Progressive Fibrosis Is Driven by Genetic Predisposition, Allo-immunity, and Inflammation in Pediatric Liver Transplant Recipients.

AIM: To determine predisposing factors of idiopathic allograft fibrosis among pediatric liver transplant recipients. BACKGROUND: Protocol biopsies (PB) from stable liver transplant (LT) recipient children frequently exhibit idiopathic fibrosis. The relation between allograft inflammation, humoral immune response and fibrosis is uncertain. Also the role of HLA-DRB1 genotype has not been evaluated, though it's associated with fibrosis in autoimmune hepatitis. PATIENTS AND METHODS: This observational study, included 89 stable LT recipient transplanted between 2004-2012 with mean follow-up of 4.3years, 281 serial PBs (3.1 biopsy/child) and human leukocyte antigen (HLA) antibody data. PBs were taken 1-2, 2-3, 3-5, 5-7, and 7-10years post-LT, and evaluated for inflammation and fibrosis using liver allograft fibrosis score (LAFSc). The evolution of fibrosis, inflammation and related predisposing factors were analysed. FINDINGS: HLA-DRB1*03/04 allele and Class II DSA were significantly associated with portal fibrosis (p=0.03; p=0.03, respectively). Portal inflammation was predisposed by Class II DSA (p=0.02) and non-HLA antibody presence (p=0.01). Non-portal fibrosis wasn't predisposed by inflammation. Lobular inflammation was associated with non-HLA antibodies. INTERPRETATION: We conclusively demonstrated that allograft inflammation results in fibrosis and is associated with post-LT Class II DSA and non-HLA antibodies. The HLA-DRB1*03/04 allele caused genetic predisposition for fibrosis. FUNDING: None.

[Influence of chest physiotherapy on gastro-œsophageal reflux in children]. / Influence de la kinésithérapie respiratoire sur le reflux gastro-œsophagien chez l'enfant.

INTRODUCTION: Chest physiotherapy is regularly prescribed for children, particularly in cystic fibrosis. Gastro-oesophageal reflux is common in this disease and is associated with certain chest physiotherapy manoeuvres. AIM OF THE STUDY: To evaluate the influence of two chest physiotherapy techniques on gastro-oesophageal reflux in children. MATERIAL AND METHOD: Twenty-nine children were investigated by routine pHmetry. During the examination, they performed two chest physiotherapy manoeuvres in a seated position for 10 minutes each with a 5 minutes rest between them. The two manoeuvres used were a slow expiration technique (ELPr) and positive expiratory pressure (PEP). It was a prospective study and the order of manoeuvres was randomised. The pH traces were analysed blindly when all the studies had been completed. RESULTS: In the sample, 21% of children had gastro-oesophageal reflux during the physiotherapy session. No relationship was found between reflux during physiotherapy and pathological reflux (P=0.411) nor the physiotherapy technique used (P=0.219). CONCLUSION: The use of these two chest physiotherapy techniques in children in a seated position can produce gastro-oesophageal reflux.

Raised immunoglobulin A and circulating T follicular helper cells are linked to the development of food allergy in paediatric liver transplant patients.

BACKGROUND: Post-transplant food allergy (LTFA) is increasingly observed after paediatric liver transplantation (LT). Although the immunopathology of LTFA remains unclear, immunoglobulin (Ig) E seems to be implicated. OBJECTIVE: To study humoral and cellular immunity in paediatric LT patients in search for factors associated with LTFA, and compare with healthy controls (HC) and non-transplant food-allergic children (FA). METHODS: We studied serum Ig levels in 29 LTFA, 43 non-food-allergic LT patients (LTnoFA), 21 FA patients and 36 HC. Serum-specific IgA and IgE against common food allergens in LTFA, IgA1 , IgA2 and joining-chain-containing polymeric IgA (pIgA) were measured. Peripheral blood mononuclear cells were analysed by flow cytometry for B and T cell populations of interest. RESULTS: Serum IgA and specific IgA were higher in LTFA compared to LTnoFA. LTFA patients had the highest proportion of circulating T follicular helper cells (cTfh). The percentage of cTfh correlated positively with serum IgA. Unique in LTFA was also the significant increase in serum markers of mucosal IgA and the decrease in the Th17 subset of CXCR5(-) CD4(+) cells compared to HC. Both LT patients exhibited a rise in IgA(+) memory B cells and plasmablasts compared to HC and FA. CONCLUSIONS: LT has an impact on humoral immunity, remarkably in those patients developing FA. The increase in serum markers of mucosal IgA, food allergen-specific IgA and cTfh cells observed in LTFA, point towards a disturbance in intestinal immune homoeostasis in this patient group.

