέση με τα sham. Στο πείραμα 2 χρησιμοποιήθηκαν εννέα BDL και εννέα sham πειραματόζωα. Τρία πειραματόζωα από τις δύο παραπάνω ομάδες θανατώθηκαν την πρώτη, την πέμπτη και τη δέκατη μετεγχειρητική ημέρα. Τα επίπεδα οκκουδίνης σε αυτά τα πειραματόζωα συσχετιστήκαν με τις τιμές τηςinvivo μέτρησης των ελευθέρων ριζών οξυγόνο. Τα αποτελέσματα ανέδειξαν ότι η τιμή της οκκλουδίνης στα BDL ζώα ήταν σημαντικά μειωμένη σε σχέση με τα sham όλες τις χρονικές στιγμές που έγιναν οι μετρήσεις, ενώ οι χαμηλότερες τιμές καταγράφηκαν στα πειραματόζωα που παρέμειναν υπό αποφρακτικό ίκτερο για δέκα ημέρες. Επιπλέον καταδείχθη ότι η χρονικά συσχετιζόμενη μείωση των επιπέδων της οκκλουδίνης στα ενδοθηλιακά κύτταρα του εγκεφάλου συσχετίζεται με τα αυξανόμενα επίπεδα ελευθέρων ριζών οξυγόνου της ημέρες μετά το χειρουργείο, φανερώνοντας τη σχέση μεταξύ αυτών των δύο φαινομένων. Συμπερασματικά, η παρούσα μελέτη πρώτη παραθέτει στοιχεία που προτείνουν την εμπλοκή της οκκλουδίνης στην παθοφυσιολογία της ηπατικής εγκεφαλοπάθειας σε συνθήκες εξωηπατικής χολόστασης. Αυτό γίνεται μέσω της μείωσης των επιπέδων τις οκκλουδίνης στις αποφρακτικές συνδέσεις των ενδοθηλιακών κυττάρων του εγκεφάλου υπο την επίδραση του χολοστατικού ικτέρου, η οποία οδηγεί σε άρση του αιματο-εγκεφαλικού φραγμού. / Hepatic encephalopathy in a complicate neuro-psychiatric syndrome which is common under hepatic cirrhosis. Various pathological and toxic lesions can deteriorate liver function in such way, so hepatic encephalopathy can be inflicted. Oxidative stress is involved in pathogenesis of hepatic encephalopathy in several protocols. Moreover the development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study aimed to detect alterations of tight junction-associated occludin in rat brain capillaries. To accomplish that we have used a rats bile duct ligation experimental model (BDL). In experiment 1, occludin expression was evaluated by Western blot analysis. In this experiment were used five BDL and five sham rats. The experimental animals were sacrificed ten days after the operation. Western blot analysis revealed significant decrease of occlidins amount in BDL rats compared to the sham rats. In experiment 2, nine BDL and nine sham animals were used. Three animals from each group were sacrificed during the first, fifth and tenth post-operate day. The results of occludin level to these animals were associated with the in vivo superoxide radical production. The results indicated that occludin level in BDL animals, as opposed to sham-operated, was significantly reduced at every time point studied, being lowest in the rats remaining on BDL condition for 10 days. Moreover, it was demonstrated that the time-dependent reduction of occludin level in the brain endothelial was significantly correlated with the time dependent increase of brain superoxide radical level, implying a relationship between these two abnormalities. In conclusion, the evidence presented herein suggests for first time the implication of occludin in pathophysiology of hepatic encephalopathy under extra-hepatic cholestasis. This phenomenon occur due to the reduce of occludin level to cerebral endothelial cells’ tight junctions under cholestatic jaundice, which drives to lift of brain-blood barrier.

