A validated HPLC method for the monitoring of thiopurine metabolites in whole blood in paediatric patients with inflammatory bowel disease.

Therapeutic drug monitoring (TDM) of major metabolites of thiopurine drugs is a widely used tool for assessing treatment efficacy and toxicity in patients with inflammatory bowel disease (IBD). We report the laboratory and clinical validation of a simple and reliable high performance liquid chromatography (HPLC) method for the measurement of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) on paediatric patients with IBD. The aim of this paper is to develop and validate a method for the measurement of 6-TGN and 6-MMP applicable to routine practice and to evaluate the usefulness of the TDM of thiopurine drugs in children with IBD attending our Gastroenterology Unit. The HPLC method was validated following international guidelines starting from red blood cells (RBC) and whole blood (WB). A comparison between RBC and WB was assessed. The usefulness of TDM was then evaluated using the new method from WB in 47 paediatric patients with IBD treated with thiopurine drugs. WB and RBC resulted in interchangeable matrices. The majority of patients had the metabolite levels inside the therapeutic ranges. A moderate correlation was found between 6-MMP concentration and the dose of thiopurines. A higher percentage of non responders was found among patients with lower levels of 6-TGN. Toxicity was found in eight patients and was evaluated in respect to the metabolite concentration. The described HPLC method is applicable to routine practice and it is suitable for its use in multicentric studies. Our results of TDM on paediatric IBD patients can contribute to clarify its role in their therapeutic management.

Megacystis, megacolon, and malrotation: a new syndromic association?

Chronic intestinal pseudo-obstruction (CIPO) can occur as a consequence of neuropathies including diffuse Intestinal Neuronal Dysplasia (IND), a relatively rare enteric nervous system (ENS) abnormality. Although various authors reported of diffuse IND associated either with intestinal malrotation or megacystis, the co-existence of these three entities in the same patient has never been described before. The aim of this paper is to report for the first time in literature a series of patient with such association, focusing on one who carries a de novo duplication of chromosome 12, suggesting a new syndromic association (megacolon, megacystis, malrotation).

TPMT genotype and the use of thiopurines in paediatric inflammatory bowel disease.

BACKGROUND: Thiopurines are used in the treatment of inflammatory bowel disease. They are metabolised via methylation by thiopurine-S-methyltransferase (TPMT), which displays a genetically determined polymorphic activity. Subjects with reduced TPMT activity have a higher concentration of active thiopurine metabolites and may be at increased risk of bone-marrow suppression. AIMS: To evaluate the relevance of TPMT genotyping in the management of thiopurines therapy in inflammatory bowel disease patients. PATIENTS AND METHODS: Adverse effects and clinical response were determined retrospectively and correlated with TPMT genotype in 70 paediatric inflammatory bowel disease patients. RESULTS: Nineteen patients (27.1%) developed adverse effects; of the 51 who did not, 34 (66.7%) responded to treatment. Five patients (7.1%) were heterozygous for a variant TPMT allele; two of these (40%) were intolerant to thiopurines, compared to 17 of the 65 patients (26.2%) with a wild type gene (O.R. 1.88, 95% CI 0.29-12.2, p=0.61); among the 34 responders, the median dosage of the drug required to obtain remission was lower for mutated than for wild type patients (1.6mgkg(-1)day(-1) versus 2.0mgkg(-1)day(-1), p=0.043). CONCLUSIONS: There was no significant association between adverse effects of thiopurines and TPMT heterozygous genotype, but TPMT genotyping could be useful in establishing the most appropriate dose of thiopurines to start treatment.

Usefulness of omega-3 fatty acid supplementation in addition to mesalazine in maintaining remission in pediatric Crohn's disease: a double-blind, randomized, placebo-controlled study.