MR enterography in children with Crohn disease: results from the Belgian pediatric Crohn registry (Belcro).

INTRODUCTION: Magnetic Resonance enterography (MRE) is an imaging modality avoiding ionizing radiation and the discomfort associated with enteroclysis. The results of MRE at diagnosis in the patients of the Belgian pediatric Crohn registry (Belcro) are compared to endoscopical and histological results. METHODS: Results of MRE, endoscopy and histology were obtained from the medical charts and assigned to one of the following segments: jejunum, ileum, ascending colon, transverse colon, descending colon or rectosigmoid. MRE images were reviewed in a blinded way by 4 radiologists with specific interest in pediatric MRE. RESULTS: From the Belcro registry, twenty-two patients underwent a MRE during their work-up for Crohn disease. The results of endoscopy, histology and MRE were concordant (either all negative or positive) in the ileum in 16/18 patients and in the rectosigmoid, descending colon, transverse colon and ascending colon in resp 9, 8, 8 and 8/22 patients. In the non-concordant cases (MRE colon negative but endoscopy and/or histology positive), MRE could not reflect the subtle endoscopic or histologic lesions such as erosions that were described.In 4 cases where ileocaecal valve intubation was impossible ileal MRE findings were abnormal. MRE detected ileal stenosis, jejunal lesions and fistula in resp 4/22, 3/22 en 2/22 patients. The 100% and 75% interobserver agreement was resp 50-82% and 773-100% according to the different intestinal segments. CONCLUSIONS: MRE is a promising imaging modality avoiding radiation in Crohn disease. It should probably become the technique of first choice for the evaluation of extensive small bowel disease in children with Crohn disease.

Profile of pediatric Crohn's disease in Belgium.

AIM: A Belgian registry for pediatric Crohn's disease, BELCRO, was created. This first report aims at describing disease presentation and phenotype and determining associations between variables at diagnosis and registration in the database. METHODS: Through a collaborative network, children with previously established Crohn's disease and newly diagnosed children and adolescents (under 18 y of age) were recruited over a 2 year period. Data were collected by 23 centers and entered in a database. Statistical association tests analyzed relationships between variables of interest at diagnosis. RESULTS: Two hundred fifty-five patients were included. Median age at diagnosis was 12.5 y (range: 1.6-18 y); median duration of symptoms prior to diagnosis was 3 m (range: 1-12 m). Neonatal history and previous medical history did not influence disease onset nor disease behavior. Fifty three % of these patients presented with a BMI z-score < -1. CRP was an independent predictor of disease severity. Steroids were widely used as initial treatment in moderate to severe and extensive disease. Over time, immunomodulators and biological were prescribed more frequently, reflecting a lower prescription rate for steroids and 5-ASA. A positive family history was the sole significant determinant for earlier use of immunosuppression. CONCLUSION: In Belgium, the median age of children presenting with Crohn's disease is 12.5 y. Faltering growth, extensive disease and upper GI involvement are frequent. CRP is an independent predictive factor of disease activity. A positive family history appears to be the main determinant for initial treatment choice.

[Pigmented villonodular synovitis of the knee]. / Le cas clinique du mois. Synovite villonodulaire pigmentée du genou.

This case report is concerned with a 30 year old patient diagnosed with pigmented villonodular synovitis (PVNS) in the knee. The patient underwent an orthopedic surgical operation to remove the lesions. PVNS is a rare proliferative disorder, mostly benign and affecting the knee; its aetiology remains unclear. It represents a medical challenge because of non-specific symptoms that delay the diagnosis with a very high rate of recurrence. MRI imaging is necessary to explore the lesions, but the final diagnosis can only be made after anatomopathologic analysis of the excised lesions. When multiple lesions are present, the treatment consists of their excision by arthrotomy, or by arthroscopy if the disease is localized.

Acquired antithrombin type IIb deficiency after liver transplantation: a case report.