ΣΚΟΠΟΣ: Σήμερα είναι πλέον γνωστό ότι το χολοστατικό ήπαρ είναι πιο ευαίσθητο στην ισχαιμία από το φυσιολογικό ήπαρ. Η ηπατική αναγέννηση μετά μερική ηπατεκτομή (ΡΗχ) ρυθμίζεται από διάφορους παράγοντες οι οποίοι ενεργοποιούν ή αναστέλλουν τον πολλαπλασιασμό των ηπατοκυττάρων. Ένα από τα κυτταρικά στοιχεία που συμβάλλει στην αναγέννηση του ήπατος είναι το ηπατοκύτταρο με χολαγγειακή μορφολογία (DΗ). Από την άλλη μεριά, η απόπτωση φαίνεται να παίζει ένα σημαντικό ρόλο στον κυτταρικό πολλαπλασιασμό και στην ηπατική αναγέννηση. Στην παρούσα μελέτη, μετά από Ρηχ σε φυσιολογικούς ποντικούς και σε άλλους με ίκτερο, με ή χωρίς επιπλέον ισχαιμία, ερευνήσαμε: α) τα ιστοπαθολογικά χαρακτηριστικά της ισχαιμικής ηπατικής βλάβης και αναγέννησης, β) το δυνητικό ρόλο των ηπατοκυττάρων με χολαγγειακή μορφολογία κατά τη διάρκεια της ηπατικής αναγέννησης και γ)την έκφραση των γονιδίων bcl-2 και bax, τα οποία σχετίζονται με την απόπτωση, καθώς και την παρουσία της απόπτωσης. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Η μελέτη συμπεριέλαβε 140 ποντικούς Wistar οι οποίοι χωρίστηκαν σε 14 ομάδες: Ι –control, II - εγχείρηση sham. III-IV-V-παροδικός αγγειακός αποκλεισμός ηπατικής αρτηρίας και πυλαίας φλέβας (ολική ηπατική ισχαιμία) (TLI) για 15-30-60 λεπτά αντίστοιχα. VI - απολίνωση του κοινού χοληδόχου πόρου (BDL) για 10 ημέρες, VII-VIII-ΙΧ-BDL-ΤLI-ΡΗχ. Χ-μερική ηπατεκτομή (ΡΗχ 68%}, XI- ΡΗχ + TLI (30 min), XII-BDL+ΡΗχ, XIII-BDL+ ΡΗχ+TLI (30 min) και XIV- BDL+TLI (30 min) + ΡΗχ. Τα ζώα θυσιάστηκαν 24 και 48 ώρες μετά από Ρηχ, με εξαίρεση τις περιπτώσεις όπου η κατάσταση υγείας ήταν κρίσιμη. Η μελέτη του ηπατικού ιστού περιελάμβανε: (α) Η&Α stain, (β) υβριδισμό in situ (ανίχνευση mRΝΑ των bcl-2 και bax σε παραφίνη, (γ) ανάλυση Western Blot για τη μελέτη των πρυτεϊνικών επιπέδων των bcl-2 και bax, (δ) in situ υβριδισμός (TUNEL) για την ανίχνευση παραγόντων απόπτωσης, (ε) ανοσοϊστοχημικά stains σε παραφίνη (μέθοδος στρεπταβιδίνης-βιοτίνης) με σκοπό να μελετηθεί η έκφραση των ακόλουθων αντιγόνων: κυτοκερατίνη 7 και 19 (χολικός φαινότυπος), ηπατοκυτταρίκή και α1-σντιθρυψίνη (ηπατοκυτταρικός φαινότυπος), βιμεντίνη, CD34, α-φετοπρωτείνη, GST-π(γλουταθειονιν-S-τρανσφεράση-π). Κι67 (κυτταρικός πολλαπλασιασμός), bcl-2 και bax (γονίδια απόπτωσης). ΑΠΟΤΕΛΕΣΜΑΤΑ: Ήπαρ από ποντικούς α)με TLI ανέπτυξε βλάβες ηπατικής ισχαιμίας, β)με BDL βλάβες ηπατικής απόφραξης, γ)με BDL+TLI ανέπτυξαν βλάβες ηπατικής ισχαιμίας μαζί με αλλαγές λόγω απόφραξης των χοληφόρων. Το ποσοστό του επηρεασμένου ηπατικού παρεγχύματος (%ηπατικής νέκρωσης) ήταν υψηλότερο στις ομάδες με TLI+BDL. Σε αυτές τις ομάδες, ο ηπατοκυτταρικός δείκτης (δείκτης πολλαπλασιασμού) του Κi67 ήταν μικρότερος από τον αντίστοιχο της ομάδας χωρίς TLI. Το εύρημα αυτό υποδηλώνει ότι το χολοστστικό ήπαρ είναι πιο ευαίσθητο στην ισχαιμία κι εκδηλώνει μειωμένη αναγεννητική ικανότητα, συγκρινόμενο με μη χολοστατικό ήπαρ. Βιοψίες ήπατος έδειξαν χαρακτήρες ηπατικής αναγέννησης προερχόμενης από τη ζώνη 2, η οποία επεκτεινόταν στη ζώνη 1 και κάποιες φορές στη ζώνη 3. Ανοσοϊστοχημικές αναλύσεις ανέδειξαν κύτταρα θετικά στο ΑΕ1 και κύτταρα θετικά στο ΗΕΡΡΑΡ. Σε όλες τις ομάδες με TLI τα ηπατοκύτταρα χολαγγειακής μορφολογίας εμφανίζονταν κυρίως σε περιοχές περι-πυλαίες. Εξέφραζαν κυτοκερατίνες 19 και 7, ηπατοκυτταρίκή και α1-αντιθρυψίνη ενώ εμφάνιζαν και συνδυασμένη έκφραση CΚ7/CΚ19 και ηπατοκυτταρική ή ΑΕ1/CΚ19 και α1-αντιθρυψϊνη. Το ποσοστό των κυττάρων αυτών ήταν ευθέως ανάλογο με το χρόνο επιβίωσης των ζώων (r=0,354, ρ<0,05). Όσον