AIM: To assess the value of long-chain omega-3 fatty acids (FAs) supplementation in addition to amino-salicylic-acid (5-ASA) in pediatric patients with Crohn's disease (CD). METHODS: Thirty-eight patients (20 males and 18 females, mean age 10.13 years, range 5-16 years) with CD in remission were randomized into two groups and treated for 12 mo. Group I (18 patients) received 5-ASA (50 mg/kg/d)+ omega-3 FAs as triglycerides in gastro-resistant capsules, 3 g/d (eicosapentanoic acid, EPA, 400 mg/g, docosahexaenoic acid, DHA, 200 mg/g). Group II (20 patients) received 5-ASA (50 mg/kg/d)+olive oil placebo capsules. Patients were evaluated for fatty acid incorporation in red blood cell membranes by gas chromatography at baseline 6 and 12 mo after the treatment. RESULTS: The number of patients who relapsed at 1 year was significantly lower in group I than in group II (P<0.001). Patients in group I had a significant increase in the incorporation of EPA and DHA (P<0.001) and a decrease in the presence of arachidonic acids. CONCLUSION: Enteric-coated omega-3 FAs in addition to treatment with 5-ASA are effective in maintaining remission of pediatric CD.

Congenital esophageal stenosis diagnosed in an infant at 9 month of age.

Esophageal stenosis is a relatively uncommon condition in pediatrics and requires an accurate diagnostic approach. Here we report the case of a 9-month old female infant who presented intermittent vomiting, dysphagia and refusal of solid foods starting after weaning. She was treated for gastroesophageal reflux. At first, radiological investigation suggested achalasia, while esophagoscopy revelaed a severe congenital esophageal stenosis at the distal third of the esophagus. She underwent four endoscopic balloon dilatations that then allowed her to swallow solid food with intermittent mild dysphagia. After 17 months of esomeprazole treatment off therapy impedance-pH monitoring was normal. At 29 months of follow-up the child is asymptomatic and eats without problems.Infants with dysphagia and refusal of solid foods may have undiagnosed medical conditions that need treatment. Many disorders can cause esophageal luminal stricture; in the pediatric age the most common are peptic or congenital. Careful assessment with endoscopy is needed to diagnose these conditions early and referral to a pediatric gastroenterologic unit may be necessary.

Paediatric ulcerative colitis surgery: Italian survey.

BACKGROUND AND AIMS: Recent epidemiological studies showed an increase in ulcerative colitis among children, especially in its aggressive form, requiring surgical treatment. Although medical therapeutic strategies are standardized, there is still no consensus regarding indications, timing and kind of surgery. This study aimed to define the surgical management of paediatric ulcerative colitis and describe attitudes to it among paediatric surgeons. METHODS: This was a retrospective cohort study. All national gastroenterology units were invited to participate. From January 2009 to December 2013, data on paediatric patients diagnosed with ulcerative colitis that required surgery were collected. RESULTS: Seven units participated in the study. Seventy-one colectomies were performed (77.3% laparoscopically). Main surgical indications were a severe ulcerative colitis attack (33.8%) and no response to medical therapies (56.3%). A three-stage strategy was chosen in 71% of cases. Straight anastomosis was performed in 14% and J-pouch anastomosis in 86% of cases. A reconstructive laparoscopic approach was used in 58% of patients. Ileo-anal anastomosis was performed by the Knight-Griffen technique in 85.4% and by the pull-through technique in 9.1% of patients. Complications after colectomy, after reconstruction and after stoma closure were reported in 12.7, 19.3 and 35% of cases, respectively. CONCLUSIONS: This study shows that there is general consensus regarding indications for surgery. The ideal surgical technique remains under debate. Laparoscopy is a procedure widely adopted for colectomy but its use in reconstructive surgery remains limited. Longer follow-up must be planned to define the quality of life of these patients.

Circadian variations of autologous mixed lymphocyte reactions and endogenous cortisol.

The degree of proliferation of human T cells stimulated with autologous PHA-T cells and with autologous non-T cells displays circadian variations. The highest proliferation occurs with cells isolated from blood drawn at 8 a.m. in mixed lymphocyte reactions (MLR) with autologous PHA-T cells and from blood drawn at 8 p.m. in MLR with autologous non-T cells. The circadian variations of autologous MLRs appear to reflect changes in the proliferative response of T cells. In autologous MLRs with non-T cells as stimulators the extent of proliferation was inversely correlated with the level of endogenous cortisol. The circadian variations of autologous MLRs do not reflect non-specific changes in the proliferative and stimulatory properties of T and non-T cells, since circadian variations were not observed in the proliferative response of T cells to mitogens and in allogeneic MLRs. Circadian variations of autologous MLRs must be taken into account when analyzing abnormalities of these reactions in pathological conditions.

