Viruses as a source of antiviral peptide-based drugs

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016As patologias causadas por infeções contribuem consideravelmente para o número total de mortes que ocorrem mundialmente. Segundo a organização mundial de saúde, o vírus da Imunodeficiência Humana do tipo 1 (VIH-1) é responsável por cerca de 2.7% destas infeções. O VIH-1 foi identificado em 1981 e pertence, de acordo com a classificação de Baltimore, ao grupo dos vírus de cadeia simples de RNA com DNA intermediário (ssRNA-RT), fazendo parte da família Retroviridae e género Lentivirus. Este retrovírus é o responsável pela síndrome da imunodeficiência adquirida (SIDA) e a sua infeção é caracterizada por causar uma rápida depleção na população de linfócitos T CD4+ do hospedeiro, tornando-o imunocomprometido e vulnerável a infeções oportunistas. Apesar dos esforços da comunidade científica ao longo dos anos, ainda não existe uma vacina ou tratamento totalmente eficaz. Atualmente os tratamentos disponíveis incluem vários medicamentos aprovados para uso clínico e aumentaram significativamente a esperança de vida dos pacientes infetados. O tratamento anti-retroviral (TARV) consiste num regime triplo de fármacos dirigidos a, pelo menos, dois alvos moleculares distintos e hoje em dia, é aceite como a forma mais eficaz de controlar a virémia. No entanto, o potencial deste tipo de terapia é limitado pelo desenvolvimento da resistência do VIH-1 aos fármacos. Assim, o estudo continuado de novos medicamentos eficazes é fundamental para combater estirpes que se tornam resistentes. O desenvolvimento de novos medicamentos peptídicos tem vindo a aumentar nos últimos anos. Os péptidos, tipicamente constituídos por sequências de 2 a 50 resíduos de aminoácidos, podem ser encontrados naturalmente em diferentes organismos onde desempenham várias funções nomeadamente como parte integrante do sistema imunitário. Nos últimos anos tem sido demonstrado que vários péptidos possuem propriedades anti-VIH-1, onde se incluem péptidos que derivam de proteínas do VIH e péptidos antimicrobianos (AMPs). Os AMPs fazem parte da imunidade inata de diversos organismos multicelulares e possuem um largo espetro de ação contra bactérias, fungos e vírus. As defensinas e as catelicidinas são as principais famílias de AMPs com ação conhecida contra o VIH-1. Adicionalmente, são vários os péptidos derivados de proteínas do VIH-1 que possuem atividade antiviral. De entre estes o que mais se destaca é o T20 por ser o único membro da classe dos inibidores de fusão aprovado para uso clínico. Este péptido deriva da glicoproteína do envelope gp41 e é um exemplo de sucesso no desenvolvimento de péptidos antivirais. Os AMPs de origem natural são normalmente encontrados em microorganismos, plantas e animais. No entanto, trabalhos de investigação recentes do nosso laboratório mostraram que sequências de péptidos com atividade antibacteriana podem também ser encontradas em proteínas virais, o que indica que estas proteínas são uma fonte inexplorada de péptidos bioativos. Assim, o trabalho aqui apresentado pretendeu explorar a hipótese de proteínas virais serem também uma fonte de novas sequências de péptidos antivirais (AVPs) e assim contribuir para o desenvolvimento de novos péptidos com atividade anti-HIV-1. A identificação de sequências de aminoácidos com potencial atividade antiviral foi realizada com recurso à ferramenta bioinformática AVPpred utilizando todas as proteínas estruturais de vírus anotadas na Uniprot. Tendo em conta todas as sequências com potencial atividade antiviral, obtidas de acordo com a classificação dos modelos de avaliação da ferramenta AVPpred, treze péptidos foram escolhidos para serem testados experimentalmente contra a estirpe laboratorial VIH-1NL4.3. De forma a entender a influência da carga de cada péptido na sua previsão enquanto AVP, foi necessário traçar um gráfico da carga dos péptidos positivos em função da sua previsão enquanto potencial péptido antiviral. Apesar de empiricamente ser visível uma correlação entre estas duas variáveis, estatisticamente esta não foi significativa. Assim, através desta análise entende-se que a carga dos péptidos antivirais não deverá ter uma influência direta na sua atividade antiviral. Através da realização de ensaios de infeção viral com células suscetíveis na presença de diferentes concentrações dos vários péptidos, foi possível determinar para cada péptido a concentração capaz de inibir cinquenta por cento (IC50) da infeção viral. Estes ensaios foram realizados com células cuja expressão de luciferase é regulada pela proteína viral trans-ativadora da transcrição (Tat). Esta proteína ao ligar-se ao promotor Long Terminal Repeat (LTR) inicia a sua transcrição e, permite a quantificação da infeção viral pela intensidade de luminescência observada, após a conversão de luciferina a oxi-luciferina que é catalisada enzimaticamente pelo luciferase. Através dos resultados obtidos foi possível observar que seis péptidos revelaram uma baixa atividade com um IC50 superior a 100 μM; cinco péptidos apresentaram atividade moderada com um IC50 entre os 12.5 μM e os 100 μM; e os péptidos vAVP 1472 e vAVP 1137 revelaram uma elevada atividade antiviral com um IC50 de 61.6 nM e de 5.63 μM, respetivamente. As curvas de dose-resposta obtidas na determinação da atividade antiviral dos dois péptidos com elevada atividade foram distintas sendo que, para a concentração mais elevada de péptido testada, o vAVP 1472 inibiu na totalidade a infeção viral, enquanto o vAVP 1137 só foi capaz de inibir 80% da infeção viral. Esta diferença pode ser explicada de forma empírica pelo passo do ciclo replicativo do vírus em que os péptidos atuam. O péptido vAVP 1472 ao inibir a infeção nos primeiros passos do ciclo replicativo, antes da integração do genoma viral, não permite a expressão de Tat, pelo que, não é possível haver produção de luciferase, o que se traduz num sinal praticamente nulo. Por outro lado, o péptido vAVP 1137 ao inibir a infeção após o passo de integração do genoma viral, possibilita que haja uma produção residual de luciferase, mesmo estando o péptido a inibir a infeção eficazmente. Comparando os resultados bioinformáticos com os resultados obtidos através dos ensaios antivirais foi possível observar algumas disparidades. Enquanto que a previsão obtida pela ferramenta AVPpred dava os treze péptidos com antivirais, nos resultados dos ensaios in vitro para três dos péptidos testados o seu valor de IC50 foi indeterminado. Os resultados obtidos neste trabalho permitiram identificar dois péptidos derivados de proteínas virais com elevada atividade anti-VIH-1: o péptido vAVP 1472, que provém da proteína da cápside do vírus da Dengue tipo 3, e o péptido vAVP 1137, que provém da proteína da cápside do Coronavírus humano. Atualmente existem duas bases de dados de péptidos antivirais, a HIPdb e a AVPdb, que contêm todas as sequências peptídicas conhecidas de diferentes origens, cuja atividade foi descrita contra o VIH-1, ou outros vírus, respetivamente. Através da análise da informação disponível nestas bases de dados foi possível constatar que a maioria dos péptidos de origem viral é ativo, ou a sua atividade apenas foi descrita contra o vírus de origem. De fato os resultados obtidos para o péptido vAVP 3400 estão de acordo com este princípio. Este péptido que tinha sido descrito previamente como tendo elevada atividade antiviral contra o seu vírus de origem, o vírus sincicial respiratório, VSR, demonstrou apenas atividade moderada contra o VIH-1. No entanto os resultados obtidos com os péptidos que demonstraram elevada atividade anti-VIH-1, o vAVP 1472 e o vAVP 1137, mostram que é possível encontrar péptidos com elevada atividade contra vírus diferentes do vírus que contém as proteínas de onde estes péptidos são derivados. Com base nos resultados obtidos podemos concluir que as proteínas virais têm de fato potencial para serem usadas como fonte de péptidos bioativos, com destaque para a identificação de dois péptidos com elevada atividade anti-VIH-1.The annual number of infections observed worldwide contributes greatly to the total number of deaths, in which the human immunodeficiency virus type 1 (HIV-1) is responsible for 2.7% of them. Even after the breakthroughs observed since its discovery in the early 80’s, currently, about thirtyseven million people live with the virus. According to Baltimore classification, this retrovirus belongs to the group of viruses containing a single strand of RNA with DNA intermediate (ssRNART), and it is responsible for the depletion of the host lymphocytes T CD4+, leading to an immunocompromised state, which is denominated by acquired immune deficiency syndrome (AIDS). The therapies used throughout the years faced different scenarios, from the use of a single inhibitor to the combination of three inhibitors targeting at least two different steps of the viral cycle in single day pill known as highly active antiretroviral therapy (HAART). Despite the improvement observed, the constant development of new drug resistant strains to the presently used inhibitors demand an incessant search for alternative treatments. Peptides appear as good substitutes to small molecules, given their high specificity, and low toxicity. Regarding