αφορά την απόπτωση, προ ηπατεκτομής, τα επίπεδα του bcl-2 ( πρωτεΐνη ή mRΝΑ) ήταν υψηλότερα σε ποντικούς με ίκτερο απ' ό,τι στους ποντικούς controls. Επιπλέον τα επίπεδα του bax (πρωτεΐνη ή mRΝΑ) και ο σωματικός δείκτης απόπτωσης (ΑΒΙ) ήταν υψηλότερα σε χολοστατικό ήπαρ. Μετά ηπατεκτομή, παρατηρήθηκε πρώιμη ελάττωση των επιπέδων πρωτεϊνών και mRΝΑ του γονιδίου bcl-2 και μια ύστερη αύξηση του προ-αποπτωτικού γονιδίου bax και του ΑΒΙ, σε σύγκριση με τα controls. ΣΥΜΠΕΡΑΣΜΑΤΑ: Σε περιπτώσεις αποφρακτικού ίκτερου (χολαγγειοπάθεια), η ηπατική ισχαιμία καθιστά το όργανο πιο ευαίσθητο σε ενδεχόμενη βλάβη, καθώς προκαλεί ευρύτερη νέκρωση παρεγχύματος και ελαττώνει το ρυθμό πολλαπλασιασμού των ηπατοκυττάρων, Η διεργασία αναγέννησης του ήπατος επιτελείται κυρίως μέσω του πολλαπλασιασμού μη νεκρωτικών κυττάρων τα οποία εκφράζουν ηπατοκυτταρικά ή χολαγγειακά επιθηλιακά χαρακτηριστικά. Σε τέτοιες περιπτώσεις, το κύριο στοιχείο της ηπατικής αναγέννησης αποτελεί η παρουσία των ηπατοκυττάρων με χολαγγειακή μορφολογία (κύτταρα που εκδηλώνουν φαινότυπο ενδιάμεσο μεταξύ κυττάρων χολαγγειακής και ηπατικής προέλευσης) και η διαφοροποίηση τους σε ώριμα ηπατοκύτταρα. Τέλος, η απόπτωση έχει ρόλο στο χολοστατικό ήπαρ με ή χωρίς επιπλέον ισχαιμία και πιστεύεται ότι μπορεί να συμβάλλει στην παρουσία κατεσταλμένης αναγεννητικής απόκρισης που παρατηρήθηκε σε ήπαρ ποντικών με ίκτερο μετά από μερική ηπατεκτομή. / AIM: Today, it is known that cholestatic liver is more susceptible to ischemia than normal liver. Liver regeneration after partial hepatectomy (PHx) is regulated by several factors that activate or inhibit hepatocyte proliferation. One of the cellular elements that contributes to liver regeneration is ductular hepatocyte (DH). On the other hand, apoptosis seems to play an important role in cellular proliferation and liver regeneration. In this study and. after PHx. in normal and jaundiced rats with or without superimposed ischemia, we assessed a) the histopathologic features of hepatic Ischemic damage and liver regeneration, b) the potential role of ductular hepatocytes during liver regeneration and c) the expression apoptosis-asscciated genes bcl-2 and bax. and the presence of apoptosis, MATERIALS £ METHODS: The study comprised 140 male Wistar rats, assigned randomly in 14-groups: l-controls. ll-sham. Ill-IV-V-cIamping of hepatic artery and portal vein (total liver Ischemia-TLI) for 15-30-60 min respectively. Vl-common bile duct ligation (BDL) for lOdays. VII-VIII-IX-BDL-TLI-X-partial hepatectomy(PHx-68%). XI-PHx+TLI(30min). XU-BDL+PHx. XIII-BDL+PHx+TLI(30min) and XIV-BDL+TLI (30min)+PHx. Animals were sacrificed 24 and 48hrs after PHx unless their condition was critical. Liver tissue evaluation Included: (a) H&E stain, (b) in situ hybridization (detection of bcl-2 and bax mRNA) in paraffin sections, (c) Western blot analysis for the evaluation of bcl-2 and bax protein levels, (d) in situ hybridization (TUNEL) for the detection of apoptotic bodies, and (e) immunohistochemical stains (streptavidin-biotin method) in paraffin sections, in order to evaluate the expression of the following antigens: cytokeratins 7 and 16 (biliary phenotype), Hepatocyte and a 1-antitrypsin (hepatocytic phenotype), vimentin, CD34, alpha-fetoprotein, GST-pi (glutahione-S-trarsferase-pi), Ki67 cell proliferation), bcl-2 and bax (apoptotlc genes). RESULTS: Sections from rats a) with TLI developed changes of liver ischemia, b) with BDL changes of liver obstruction and c) with