Report on two patients with Costello syndrome and sialuria.

We report on 2 unrelated patients with Costello syndrome. The first is a 5-year-old girl with "coarse" face, nasal papillomata, redundant skin of feet and hands, hyperextensible hand and finger joints, curly hair, feeding problems due to oral motor apraxia, growth and psychomotor retardation. The second is a 3-year-old boy with "coarse" face, loose skin on hands and feet, curly hair, oral motor apraxia, severe growth and psychomotor retardation. In both patients urine sialic acid levels were found to be repeatedly high. The meaning of this biochemical abnormality is discussed.

Arthrogryposis, cholestatic pigmentary liver disease and renal dysfunction: report of a second family.

We report on a boy, born to consanguineous parents, who had arthrogryposis, cholestatic liver disease, and renal dysfunction. The child died at age 2 months, and autopsy showed pigmentary storage disease in liver cells, nephrocalcinosis, and rarefaction of motor neuron cells in the anterior horns of spinal cord. This association, reported in 1979 by Nezelof et al., is a distinctive syndrome. The possibility of an autosomal recessive or an X-linked inheritance is discussed.

Azathioprine in paediatric inflammatory bowel disease: an Italian multicentre survey.

AIM: To assess the efficacy and safety of azathioprine in a paediatric population with inflammatory bowel disease. PATIENTS AND METHODS: One hundred and twenty-three Italian children treated with azathioprine were studied retrospectively. The treatment duration and causes of its discontinuation, side-effects and variation in corticosteroid dose were assessed. RESULTS: The mean age at inflammatory bowel disease diagnosis was 9.8 +/- 3.6 years, and at the start of azathioprine therapy 11.8 +/- 4.3 years. The mean duration of treatment was 19 +/- 16 months. Fifty patients (41%) stopped treatment due to surgery (12%), prolonged remission (11%), non-response (7%), severe side-effects (7%) and poor compliance (3%). Of the 73 patients (59%) remaining on azathioprine, 11 had never been treated with corticosteroids, 27 were able to stop them and 35 were still on a very low daily dose (91% < 0.3 mg/kg). The difference in the daily corticosteroid dose between the beginning of azathioprine treatment (1 +/- 0.6 mg/kg) and the conclusion of the study (0.18 +/- 0.16 mg/kg) was statistically significant. Side-effects were recorded in 48 of the 123 patients (39%), but only eight required discontinuation of azathioprine. CONCLUSIONS: Azathioprine was efficacious in 70% of patients, but ineffective in 20% and induced severe toxicity in 7%. Corticosteroids were stopped or markedly reduced in 62% of patients, but they were never given in 9%.

Response to infliximab is related to disease duration in paediatric Crohn's disease.

BACKGROUND: Infliximab is an effective therapy in adult patients with refractory and fistulizing Crohn's disease. Experience in children is still limited. AIM: : To evaluate the experience in 22 children and adolescents treated with infliximab with refractory and/or fistulizing Crohn's disease, and to compare duration of response in children between early Crohn's disease and late Crohn's disease. METHODS: The experience in 22 children and adolescents treated with a total of 73 infusions was evaluated retrospectively. Treatment indication was refractory Crohn's disease in 9/22 patients, fistulizing Crohn's disease in 7/22 patients and both these conditions in 6/22. All patients with refractory Crohn's disease had late Crohn's disease (> 1 year), whereas 6/13 patients with fistulas had early disease (< 1 year). RESULTS: Mean Paediatric Crohn's Disease Activity Index (PCDAI) decreased from 41.2 to 16.2 at 4 weeks (P < 0.01), and to 15.4 at 18 weeks (P < 0.01). Mean PCDAI at 18 weeks in children with early Crohn's disease and late Crohn's disease was 5.5 and 18.1, respectively (P < 0.05). Complete closure of fistulas was obtained in 5/6 children with early Crohn's disease and in 2/7 children with late Crohn's disease. Immediate adverse reactions were observed in two children. CONCLUSIONS: Infliximab is a highly effective treatment in children and adolescents with both severe refractory or fistulizing Crohn's disease. Children with early Crohn's disease have a higher chance of prolonged response to infliximab than children with late Crohn's disease.