the combat of viral infections, antiviral peptides (AVPs) have been discovered in the past years. Although the majority are also known as antimicrobial peptides (AMPs), which are characterized by their broad spectrum of action (from bacteria to virus, passing through fungi) with different mechanisms depending on the targeted pathogen, AVPs can also be found in proteins from the virus itself. The Denfensins, and Cathelecidins are two families of AMPs with anti-HIV-1 activity. On the other hand, one of most successful AVPs, the enfuvirtide, is derived from the HIV- 1 envelope protein gp41 and its currently the only anti-HIV-1 fusion inhibitor peptide in the market. Moreover, a recently published research from our lab uncovered the potential of viral proteins as an unexplored source for bioactive peptides. Therefore, the present work intended to scan viral proteins as a source of novel antiviral peptides sequences with anti-HIV-1 activity. The presence of small regions with antiviral propensity (20 residues long) in multifunctional viral proteins was examined using a trained online tool named AVPpred. From these, a pool of thirteen peptides, containing the best single protein derived peptides, was selected according to their antiviral potential determined by the AVPpred models. Moreover, given the importance of the net charge of the peptides to the AMP activity, this property was studied for its influence in AVP prediction. Therefore, a plot of the net charge of each peptide against its antiviral predicted value was made. Empirically was observed a correlation between the two variables. However, statistically the correlation was irrelevant. Finally, the antiviral potential of these was experimentally analyzed to validate the viability of using viral proteins as an antiviral peptidic source. The pseudovirus HIV-1NL4.3 was used to calculate the concentration of peptide needed to inhibit fifty percent (IC50) of the viral infection in a single cycle assay. From the tested peptides, six had low activity (IC50 ≥ 100 μM); five had moderate activity (100 μM > IC50 > 12.5 μM); and the peptides vAVP 1472 and vAVP 1137 were highly active against HIV-1NL4.3 and appeared to have different mechanism of action. The mode of action of these last two peptides was proposed based on their dose-response curves. Despite the high activity of the vAVP 1137, the maximum concentration of peptide used was able to inhibit only 80% of HIV-1 infection, whereas the maximum concentration of vAVP 1472 used was able to completely inhibit the HIV-1 infection. This difference can be explained by different inhibition targets of the peptides in different steps of the viral cycle. The single cycle assay was conducted using cells expressing luciferase, which is regulated by the trans-activator of transcription (Tat) protein, and allows to quantify the efficacy of viral infection. The amount of luciferase expressed was measured by its enzymatic conversion of luciferin to oxyluciferin, which irradiates bioluminescence. The inhibition of the viral infection at early steps of its cycle (before integration) disables the expression of Tat and therefore the expression of luciferase, whereas the inhibition of the viral infection at late steps (post integration) enables a basal expression of luciferase, even with a successful inhibition of viral infection. A comparison between the prediction of AVP by AVPpred and the results obtained through the in vitro assays was made. In opposite to the prediction, three peptides did not reveal any anti-HIV-1 activity. Instead, these peptides promoted the viral infection in a dose dependent manner. The results obtained with the present work showed that the peptide derived from the capsid protein of Dengue virus type 3, vAVP 1472, and the peptide derived from the capsid protein of Human coronavirus, vAVP 1137 were highly active against HIV-1. From two databases present in the literature, containing peptides from diverse origins with described activity against HIV-1 (HIPdb), and other viruses (AVPdb) was observed that the majority of the peptides derived from viral proteins targeted their virus of origin. The results obtained for the vAVP 3400 supported this observation, since the peptides was already described with anti-respiratory syncytial virus activity (the virus of origin), and its activity against HIV-1 was moderate. Nevertheless, with the results obtained for the highly active peptides, the spectrum of action of these peptides was increased. In conclusion, the potential of using viral proteins as antiviral peptides was confirmed, giving more insights for the use of antiviral peptides