BDL+TLI developed changes of ischemia together with changes of biliary obstruction. The total liver parenchymal area affected (% liver necrosis} was higher in groups with TLI+BDL. In these groups the Ki67 hepatocytic index (proliferation index) was lower compared to the groups without TLI. This finding implies that cholestatic livers are more vulnerable to ischemia and also display impaired generative capabilities, compared to non-cholestatic ones. Liver biopsies exhibited features of liver regeneration that originated from zone 2, extended to zone 1 and occasionally to zone 3. Immunonistochemical stains revealed cells positive to AE1 and cells positive to HEPPAR. In all groups with TLI, DHs appeared mainly in periportal areas. They expressed cytokeratirs-19 and 7. hepatocyte and al-antrtrypsm and co-expressed CK7/CK1S and Hepatocyte or AE1/CK1S and a 1-antitrypsin The percentage of DHs was directly correlated with the time that animals survived (r=0,354, p<0,05). Regarding apoptosis, before hepatectomy, bcl-2 (protein or mRNA} levels were higher in jaundiced rats vs controls. Furthermore, bax (protein or mRNA) levels and apoptotic body index (ABl) were higher in cholestatic livers. After hepatectomy, there was an early decease in the protein and mRNA levels of antiapoptotic gene bcl-2 and a late increase of proapoptotic gene bax and the A8I. compared to controls. CONCLUSIONS: In cases with obstructive jaundice (cholangiopathy), liver ischemia makes the organ more vulnerable to damage because it causes greater parenchymal necrosis and decreases the degree of hepatocyles proliferation. Liver regenerative process is mediated mainly by proliferation of non-necrotic cells that express hepatocellular or ductular epithelial features. In such cases a main element of liver regeneration is the presence of DHs, cells that display a phenotype intermediate between ductular and hepatocytic origin} and their differentiation to mature hepatocytes. Finally, apoptosis takes place in cholestatic livers with or without superimposed ischemia and may contribute in the impaired regenerative response observed in livers of jaundiced rats after partial hepatectomy.

Present work has been focused on the importance of the products of the heme catabolic pathway, in particular under conditions of unconjugated hyperbilirubinemias (neonatal jaundice and Crigler-Najjar syndrome (CNS)). The second part of the project was focused on the improvement of some pharmacological approaches used in the treatment of these diseases, as well as on studies of bilirubin products that are formed during the treatment by phototherapy (PT). Neonatal jaundice is one of the most common complications in neonates. Currently, there is no efficient pharmacotherapy and the treatment with blue light is used as a gold standard for severe neonatal jaundice. However, the absolute safety of PT has still not been confirmed. In this context, it is important to note that some neonatologists start the PT before serum bilirubin levels reach the recommended values and that patients with CNS type I (CNSI) are forced to be on life-long PT (unless undergoing liver transplantation). The focus of the present project was to study biological effects of bilirubin photoisomers (PI) in an in vitro model of the human neuroblastoma SH-SY5Y cells that are used for studies of the neuronal metabolism. In further studies performed on animal model of hyperbilirubinemic rats and mice, we investigated a suitable gene...