The use of ciclosporin in paediatric inflammatory bowel disease: an Italian experience.

AIM: To asses the efficacy and safety of ciclosporin in a paediatric population with inflammatory bowel disease. PATIENTS AND METHODS: Twenty-three Italian children treated with ciclosporin were studied retrospectively. The indications for treatment were severe unresponsive colitis, chronic active colitis or severe fistulizing Crohn's disease. The treatment duration, follow-up and causes of drug discontinuation were assessed. RESULTS: Sixteen patients were treated intravenously for a mean time of 10 +/- 7 days (1-24 days) and 19 orally for a mean time of 133 days (17-660 days). The mean follow-up of all patients was 13.2 months. Ciclosporin was totally ineffective, being discontinued for surgery, in nine of 23 patients (39%); it was discontinued for partial response in three patients (13%). During treatment, clinical remission was achieved in eight children (35%) and maintained after drug withdrawal in four (17%). In severe unresponsive colitis, urgent colectomy was avoided in 12 (85%) of 14 patients who tolerated the drug. Side-effects appeared in six of 23 patients (26%), and three (13%) required ciclosporin to be discontinued due to neurotoxicity. CONCLUSIONS: Ciclosporin shows disappointing long-term results in the treatment of refractory inflammatory bowel disease, but can play an important role in preventing urgent surgery in unresponsive severe colitis. Severe side-effects can occur.

Scanning electron microscopy study of small bowel biopsies in chronic diarrhoea in childhood.

In this study we have compared the results of Scanning Electron Microscopy (SEM) with Light- and Stereomicroscopy in a series of small bowel biopsies in children. In 9 cases displaying features of partial or subtotal atrophy, Light and Dissecting-Microscopy yielded similar results. The distinction between coeliac and non-coeliac chronic diarrhoea was only possible on clinical grounds, and by the immunological detection of specific antibodies. On SEM however coeliac patients showed characteristic alterations consisting of: absence of villi; prominent crypt outlets resulting in a mosaic appearance; concentric furrows running all around the openings; and downy brush feature at high power. The microvilli were loosely distributed and had an irregular pleomorphic outline; they often displayed a drumstick swelling of the tip and were bent. In contrast, non-coeliac chronic diarrhoea cases were characterized by a thick mucous layer on the mucosal surface, that made it impossible to visualize further changes. Peculiar vascular changes in lymphangiectasia and in sickle beta thalassemia could be detected only by Light Microscopy. In addition, in the lymphangiectasia case SEM allowed the detection of enteroadherent bacteria; and in the lambliasis case, of pseudomembranes. Absence of glycocalyx was noted both in controls and in patients. The results of this study point to a diagnostic utility of SEM particularly in the differential diagnosis of chronic diarrhoea; moreover they suggest that enteroadherent bacteria may not be pathogenic and that the absence of glycocalyx is not specific for allergic enteropathy as previously claimed.

Increased gut permeability in juvenile chronic arthritides. A multivariate analysis of the diagnostic parameters.