Motricidade Infantil: estratégias para recreios escolares ativos

O presente relatório encontra-se inserido no âmbito da unidade curricular de Estágio em Motricidade Infantil enquanto Fisiologista do Exercício realizado na Cooperativa de Ensino a Torre e Ginásio Clube Português com a duração de um ano letivo académico. Com este documento, pretende dar-se a conhecer ao leitor a problemática da promoção da atividade física e implementação de programas de exercício físico na criança, assim como o enquadramento da prática profissional de um fisiologista do exercício nesta área, reforçando o papel do exercício no desenvolvimento de competências motoras e de comportamentos ativos nesta população. Posteriormente, é relatada a experiência nos diferentes contextos observados ao longo do estágio. Para cada local, é efetuada a sua caracterização e a descrição do funcionamento de cada tipo de aula. No caso da Cooperativa de Ensino a Torre, as aulas estão inseridas no programa educativo da escola, enquanto no Ginásio Clube Português, as aulas são extracurriculares e dizem respeito a uma classe de iniciação focada essencialmente na formação para a progressão gímnica dos seus alunos. Assim, este registo tem como objetivo apresentar o trabalho realizado ao longo de um ano letivo de estágio, expondo as várias tarefas propostas e executadas, o conhecimento adquirido, os desafios complementares realizados e ainda o contributo pessoal desenvolvido para uma das instituições e a sua aplicação futura.This report is part of the curricular unit of internship in Childhood Motricity as Exercise Physiologist held at Cooperativa de Ensino a Torre and Ginásio Clube Português for the duration of one academic year. With this document, the aim is to make the reader aware of the problem of promoting physical activity and the implementation of physical exercise programs for children, as well as the professional practice framing of an exercise physiologist in this area, reinforcing the role of exercise in the Development of motor skills and active behaviors in this population. Subsequently, experience is reported in the different contexts observed throughout the internship. For each place, its characterization and the description of the operation of each type of class is carried out. In the case of Cooperativa de Ensino a Torre, the classes are included in the school's educational program, while at GCP, the classes relate to an extracurricular class focused essentially on gymnastic for beginners. The purpose of this report is to present the work carried out during a one-year internship, exposing the various tasks proposed and performed, the knowledge acquired, the complementary challenges and the personal contribution made to one of the institutions and their application in the future