Introdução: O acesso biliar ecoguiado é um método de drenagem alternativo à drenagem percutânea transhepática (DPTH) e à cirurgia em pacientes com obstrução biliar distal incurável que falharam drenagem por Colangiopancreatografia Endoscópica Retrógrada (CPRE). Nos casos em que a drenagem ecoguiada anterógrada transpapilar (ou transanastomótica) e o rendez-vous ecoguiado não podem ser realizados como primeira opção, a coledocoduodenostomia (CDT) e a hepaticogastrostomia (HPG) ainda podem ser realizadas em pacientes selecionados. Estas duas vias de drenagem não anatômicas criam uma fístula entra a via biliar e o estômago ou duodeno. Não há dados na literatura que determinem superioridade de uma ou outra técnica. Objetivo: Comparar o sucesso técnico, sucesso clínico e fatores associados entre as duas vias de drenagem em pacientes com obstrução da via biliar distal maligna incurável que não lograram sucesso na drenagem por CPRE ou rendez-vous ecoguiado. Métodos: Entre abril de 2010 e dezembro de 2013, 49 pacientes com obstrução biliar distal maligna incurável que falharam CPRE e rendez-vous ecoguiado foram randomizados para CDT ou HPG. Dados referentes ao sucesso técnico, sucesso clínico, tempo de procedimento, complicações, qualidade de vida e sobrevida foram coletados até três meses após o procedimento. Todos os procedimentos foram realizados em um centro terciário de endoscopia pelo mesmo endoscopista. Próteses biliares parcialmente recobertas (Boston Scientific, Wallflex, 10 mm, 8 cm ou 6 cm) foram utilizadas em todos os pacientes com sucesso técnico. Nos casos de HPG a punção ecoguiada foi intra-hepática no ducto hepático esquerdo. Nos casos de CDT a punção foi extra-hepática no segmento distal não obstruído do colédoco. Após a punção foi realizada colangiografia com introdução de um fio guia hidrofílico de 0,035 polegada. Dilatação com cateter e um dispositivo de needle knife foi realizada para permitir introdução do sistema de disparo da prótese biliar com 8,5 Fr. Resultados: Quarenta e nove procedimentos foram realizados (25 HPG e 24 CDT). Todos os pacientes tinham dilatação da via biliar intra e extra-hepática. A taxa de sucesso técnico foi de 96 % para HPG e de 91% para CDT (p = 0,609). A taxa de sucesso clínico foi de 91% para o grupo HPG e de 77% para o grupo CDT (p = 0,234). No grupo da HPG 5 pacientes (20%) tiveram complicações (3 sangramentos, 2 biliomas e uma bacteremia). No grupo da CDT 3 pacientes (12,5%) tiveram complicações (1 bilioma, 1 sangramento e 1 perfuração). Somente o caso da perfuração necessitou tratamento cirúrgico. As outras complicações foram tratadas clinicamente. O tempo de procedimento médio foi de 47,83 min para a HPG e de 48,88 min para a CDT (p = 0,843). Conclusão: O presente estudo não demonstrou diferença estatisticamente significante em relação ao sucesso técnico, sucesso clínico, complicações e tempo de procedimento entre os dois grupos estudados. Mais estudos são necessários para elucidar o papel de cada via de drenagem / Background: EUS-guided biliary access is an alternative for percutaneous access or surgery in patients with malignant unresectable distal biliary obstruction and failed ERCP. When rendezvous or anterograde transpapillary/transanastomotic intervention fails as primary drainage options, a Choledochoduodenostomy (CDT) or a Hepaticogastrostomy (HGT) can still be performed in selected patients. This procedure creates a new \" \" y I w one route or the other should be recommended. Aim: To compare technical and clinical success and possible associated factors between the two different drainage routes CDT and HGT in patients with distal unresectable malignant biliary obstruction that failed standard ERCP and EUS-guided rendez vouz (RV) maneuver. Methods: Between April/2010 and December/2013 49 consecutive jaundiced patients with distal unresectable malignant biliary obstruction that failed previous ERCP and EUS-guided RV maneuver were elected randomly to undergo either EUS-guided CDT or HGT. Data including indications, clinical and technical success, procedural times and complications with a three-month follow-up were prospectively collected in a database. All procedures were performed in a tertiary center by the same endoscopist. A partially covered SEMS (Boston Scientific, Wallflex, 10 mm, 8 cm or 6 cm) was used in all technically successful procedures. After puncture of left hepatic duct in case of HGT or the distal unobstructed segment of common bile duct in case of CDT a cholangiogram was obtained followed by advancement of a 0,035-inch guide wire into the biliary system. Bougies and wire-guided needle-knife were used to perform track dilation to allow passage of an 8.5 Fr stent delivery system. Results: Forty-nine cases (25 HGT and 24 CDT) were performed. All patients had intra and extra hepatic biliary dilation. Technical success rate was 96 % for HGT and 91% for CDT (p = 0.609). Clinical success rate was 91% for HPG and 77% for CDT (p = 0.234). In the HGT group five patients (20%) had complications (3 bleeding, 2 bilomas and 1 bacteremia). In the CDT group 3 patients (12.5%) had complications (1 biloma, 1 bleeding and 1 perforation). Only the perforation patient required surgery. All other complications were managed clinically. The median procedural time was 47.83 min for HGT and 48.88 min for CDT (p = 0.843). Conclusion: No significant difference was found in regards to technical or clinical success, complications and procedure time between the two drainage routes. More studies are needed to clarify situations in which the CDT or the HGT should be advocated