OBJECTIVE: This study was aimed at evaluating intestinal permeability (IP) in patients with oligoarticular juvenile idiopathic arthritis (o-JIA), spondyloarthropathy (SpA) associated with inflammatory bowel disease (IBD) and other forms of juvenile-onset chronic arthritiis (OIA) using the lactulose/mannitol (L/M) test in comparison with other non-invasive parameters of gut involvement. METHODS: A series of 26 children affected with o-JIA and 14 with either SpA/IBD or OIA were assessed for IP. The urinary L/M ratio was measured by gas chromatography. The erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and faecal alpha 1 antitrypsin concentrations were also evaluated. Ten o-JIA patients displayed active arthritis while in 16 the disease was under control. Among the OIA patients, 11 were affected with psoriatic arthritis and the remaining 3 with chronic reactive arthritis. 14 patients with SpA-IBD had active synovitis or spine inflammation. 14 eo-pJCA and 22 OIA and SpA-IBD patients, respectively, were receiving NSAID therapy. RESULTS: The mean L/M ratios for the Spa-IBD (0.07 +/- 0.02, mean +/- SD), OIA (0.05 +/- 0.02) and o-JIA (0.04 +/- 0.02) patients were significantly higher (p < 0.001, p = 0.022 and p = 0.01, respectively) than those found in controls (0.02 +/- 0.01). Logistic regression analysis disclosed a positive correlation between the L/M ratio and the presence of gastrointestinal manifestations (p = 0.011). The type of disease (p = 0.28), the disease activity in the JCA patient group (p = 0.24) and NSAID administration (p = 0.210) did not seem to significantly influence the L/M ratio. CONCLUSIONS: All of the subtypes of juvenile chronic arthritides that we studied displayed an increased IP. Hence, gut wall inflammation (albeit asymptomatic) may also be present in o-JIA patients. The SpA-IBD patients with gastrointestinal symptoms displayed the highest mean L/M ratio values. The L/M test seemed to correlate with histopathological features of the gut mucosa. The L/M ratio was shown to be a highly sensitive but poorly specific test for predicting gut inflammatory disease compared to other non-invasive screening tests.

99mTc-white cell scanning to detect gut inflammation in children with inflammatory bowel diseases or spondyloarthropathies.

OBJECTIVE: Gut inflammation is a common feature shared by inflammatory bowel diseases (IBD) and the spondyloarthropathies (SpA). The aim of the present study was to compare the reliability of a number of non-invasive investigations for the detection of an inflammatory process of the intestine. METHODS: Forty-two children were studied: (i) patients with a previous diagnosis of IBD (group A); (ii) patients with suspected IBD (group B); and (iii) patients with predominantly rheumatological manifestations associated with gastrointestinal symptoms (group C). All the patients were studied using 99mTechnetium-HMPAO labelled white cell scanning (99mTc-WCS), and abdominal ultrasound (US). In addition the ESR, serum class A immunoglobulins, faecal occult blood (OB) and faecal alpha 1-antitripsin level (F alpha 1-AT) were determined. Colonoscopy plus multiple biopsies and radiological study of the intestine were used as gold standards. RESULTS: 99mTc-WCS showed the highest sensitivity (85%) and specificity (100%) in detecting the presence of gut inflammation. This was followed by ESR and faecal occult blood (63% sensitivity, 44% specificity), F alpha 1-AT (43% sensitivity, 44% specificity) and IgA (42% sensitivity, 88% specificity). Ultrasound was informative in 28% of the active/affected patients, with a specificity of 75%. CONCLUSION: Although ileo-pancolonscopy remains the gold standard for the histological characterisation of gut inflammation, 99mTc-WCS represents the most reliable non-invasive test for its detection.

A girl with severe fistulizing Crohn's disease.

We report the case of a girl with a severe perianal fistulizing Crohn's disease who during intravenous infusion of cyclosporine developed headache, general seizures and cortical blindness. Head magnetic resonance imaging showed spread, cortical-subcortical nonenhancing signal changes. Full neurological recovery was achieved in 24 hours. Cyclosporine was stopped and a single dose of infliximab was infused with rapid improvement of the fistula that at 7 months' follow-up is still closed.

Severe attack of ulcerative colitis in children: retrospective clinical survey.