Laparoscopic Upper-pole Nephroureterectomy In Infants

Objective: Report the results of laparoscopic upper-pole nephroureterectomy in infants. Materials and Methods: Six consecutive infants underwent 7 laparoscopic upper-pole nephroureterectomy. Pre and post-operative evaluation included renal sonography, voiding cystourethrogram and renal scintigraphy. All infants showed upper-pole exclusion. Surgery was performed through a transperitoneal approach with full flank position in all infants. Three or 4 ports were used according to the necessity of retracting the liver. The distal ureter was ligated close to the bladder whenever reflux was present and the dysplastic upper-pole was divided with the help of an electrocautery. Data regarding operative time, postoperative use of analgesics, time to resume oral feeding, hospital stay and tubular function were collected and analyzed. Results: All procedures were concluded as planned. Mean operative time was 135 min. One patient underwent staged bilateral upper-pole nephrectomy. There were no complications and the postoperative hospital stay was 48 hours in 5 procedures and 24 hours in 2 procedures. Pain medication was required only in the first day. Renal tubular function showed improvement in half of the cases. Conclusion: Laparoscopic partial nephrectomy is a safe and feasible procedure in infants. Due to the magnification provided by the lenses, a better vision of the structures is achieved, facilitating selective dissection of vascular upper-pole, renal parenchyma and distal ureter. This approach is less damaging to the lower pole, and is associated to low morbidity and a short hospital stay.3318791Peters, C.A., Laparoscopic and robotic approach to genitourinary anomalies in children (2004) Urol Clin North Am, 31, pp. 595-605Robinson, B.C., Snow, B.W., Cartwright, P.C., De Vries, C.R., Hamilton, B.D., Anderson, J.B., Comparison of laparoscopic versus open partial nephrectomy in a pediatric series (2003) J Urol, 169, pp. 638-640Steyaert, H., Valla, J.S., Minimally invasive urologic surgery in children: An overview of what can be done (2005) Eur J Pediatr Surg, 15, pp. 307-313Koyle, M.A., Woo, H.H., Kavoussi, L.R., Laparoscopic nephrectomy in the first year of life (1993) J Pediatr Surg, 28, pp. 693-695Valla, J.S., Breaud, J., Carfagna, L., Tursini, S., Steyaert, H., Treatment of ureterocele on duplex ureter: Upper pole nephrectomy by retroperitoneoscopy in children based on a series of 24 cases (2003) Eur Urol, 43, pp. 426-429Jordan, G.H., Winslow, B.H., Laparoendoscopic upper pole partial nephrectomy with ureterectomy (1993) J Urol, 150, pp. 940-943Horowitz, M., Shah, S.M., Ferzli, G., Syad, P.I., Glassberg, K.I., Laparoscopic partial upper pole nephrectomy in infants and children (2001) BJU Int, 87, pp. 514-516Gill, I.S., Delworth, M.G., Munch, L.C., Laparoscopic retroperitoneal partial nephrectomy (1994) J Urol, 152, pp. 1539-1542Borzi, P.A., A comparison of the lateral and posterior retroperitoneoscopic approach for complete and partial nephroureterectomy in children (2001) BJU Int, 87, pp. 517-520Desgrandchamps, F., Gossot, D., Jabbour, M.E., Meria, P., Teillac, P., Le Duc, A., A 3 trocar technique for transperitoneal laparoscopic nephrectomy (1999) J Urol, 161, pp. 1530-1532Hulbert, W.C., Rabinowitz, R., Prenatal diagnosis of duplex system hydronephrosis: Effect on renal salvage (1998) Urology, 51, pp. 23-26El-Ghoneimi, A., Farhat, W., Bolduc, S., Bagli, D., McLorie, G., Khoury, A., Retroperitoneal laparoscopic vs open partial nephroureterectomy in children (2003) BJU Int, 91, pp. 532-535Jednak, R., Kryger, J.V., Barthold, J.S., Gonzalez, R., A simplified technique of upper pole heminephrectomy for duplex kidney (2000) J Urol, 164, pp. 1326-1328Borzi, P.A., Yeung, C.K., Selective approach for transperitoneal and extraperitoneal endoscopic nephrectomy in children (2004) J Urol, 171, pp. 814-816Guillonneau, B., Ballanger, P., Lugagne, P.M., Valla, J.S., Vallancien, G., Laparoscopic versus lumboscopic nephrectomy (1996) Eur Urol, 29, pp. 288-291Janetschek, G., Seibold, J., Radmayr, C., Bartsch, G., Laparoscopic heminephroureterectomy in pediatric patients (1997) J Urol, 158, pp. 1928-193

Cross-sectional study comparing different therapeutic modalities for cystic lymphangiomas in children