In 2007, the Canadian Paediatric Society (CPS) released a guideline aimed at preventing complications of neonatal jaundice through universal screening and guidelines for follow-up and treatment. This thesis investigates the impact of implementation of the CPS guideline on health services utilization at a population level in Ontario. First, we surveyed all Ontario hospitals providing maternal-newborn services to determine if and when they had implemented universal bilirubin screening, and to gather information about the organization of services to provide follow-up and treatment, and about the factors that influenced screening implementation. Then we conducted two population-based cohort studies using linked administrative health data to evaluate the association between 1) the implementation of universal bilirubin screening and phototherapy use (during and following birth hospitalization) length of stay (LOS), jaundice-related emergency department (ED) visits and readmissions; and 2) universal bilirubin screening implementation and access to recommended follow-up care by socio-economic status (SES). By 2012, the majority of Ontario hospitals had implemented universal bilirubin screening. There is heterogeneity in how hospitals organize services, but a notable trend towards hospital-based post-discharge care. Screening was associated with an increase in phototherapy during hospitalization at birth (relative risk (RR) 1.32, 95% confidence interval (CI) 1.09-1.59), and a decrease in jaundice-related ED visits (RR 0.79, 95% CI 0.64-0.96), but no statistically significant difference in phototherapy after discharge, length of stay, or jaundice-related readmissions after accounting for pre-existing temporal trends in healthcare service use and other patient socio-demographic and hospital characteristics. Implementation of the universal bilirubin screening in Ontario was associated with a modest increase in rates of early follow-up (adjusted RR 1.11, CI 1.0014-1.22, p=0.0468), but most babies were not seen within the recommended timeframe. Babies of lowest SES were least likely to receive recommended follow-up, and disparities in follow-up increased following universal bilirubin screening implementation. En 2007, la Société canadienne de pédiatrie (SCP) a publié une directive visant à la prévention des complications de l'ictère néonatal par le dépistage universel et des lignes directrices pour le suivi et le traitement. Cette thèse étudie l'impact de la mise en œuvre de la directive SCP sur l'utilisation des services de santé à niveau de population de l'Ontario. Tout d'abord, nous avons interrogé tous les hôpitaux de l'Ontario offrant des services de santé maternelle-nouveau-né afin de déterminer si et quand ils avaient mis en œuvre le dépistage universel de la bilirubine, et à recueillir des informations sur l'organisation des services pour assurer un suivi et de traitement, et sur les facteurs qui ont influencé la mise en œuvre de dépistage. Ensuite, nous avons mené deux études de cohorte basée sur la population à partir de données administratives sur la santé pour évaluer 1 ) l'association entre la mise en œuvre du dépistage de la bilirubine universel et la photothérapie utilisation lors de l'hospitalisation à la naissance, la photothérapie après avoir sortie de l'hôpital, la durée du séjour, le service des urgences liées à la jaunisse et des réadmissions liées à la jaunisse; et 2 ) l'association entre la mise en œuvre du dépistage universel et l'accès aux soins de suivi recommandés et si cela différait entre les quintiles de statut socioéconomique. En 2012, la majorité des hôpitaux de l'Ontario a mis en œuvre le dépistage universel de la bilirubine. Il existe une hétérogénéité de la façon dont les hôpitaux organisent des services, mais une tendance notable vers les soins post-décharge en milieu hospitalier. Le dépistage a été associé à une augmentation de la photothérapie pendant l'hospitalisation à la naissance (risque relatif (RR) de 1,32, intervalle de confiance 95 % (IC 95 %) de 1,09 à 1,59), et une diminution des visites à l'urgence liées à la jaunisse (RR 0,79, IC 95 % 0,64 à 0,96), mais aucune différence statistiquement significative dans la photothérapie après la sortie , la durée du séjour , ou réadmissions liées jaunisse - après comptabilisation des tendances temporelles pré- existants dans l'utilisation des services de soins de santé et d'autres caractéristiques socio- démographiques des patients et caractéristiques de l'hôpital. La mise en œuvre de le dépistage universel en Ontario a été associée à une légère augmentation des taux de suivi précoce (RR ajusté 1,11; IC de 1,0014 à 1,22; p = 0,0468), mais la plupart des bébés n'ont pas été vues dans les délais recommandés. Les bébés de statut socioéconomique faibles étaient moins susceptibles de recevoir de soins de suivi recommandés et les disparités dans le suivi ont augmenté suite à la mise en œuvre du dépistage universel de la bilirubine.