AIM: To obtain clinical data concerning severe attacks of ulcerative colitis in children. PATIENTS AND METHODS: A retrospective chart review of 37 children with ulcerative colitis was carried out in order to assess the prevalence, risk factors, timing of presentation, and outcome of severe attacks of ulcerative colitis. RESULTS: A total of 20 severe attacks occurred in 15 out of the 37 patients. No difference in the occurrence of severe attacks was detected in relation to age or disease extent at diagnosis. The mean interval between disease diagnosis and a severe attack was 9.1 months (range 0-30). Of the 20 severe attacks, 11 were resolved with medical treatment in a mean time of 1 1 +/- 4.6 days while 9 out of 20 needed urgent surgery in a mean time of 7.4 +/- 4.8 days. Of 10 out of the 15 patients who recovered from the first attack 4 required colectomy after a mean time of 6.7 months, another 4 are still in remission at a mean period of 40.7 months, one needed elective surgery after 25 months and one was lost to follow-up. CONCLUSIONS: Severe attacks of ulcerative colitis had a high prevalence rate [40%); age and disease extent at presentation were not predictors of their occurrence. Approximately half the attacks resolved with medical treatment alone, while the other half required emergency surgery. After successful medical treatment of the first attack, 40% of children maintained long-term remission, while 40% required early colectomy

T-lymphocytes phenotype and functions in patients with head and neck cancer.

We studied the phenotype of T-lymphocytes isolated from 18 patients with head and neck cancer, their capacity to express Ia antigens upon activation by lectins in vitro, their capacity to function either as responder or stimulator cells in autologous mixed lymphocyte reaction, and their capacity to cooperate with the normal adherent suppressor cells (NASC). The T-lymphocytes isolated from these patients have several functional defects including an impaired capacity to activate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs), a lack of a proliferative capacity in autologous MLRs, an impaired sensitivity to inhibition by NASC, and an impaired capacity to express Ia antigens upon activation by mitogens in vitro. These data indicate that, in patients with head and neck cancer, immune function is characterized by a defect in T-lymphocytes functions which concerns the process of cell to cell cooperation.

Medium-term results after cholecystectomy in patients younger than 10 years.

BACKGROUND: Gallbladder stones are a well-known and widely studied problem in children. Hematological disorders are the most common diseases that can cause cholelithiasis. However, in the last few years, the proportion of children with idiopathic cholelithiasis has increased 50%. Herein, we present a prospective study on laparoscopic cholecystectomy in a selected group of patients aged < 10 years. METHODS: Fifty-eight patients aged < 10 years underwent laparoscopic cholecystectomy for stones in the period 1992-99. The female/male ratio was 1.5, the mean age was 8 years (range, 2-10), and the mean weight was 30 kg. In all patients, parenteral nutrition, fasting state, and prolonged use of antibiotics had been suspended for > or = 6 months, and conservative treatment had been tried for > or = 12 months in the absence of symptoms of stone migration. All the patients were followed up after surgery: clinically at 1,6,12, and 36 months and by ultrasound at 1, 12, and 36 months. Liver function and hematological tests were performed in case of symptoms or if hemolytic disorders were the cause of stones. RESULTS: The mean operative time was 63 min (range, 30-120) in children undergoing cholecystectomy alone and 150 min in children undergoing associated splenectomy. There were no major complications or reoperations. Minor complications included bleeding from accessory cystic artery (n = 3) and insufflation of the omentum (n = 2). One case was converted to an open procedure due to technical problems. All the children were followed up and no complications were observed. CONCLUSION: We consider the laparoscopic approach the gold standard for cholecystectomy in children. This procedure does not have a complication rate any higher than open cholecystectomy, and patient follow-up is as good as that of open surgery. Previous abdominal surgery is not a contraindication to laparoscopy.

Results of the laparoscopic Heller-Dor procedure for pediatric esophageal achalasia.

BACKGROUND: Esophageal achalasia is not a frequent disorder in children and different treatments have been proposed during past decades. This study reviews the results of the laparoscopic Heller-Dor procedure performed in pediatric patients in two different surgical units. METHODS: We included the patients aged <14 years with a minimum follow-up of 6 months operated on in the period 1994-2001. A single longitudinal anterior esophageal myotomy (Heller) and a 180 degrees anterior gastropexy (Dor) were laparoscopically performed. The patients were checked to detect intra- or postoperative complications and recurrence. RESULTS: Twenty children were operated on. Mean follow-up was 45 months (range 6-102). Postoperative clinical score was Visick 1 in 15 cases and Visick 2 in five. CONCLUSIONS: As complication and recurrence rates are very low we consider modified Heller myotomy and Dor gastropexy through a laparoscopic approach our first choice to treat esophageal achalasia in the pediatric population.