A 3-year-old girl with multifocal hepatoblastoma was referred to our clinic for living-donor liver transplantation, the patient's father being the donor. Pretransplant evaluation revealed that the father presented partial asymptomatic antithrombin (AT) deficiency, with no inherited AT deficiency found in the girl. The genetic testing showed an AT type IIb deficiency responsible for a defect in the heparin-binding region of AT which is less thrombogenic but more common than the other AT qualitative defects. Her mother was ABO incompatible. Despite the thrombophilia on the father's side, transplantation was successfully performed under replacement therapy with intravenous AT concentrate and low-molecular-weight heparin thromboprophylaxis given to both the recipient and the donor. No thrombotic complications occurred. In the posttransplantation course, acquired partial AT deficiency was detected in the recipient, who received adjuvant chemotherapy without thrombotic complications. This case report highlights the relevance of full thrombophilic work-up before liver transplantation from a living donor, while illustrating that the procedure can be successfully performed in the case of AT deficiency on the donor's side provided that appropriate AT supplementation and thromboprophylaxis are administered to both the recipient and the donor.

Safety and cost of infliximab for the treatment of Belgian pediatric patients with Crohn's disease.

Biologicals have become an important component in the treatment of Crohn's disease in children. Their increased and long term use raises safety concerns. We describe safety and cost of infliximab in Belgian pediatric Crohn's disease patients. All patients on infliximab as part of the present or past treatment for Crohn's Disease until January 1st 2011 were selected from an existing database. Information on disease phenotype, medication and adverse events were extracted. Adverse events occurred in 25.9% of patients exposed to infliximab of which 29.6% were severe. In total 31.7% of patients stopped infliximab therapy. The main reasons for discontinuation were adverse events in 45.4% and loss of response in 30.3%. No malignancies or lethal complications occurred over this 241 patient year observation period. Immunomodulators were concomitant medication in 75% of patients and were discontinued subsequently in 38.4% of them. The cost of infliximab infusions per treated patient per year in the Belgian health care setting is approximately 9 474 euro, including only medication and hospital related costs. Even though infliximab is relatively safe in pediatric CD on the short term, close follow-up and an increased awareness of the possible adverse reactions is highly recommended. Adverse reactions appeared in 25.9% of all patients and were the main reason for discontinuation. Treatment cost has to be balanced against efficacy and modifications in disease course. In the Belgian health care system, the medication is available to all patients with moderate to severe CD.

[Long-term outcome of infliximab therapy in pediatric Crohn disease]. / Usage prolongé de l'infliximab dans la maladie de Crohn chez l'enfant.

BACKGROUND: The efficacy of infliximab (IFX) in inducing and maintaining remission in pediatric Crohn disease is currently well documented. However, the optimal treatment strategy beyond 1 year has not been established. In particular, systematic continuation of maintenance therapy and its association with immunomodulators have not yet been analyzed. OBJECTIVE: The aim of this study was to describe the long-term outcome of pediatric Crohn disease patients on IFX therapy and to evaluate the clinical response to the therapy and the effect on growth. METHODS: A single-center and retrospective chart review was conducted. The clinical maintenance response to treatment, effect on the linear growth, and long-term outcome were examined. These parameters were analyzed according to the age of the patients, duration and localization of the disease, as well as associated therapies. RESULTS: We identified 52 children with Crohn disease younger than 16 years of age at the time of diagnosis. Of these patients, 20 (38%) received a biologic therapy at a mean age of 13.9±2 years. Fifteen patients received IFX therapy and 13 (86%) were in clinical remission 10 weeks after the first infusion. Among the responders, 82% were still in remission after 1 year of therapy and 66% after 2 years. Among patients treated for more than 1 year, we observed IFX dependency in 89%. Thirty-eight percent of patients with initial IFX response showed a loss of response after a median of 30 months (range, 3-42 months). At 2 years, the median Z score for height among patients with presumed growth potential had improved slightly, from -0.7 to -0.55 DS. No serious adverse events were observed. CONCLUSION: Our results confirm the continuous efficacy of IFX in pediatric Crohn disease patients after 1 year of treatment. However, a high level of dependency was observed (89 %). A slight beneficial effect on growth was observed after 2 years of treatment.

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells.

OBJECTIVES: Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. MATERIAL AND METHODS: In vitro, we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo, we investigated co-expression of hepatocyte (albumin, alpha-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. RESULTS: We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and alpha-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro. Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. CONCLUSIONS: Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.

Cell transplantation in the treatment of liver diseases.