Introduction : La variabilité du rythme cardiaque est étudiée à partir des variations de durée des cycles cardiaques (intervalle R-R de l’électrocardiogramme). Ces variations peuvent être analysées par des méthodes linéaires (temporelles et fréquentielles) et non linéaires (théorie de l’information ou des fractales) de quantifications mathématiques et statistiques qui donnent des informations innovantes sur les signaux analysés. L’application de ces méthodes d’étude en néonatologie a démontré un intérêt pour le diagnostique précoce de l’infection néonatale tardive du prématuré mais n’avait pas été étudié dans l’infection néonatale précoce du nouveau-né à terme, dans le contexte des évènements cardio-respiratoires suivant la primo-vaccination des prématurés ou pour évaluer un effet neurologique de l’hyperbilirubinémie dans l’ictère néonatal. Notre hypothèse dans ce travail était qu’il était possible de : (i) caractériser la variabilité du rythme cardiaque en cas d’infection materno fœtale ou de méningite néonatale, (ii) mettre en évidence des facteurs prédisposant à la survenue d’évènements cardio-respiratoires post-vaccinaux, (iii) Identifier un éventuel retentissement neurologique de l’ictère néonatal par étude de la variabilité du rythme cardiaque. / The heart rate variability measures permitted to evaluate equilibrium state and perturbation in the regulation of cardio-vascular system.  These tools, based on heart rate variability analysis, helped to recognize associated disease state as early onset neonatal sepsis and non-infectious inflammatory response induced to immunization. An increase in global variability (SD), long term variability (SD, LF) and low approximated entropy (ApEn) were observed in the proven-sepsis full term infants. Importance of decrease in ApEn was correlated to the severity of sepsis assessed by blood markers. These suggest an association of sepsis with uncoordinated sympatho-vagal coactivation together with loss of adaptability. In premature infants, the risk of increase in cardio-respiratory events after the first immunization was associated with a specific pre-immunization profile: sympathetic predominance in heart rate control (high LF/HF ratio), abnormal oversimplification of heart rate variability and persistence rhythm control immaturity. Increased ApEn after immunization reflects a marginal result from adaptability of the heart rate to environmental changes without possibility to reserve in case of severe infection.

Present work has been focused on the importance of the products of the heme catabolic pathway, in particular under conditions of unconjugated hyperbilirubinemias (neonatal jaundice and Crigler-Najjar syndrome (CNS)). The second part of the project was focused on the improvement of some pharmacological approaches used in the treatment of these diseases, as well as on studies of bilirubin products that are formed during the treatment by phototherapy (PT). Neonatal jaundice is one of the most common complications in neonates. Currently, there is no efficient pharmacotherapy and the treatment with blue light is used as a gold standard for severe neonatal jaundice. However, the absolute safety of PT has still not been confirmed. In this context, it is important to note that some neonatologists start the PT before serum bilirubin levels reach the recommended values and that patients with CNS type I (CNSI) are forced to be on life-long PT (unless undergoing liver transplantation). The focus of the present project was to study biological effects of bilirubin photoisomers (PI) in an in vitro model of the human neuroblastoma SH-SY5Y cells that are used for studies of the neuronal metabolism. In further studies performed on animal model of hyperbilirubinemic rats and mice, we investigated a suitable gene...