OBJECTIVE: Here, we describe our experience with different therapeutic modalities used to treat cystic lymphangiomas in children in our hospital, including single therapy with OK-432, bleomycin and surgery, and a combination of the three modalities. METHODS: We performed a retrospective, cross-sectional study including patients treated from 1998 to 2011. The effects on macrocystic lymphangiomas and adverse reactions were evaluated. Twenty-nine children with cystic lymphangiomas without any previous treatment were included. Under general anesthesia, patients given sclerosing agents underwent puncture of the lesion (guided by ultrasound when necessary) and complete aspiration of the intralesional liquid. The patients were evaluated with ultrasound and clinical examinations for a maximum follow-up time of 4 years. RESULTS: The proportions of patients considered cured after the first therapeutic approach were 44% in the surgery group, 29% in the bleomycin group and 31% in the OK-432 group. These proportions were not significantly different. Sequential treatment increased the rates of curative results to 71%, 74% and 44%, respectively, after the final treatment, which in our case was approximately 1.5 applications per patient. CONCLUSION: The results of this study indicate that most patients with cystic lymphangiomas do not show complete resolution after the initial therapy, regardless of whether the therapy is surgical or involves the use of sclerosing agents. To achieve complete resolution of the lesions, either multiple operations or a combination of surgery and sclerotherapy must be used and should be tailored to the characteristics of each patient

Cross-sectional Study Comparing Different Therapeutic Modalities For Cystic Lymphangiomas In Children.

Here, we describe our experience with different therapeutic modalities used to treat cystic lymphangiomas in children in our hospital, including single therapy with OK-432, bleomycin and surgery, and a combination of the three modalities. We performed a retrospective, cross-sectional study including patients treated from 1998 to 2011. The effects on macrocystic lymphangiomas and adverse reactions were evaluated. Twenty-nine children with cystic lymphangiomas without any previous treatment were included. Under general anesthesia, patients given sclerosing agents underwent puncture of the lesion (guided by ultrasound when necessary) and complete aspiration of the intralesional liquid. The patients were evaluated with ultrasound and clinical examinations for a maximum follow-up time of 4 years. The proportions of patients considered cured after the first therapeutic approach were 44% in the surgery group, 29% in the bleomycin group and 31% in the OK-432 group. These proportions were not significantly different. Sequential treatment increased the rates of curative results to 71%, 74% and 44%, respectively, after the final treatment, which in our case was approximately 1.5 applications per patient. The results of this study indicate that most patients with cystic lymphangiomas do not show complete resolution after the initial therapy, regardless of whether the therapy is surgical or involves the use of sclerosing agents. To achieve complete resolution of the lesions, either multiple operations or a combination of surgery and sclerotherapy must be used and should be tailored to the characteristics of each patient.69505-

The Role Of Videolaparoscopy In The Diagnostic And Therapeutic Approach Of Nonpalpable Testis.

Evaluate the results from the first 5 years of experience with laparoscopy for diagnosis and treatment of nonpalpable testes. Medical records of 51 patients submitted to laparoscopic testicular exploration, during a 5-year period, were retrospectively analyzed. Patients' mean age was 65.7 months (median = 48) on the first procedure. The youngest patient was 10 months and the oldest was 14 years old on the first surgery. Twenty-four (47%) patients presented nonpalpable testes bilaterally, 7 (14%) only at the right side and 20 (39%) at the left, totaling 75 testicular units assessed. Patients who had their testes palpated after anesthetic induction were excluded from the study, and in all other cases, surgical management was based on the testicular position and viability. During the post-operative follow-up, surgical success was classified as palpable testis in scrotal sac, with adequate consistency and volume. Nine (12%) testes were not localized, but their vessels and deferent duct were atrophic. Two (3%) testes were intra-abdominal and atrophic, and 2 (3%) gonads, in the same patient, had a dysmorphic aspect. Nineteen (25%) testicular units were located close to the internal inguinal ring (peeping testes) and, in 22 (29%) units, the spermatic vessels and deferent duct penetrated the internal inguinal ring. Eight (10%) testes were located at a distance of less than 2 cm from the internal inguinal ring and 13 (17%) at a distance greater than 2 cm. The 2 intra-abdominal atrophic testes were removed. Inguinotomy was performed in a total of 41 (54%) cases, reaching a surgical success of 89%. Laparoscopic orchiopexy in one stage, without vascular ligation, was performed in 9 (12%) testes, which presented a distance of less than 2 cm from the internal inguinal ring, also with a surgical success index of 89%. Orchiopexy in 2 stages, with ligation of the spermatic vessels, was performed in 13 (17%) testicular units located at a distance greater than 2 cm from the internal inguinal ring, reaching 77% of good results. Videolaparoscopy is a safe and effective method for diagnosis and treatment of nonpalpable testis.29345-51; discussion 351-