The liver performs multiple functions that are essential for life, the most crucial being its role in the body metabolism. Impairment of this function, because of liver insufficiency, can be partially restored by medical management but OLT remains the ultimate therapeutic treatment. Because not always indicated or available, other alternatives are proposed such as LCT. Compared to OLT, this procedure is less invasive, less expensive, and fully reversible. More than 50 patients have thus far benefited of this technique and are reviewed here. Indications were multiple including inborn errors of metabolism, FHF, acute on chronic diseases, and decompensated end-stage cirrhosis. Documented results were encouraging, especially for metabolic disorders, with medium-term efficacy up to two yr. Related complications were exceptional. On this basis, LCT has entered its phase of clinical application and current indications and protocols are detailed. Ongoing lines of research are discussed, including cell quality, stem cell field, and rejection prevention. Further improvement of the procedure is therefore expected and should lead to broader applications of LCT.

Post-transplant lymphoproliferative disorder with supraglottic involvement.

OBJECTIVE: Transplant patients with primary Epstein-Barr virus (EBV) infection may develop post-transplant lymphoproliferative disorder (PTLD). Since many infants are seronegative at the time of transplantation, PTLD is a major concern for paediatric transplant centres. First manifestations of PTLD are frequently observed in the ENT area with adenoidal and/or tonsillar involvement. DESIGN: Retrospective study of two cases of PTLD with confirmed supraglottic involvement, their management and outcome. Only patients with pathologically and immunologically demonstrated B-cell proliferation were diagnosed as PTLD. RESULT: Two infants developed an acute stridor during PTLD respectively 8 and 10 months after orthotopic liver transplantation (OLT). These infants were seronegative for EBV at the time of transplantation. IgM anti-EBV and/or detection of EBV genome by polymerase chain reaction were positive. Laryngeal examination revealed hypopharyngeal and/or supraglottic mucosal hyperplasia. Immunostaining of laryngeal biopsy was positive for latent membrane protein-1 (LMP1). Patients were treated by a reduction in immunosuppression as far as tolerated with the intent to recover natural immune response by the patient over the proliferation of EBV-infected cells. Complete remission of PTLD was observed in these two cases. CONCLUSION: Tonsillar hypertrophy and adenoid enlargement are the most encountered features of PTLD in OLT occurring in the ENT area. Acute stridor with supraglottic involvement may also be observed in PTLD and must be promptly diagnosed as the prognosis of this disorder is related to rapid reduction in immunosuppression and consequently to the recovering of a natural immune response against the EBV infection.

Epstein-Barr virus-related lymphoproliferation in children after liver transplant: role of immunity, diagnosis, and management.

Tumor occurrence following immunosuppression remains a major concern in children after liver transplantation. More than 50% of these tumors belong to the post-transplant lymphoproliferative diseases (PTLD) and are diagnosed during childhood. These PTLD are mostly related to primary Epstein-Barr virus (EBV) infection and a heavy immunosuppressive regimen. Improvement in their prognosis was reached thanks to a better knowledge of their pathogenesis, risk factors and clinical presentation, linked probably to earlier management. However, their incidence remains stable (occurring in 5-15% of children after liver transplantation) despite different pre-emptive strategies based on these parameters. Moreover, acute graft rejection and subsequent risk of graft loss is a common side-effect of PTLD treatment. EBV viral load determination by quantitative polymerase chain reaction (PCR) is so far the only predictive marker proposed for PTLD prevention and PTLD treatment monitoring, although limited by a lack of specificity. New immunologic techniques have allowed the demonstration of a defect of the EBV-specific cellular immunity in the patients with PTLD. The level of immunity is correlated to the viral load and improves during recovery from PTLD. These recent findings add further knowledge to PTLD pathogenesis and management.

Patients at risk for development of posttransplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction.