Objetivos: A deficiência de glicose-6-fosfato desidrogenase (G6PD) está associada a um maior risco de encefalopatia bilirrubínica e de crise hemolítica aguda grave desencadeada por drogas como a primaquina e a dapsona. Conhecer a prevalência dessa deficiência enzimática em área onde a malária e a hanseníase ainda estão presentes e conhecer a prevalência das principais mutações traz subsídios para planejamento de estratégias com vistas à redução de riscos associados a esta deficiência enzimática. Métodos: Estudo descritivo transversal conduzido em uma região do centro-oeste do Brasil. Exame de triagem para deficiência de G6PD foi realizado em 3573 recémnascidos. Exame confirmatório foi necessário em 188 crianças triadas como possíveis portadores de deficiência. Nas crianças em que foi confirmada a deficiência de G6PD foi feita pesquisa das mutações G202A (G6PD A-) e C563T (G6PD Mediterrâneo) por PCR. Resultados: A deficiência de G6PD foi confirmada em 63 crianças, sendo 60 meninos (95,2%) e três meninas (4,8%). O percentual de exames falso-positivos na fase de triagem foi de 66,5%, estando o percentual de falso-positivos associado à temperatura e tempo de transporte das amostras. Entre as crianças que confirmaram deficiência de G6PD, foi mais frequente a história de anemia em familiares e de icterícia neonatal. Houve associação entre hematócrito baixo e deficiência enzimática, mas não com hemoglobina, contagem de reticulócitos ou neutrófilos. A prevalência da deficiência de G6PD (IC95%) foi de 1,76% (1,37; 2,24) entre os recém-nascidos triados e de 3,34% entre os meninos (2,58; 4,25). A mutação C563T não foi identificada em nenhuma criança, mas a mutação G202A estava presente em 58 crianças - 92,06% (IC95%: 83,29 - 97,03): 56/60 meninos e em 2/3 meninas homozigotas. Foi identificado um menino com Kernicterus portador da mutação G202A em hemizigose. Conclusão: O elevado percentual de falso-positivos na etapa de triagem, o tempo necessário entre coleta e confirmação da presença de deficiência enzimática, associado ao alto custo da triagem universal, não apoiam a inclusão da triagem de deficiência de G6PD no programa de triagem neonatal brasileiro. Na região avaliada, a prevalência observada em meninos indica que a triagem de deficiência de G6PD deva ser realizada antes do uso de drogas como a primaquina e a dapsona somente em meninos. Foi elevada a prevalência da mutação G202A, de classe III, sendo esta mutação associada a uma menor morbidade. A identificação de um menino com Kernicterus com deficiência de G6PD indica que há necessidade de se planejar estratégias para minimizar o risco dessa morbidade associada à deficiência enzimática / Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with an increased risk of bilirubin encephalopathy in neonates and acute hemolytic crisis triggered by drugs such as primaquine and dapsone. In an area where malaria and Hansen\'s disease are still present, knowing the prevalence of this enzyme defect and determining the prevalence of major mutations is important for planning strategies for reducing the risks associated with this enzyme deficiency. Methods: Sectional study was conducted in a Midwestern region of Brazil. Screening for G6PD deficiency was performed in 3,573 neonates. Confirmatory tests were necessary for 188 positively screened children. After confirmation, PCR investigation was utilized to identify the mutations. Results: G6PD deficiency was confirmed in 63 children: 60 boys (95.2%) and 3 girls (4.8%). The percentage of false-positive cases in the screening phase, 66.5% and was associated with the temperature and transportation time of the samples. Family history of anemia and jaundice was more frequent among the children with confirmed G6PD deficiency. An association between a low hematocrit and enzyme deficiency was observed. However, there was no association with hemoglobin reticulocyte or neutrophils counts. The prevalence of G6PD deficiency (CI95%) was 1.76% (1.37; 2.24) among all screened neonates and 3.34% (2.58; 4.25) among male children. The C563T mutation was not identified in any child. The G202A mutation was present in 58 children - 92.06% (CI95%: 83.29 - 97.03), 56/60 boys and 2/3 homozygous girls. One boy with a hemizygous G202A mutation was identified as having Kernicterus. Conclusion: The high percentage of false-positive results when first screening for G6PD deficiency; the long delay time between the test and result; along with the high cost of the this screening test, are all factors that do not support adding this test to the already established Brazilian neonatal screening programs. The prevalence observed among boys does indicate that screening for G6PD deficiency should be performed in this region before the use of drugs such as primaquine and dapsone only in boys. This study found a high prevalence of the G202A mutation, a Class III variant associated with lower morbidity. The identification of a G6PD deficient boy with Kernicterus reinforces the necessity for strategies to abolish the morbidity associated with this enzyme deficiency