[role Of Anorectal Manometry In The Differential Diagnosis Of Chronic Constipation In Children]

OBJECTIVES: 1. To evaluate the role of anorectal manometry in recognizing patients with histological abnormalities of the myenteric plexus as the cause of chronic constipation. 2. To correlate clinical features with the etiology of the constipation.METHODS: The etiology of the constipation was identified retrospectively from the medical records of 57 patients submitted to anorectal manometry. The test was performed in the Pediatric Gastroenterology Laboratory of the University Hospital of the State University of Campinas Medical School. The procedure was performed using a single-balloon system in which a water-perfused pressure transducer was interfaced with a polygraph and displayed on a computer screen. Manometry was performed in patients who, after 6 to 23 months of intense medical treatment using osmotic laxatives and dietary fibres (20 g/d), continued to develop fecal impaction and/or needed evacuatory enema. Regularization of bowel movements during at least 24 months follow-up or the visualization of abnormalities in myenteric plexus in rectal biopsy were used as reference points for the definitive diagnosis.RESULTS: Rectoanal reflex was present in 44 of the 57 patients (77%). Thirteen patients with a negative reflex underwent rectal biopsy which showed abnormalities of the ganglion cells in 10. Subsequent manometry performed on those three patients revealed a normal rectoanal reflex. Sensitivity, specificity, positive and negative predictive values of the manometric test were 1.0, 0.94, 0.77 and 1.0, respectively.CONCLUSIONS: Anorectal manometry is a diagnostic technique with very small possibility of error in differential diagnosis between constipation of a chronic functional nature and that which is secondary to ganglion cell abnormalities.76227-3

Does staged closure have a worse prognosis in gastroschisis?

INTRODUCTION: Correction of gastroschisis can be accomplished by primary or staged closure. There is, however, no consensus regarding the best approach or criteria to favor one method over the other has been established. OBJECTIVE: To compare the outcome of primary and staged closure in newborns with gastroschisis using intravesical pressure (IVP) as the decision criterion. PATIENTS & METHODS: We prospectively analyzed 45 newborns with gastroschisis. An IVP with a threshold of 20 cm H2O was used to indicate primary or staged closure, and the outcomes between the two methods were compared. RESULTS AND DISCUSSION: Newborns in whom primary closure was feasible were born at a lower gestational age. There was no significant difference in the frequency of complications, time to begin oral feeding, length of parenteral nutrition or length of hospital stay. Compared with previous reports, our data showed higher rates of prenatal diagnosis and cesarean delivery, a lower average birth weight, a higher rate of small gestational age babies and a more frequent association with intestinal atresia. Conversely, our data showed a lower rate of postoperative necrotizing enterocolitis and a lower average length of hospital stay. CONCLUSION: No significant difference was observed in the outcome of newborns who underwent primary closure or staged closure of gastroschisis when using an IVP below 20 cm H2O as the criterion for primary closure

BABIES WITH BRAIN DAMAGE WHO CAN NOT SWALLOW Surgical management

Background: Neonates with severe neurological impairment are often unable to swallow, necessitating gastrostomy for feeding. Because of the risk of developing severe reflux, this procedure is often associated with fundoplication. Objective: To assess the safety and efficacy of gastrostomy and Nissen fundoplication in 22 neonates with swallowing difficulties due to serious neurological impairment. Method: All children underwent an initial period of nasogastric feeding and after informed consent underwent gastrostomy and Nissen fundoplication. Results: There were no significant intraoperative complications. There were two cases of postoperative periostomy leakage. Of the 22 neonates 16 were alive four months after surgery. Six neonates died of complications due to underlying disease. Conclusion: We concluded that gastrostomy and Nissen fundoplication are safe procedures and help parents give a better care to these children.663B64164