BACKGROUND: Early diagnosis of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (PTLD) is required to detect a stage of disease that is more likely to respond to treatment. Elevated levels of EBV DNA were found in peripheral blood of patients at the onset of PTLD. METHODS: To compare plasma and peripheral blood mononuclear cells (PBMCs) as material for real-time quantitative polymerase chain reaction (RQ-PCR) measurement of Epstein-Barr viral load, we used two sets of primers and probes specific for the BAM HI-K or BAM HI-W region of the EBV genome. RESULTS: Patients with PTLD had a median viral load of 19,200 EBV genomes/microg DNA (n=9) or 3,225 EBV genomes/100 microl plasma (n=5), being significantly higher compared with immunosuppressed patients with primary (n=9) or reactivated (n=20) EBV infection or immunosuppressed patients without serological signs of active EBV infection (n=67) (P<0.001). Hence, a value of greater than 5,000 EBV genomes/microg PBMC DNA was considered as a diagnostic parameter for PTLD with a sensitivity and specificity of 1.00 or 0.89, respectively. When plasma was analyzed, however, a value of greater than 1,000 EBV genomes/100 microl plasma had both a sensitivity and specificity of 1.00 for the diagnosis of PTLD. During remission of PTLD, viral load was more effectively cleared in plasma compared with PBMCs. In plasma of nonimmunosuppressed individuals, even a qualitative detection of EBV-related sequences was sensitive and specific for the diagnosis of primary EBV infection, whereas for analysis of PBMC DNA a quantitative parameter had to be considered to differentiate healthy individuals (< 100 EBV genomes/microg PBMC DNA) from patients with primary EBV infection (>100 EBV genomes/microg PBMC DNA). CONCLUSION: Although both PBMCs and plasma were useful as material for EBV-specific RQ-PCR in immunosuppressed patients and nonimmunosuppressed individuals, the specificity of analysis seemed to be higher if plasma was taken for analysis.

Unusual evolution of an Epstein-Barr virus-associated leiomyosarcoma occurring after liver transplantation.

We report the case of a child who developed, 2 yr after orthotopic liver transplantation (OLTx) for biliary atresia, a multi-focal hepatic tumor with lymphonodular metastases, identified as an Epstein-Barr virus (EBV)-associated leiomyosarcoma. Chemotherapy was given without tumor response. Subsequently, slow growth of the tumor was observed. Immunosuppression was tapered and stopped 9 yr after transplantation. At the present time, 12 yr after the discovery of the first hepatic lesions, the patient is alive and completely symptom-free, the abdominal masses are stable, and liver function tests are completely normal. Smooth muscle tumors are increasingly recognized in children with various immunodeficiencies occurring after organ transplantation. This unusual evolution of a clinically aggressive tumor into a stable disease after restoration of immunity confirms that the immune status of the patient is a crucial factor.

Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders.

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.

Real-time polymerase chain reaction (RQ-PCR) for the monitoring of Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells.

Measurement of Epstein-Barr virus (EBV) load is useful in peripheral blood for detecting primary and reactivated EBV-infections especially in immunosuppressed patients being at high risk for developing posttransplant lymphoproliferative disorder. For quantification of EBV DNA in peripheral blood of patients two real time polymerase chain reaction (RQ-PCR) assays were developed detecting sequences specific for the BAM HI-W and BAM HI-K region of EBV. In order to determine the optimal material of peripheral blood for RQ PCR analysis, DNA preparations of whole blood, peripheral blood mononuclear cells (PBMC) and B cells from 11 healthy, EBV-seropositive individuals were analysed in parallel and compared with regard to efficiency and sensitivity. While in whole blood preparations inhibitors of RQ PCR were detected influencing sensitivity, analysis of B cells being most sensitive is limited by being too labour intensive. In contrast, analysis of DNA preparations of PBMCs is sensitive enough to frequently detect EBV-specific sequences in all individuals tested and the preparation of PBMCs itself needs only a reasonable time. Thus, longitudinal monitoring of EBV load in peripheral blood of patients is possible by RQ-PCR, the optimal material for analysis being PBMCs.

Predictive value of Epstein-Barr virus genome copy number and BZLF1 expression in blood lymphocytes of transplant recipients at risk for lymphoproliferative disease.

Epstein-Barr virus (EBV) genome numbers and RNA transcripts from the immediate-early EBV gene BZLF1 were monitored by means of polymerase chain reaction in peripheral blood lymphocytes (PBLs) of 44 children who received liver transplants. The 2 tests were compared, using several parameters to assess their value as predictors of posttransplantation lymphoproliferative disease (PTLD). All patients were infected with EBV. BZLF1 mRNA was positive in 70% of patients, with highest expression in those with largest virus load. Four patients developed PTLD that could not be unequivocally diagnosed by any of the parameters considered alone. Sensitivity of EBV genome number (>/=40,000 EBV copies/10(5) PBLs) and BZLF1 mRNA (BZLF1:glyceraldehyde-3-phosphate-dehydrogenase ratio >/=0.5) was 100%. Specificity of each of the 2 tests alone (98% and 58%, respectively) improved (to 100% and 83%, respectively) when measurement of serum IgG level was included. Because decreased virus load, but not BZLF1 mRNA expression, accurately predicted favorable responses of PTLD to therapy, monitoring of EBV genome numbers alone appears sufficient in children with liver transplants.