Establishment of a pediatric liver transplantation program: experience with 100 transplantation procedures.

OBJECTIVE: To analyze the primary factors that influence the development and consolidation of a pediatric liver transplantation program. PATIENTS AND METHODS: This was a retrospective study of 100 liver transplantation procedures performed in 84 pediatric patients between May 1990 and November 2007. The male-female ratio was 40:60. Mean (SD) age was 5 years (40 patients were younger than 2 years); cold ischemia time was 7.10 (3.1) hours; surgery time was 5.2 (2.2) hours; and time on the waiting list for transplantation was 75 (range, 1-1012) days. Indications for transplantation included cholestatic disease (43%), acute hepatic failure (AHF; 34%), metabolic disorders (14%), and cirrhosis (9%). Transplanted organs included 3 split grafts, 29 partial grafts, and 8 living-donor grafts. RESULTS: Mean graft survival was 70.4%, 59.2%, and 58.1% at 1, 3, and 5 years, respectively. Factors that influenced graft outcome were age younger than 2 years; surgery time more than 6 hours; and AHF vs cholestatic disease, metabolic disorders, and cirrhosis. There were no significant differences in long-term (51% vs 59%) and short-term (71% vs 70%) graft survival between procedures performed in 1990-1998 compared with those performed in 1999-2007; however, there was a higher percentage (P = .005) of recipients at high risk (age younger than 2 years or with AHF) in the later period. All data were consistent with those of the European Liver Transplant Registry 2007. CONCLUSIONS: A pediatric liver transplantation program can be established by a group experienced in liver transplantation.

Magnetic resonance imaging (MRI) simulation on EGEE grid architecture: a web portal design.

In this paper, we present a web portal that enables simulation of MRI images on the grid. Such simulations are done using the SIMRI MRI simulator that is implemented on the grid using MPI and the LCG2 middleware. MRI simulations are mainly used to study MRI sequence, and to validate image processing algorithms. As MRI simulation is computationally very expensive, grid technologies appear to be a real added value for the MRI simulation task. Nevertheless the grid access should be simplified to enable final user running MRI simulations. That is why we develop this specific web portal to propose a user friendly interface for MRI simulation on the grid. The web portal is designed using a three layers client/server architecture. Its main component is the process layer part that manages the simulation jobs. This part is mainly based on a java thread that screens a data base of simulation jobs. The thread submits the new jobs to the grid and updates the status of the running jobs. When a job is terminated, the thread sends the simulated image to the user. Through a client web interface, the user can submit new simulation jobs, get a detailed status of the running jobs, have the history of all the terminated jobs as well as their status and corresponding simulated image.

Prognostic significance of K-ras, p53, bcl-2, PCNA, CD34 in radically resected non-small cell lung cancers.

The aim of this study was to investigate the prognostic significance of a panel of biological parameters in patients with radically resected non-small cell lung cancers (NSCLC). 269 cases with pathological stage I-IIIA NSCLC were retrospectively analysed. Immunohistochemistry was performed to detect protein expression of p53, bcl-2, proliferating cell nuclear antigen (PCNA) and CD34. Polymerase chain reaction (PCR)/direct nucleotide sequencing method was used to detect mutations in K-ras (codons 12, 13, 61, exons 1-2). The Kaplan-Meier estimates of survival were calculated for clinical and biological variables using the Cox model for multivariate analysis. Histological subtype and the pathologic tumour extension (pT) were the most powerful clinical-pathological prognostic factors for survival (P=0.030 and P=0.031, respectively), whereas among the biological parameters, p53 overexpression (P=0.032) and K-ras mutation (P=0.078) had a negative prognostic role, as demonstrated by multivariate analysis. Conversely, bcl-2, PCNA and CD34 expression were not correlated with survival. Statistically significant associations between p53 expression and the squamous cell carcinoma (SCC) subtype, bcl-2 expression and SCC subtype, K-ras mutation and p53 negative expression, p53 and bcl-2, bcl-2 and PCNA overexpression were observed. In conclusion, some biological characteristics such as the K-ras and p53 status may provide useful prognostic information in resected NSCLC patients, in addition to the classical clinico-pathological parameters. However, further studies are needed to clarify the value of adopting biological prognostic factor into clinical practice.

Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain.

The hypothesis was tested that amino acid substitutions in specific positions within human leukocyte antigen class I heavy chain would have different impacts on transplant-related mortality (TRM) in patients receiving transplanted bone marrow from unrelated donors. One hundred patients and their unrelated donors were typed by sequence-based typing for the human leukocyte antigen (HLA)-A, -B, and -C loci. All pairs were matched for DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Forty pairs were also matched at class I, and 60 pairs had one or more mismatches at class I loci. It was found that substitutions at positions 116 and 114 of class I heavy chain significantly increased the risk for TRM in univariate and bivariate Cox analyses. Conversely, no association between number of multiple mismatches or number of amino acid substitutions and TRM was seen when positions 116 and 114 were adjusted for. Variables predictive of TRM in multivariate Cox analysis were number of cells infused, diagnosis (chronic myeloid leukemia [CML] or non-CML), and amino acid substitution at position 116 or 152. The only variable predictive of severe acute graft-versus-host disease (GVHD) in multivariate Cox analysis was substitution at position 116. Actuarial risk for acute GVHD grade III-IV, TRM, and relapse in pairs with substitutions at position 116 (n = 37) compared to other pairs (n = 63) was, respectively, 36% versus 14% (P =.01), 59% versus 28% (P =.001), and 25% versus 31% (P =.4). In conclusion these data suggest that substitutions at position 116 of class I heavy chain increase the risk for acute GVHD and TRM in patients who receive transplanted bone marrow from unrelated donors.

Improved cardiac function in patients with obstructive jaundice after internal biliary drainage: hemodynamic and hormonal assessment.

OBJECTIVE: To investigate myocardial function in patients with obstructive jaundice before and after internal biliary drainage. SUMMARY BACKGROUND DATA: Increased plasma levels of atrial natriuretic peptide (ANP) have been found in patients with biliary obstruction. METHODS: Thirteen patients with newly diagnosed obstructive jaundice and no previous heart, lung, or renal disease were studied using a Swan-Ganz catheter. Hemodynamic measurements were taken before and 4 days after internal biliary drainage. Levels of ANP and brain natriuretic peptide (BNP) were obtained and liver function tests were also determined. RESULTS: Plasma levels of ANP and BNP were increased twofold to fourfold in the basal state and declined after biliary drainage. Independent variables predicting left ventricular systolic work were total bilirubin concentrations, duration of jaundice, and BNP. In addition, bilirubin concentrations correlated with pulmonary vascular resistance, mean arterial pulmonary pressure, and right ventricular systolic work. Internal biliary drainage resulted in an improvement in left ventricular systolic work. A correlation was found between decreasing ANP concentrations and increasing cardiac output. CONCLUSIONS: Increased plasma levels of natriuretic peptides in patients with obstructive jaundice may reflect a subclinical myocardial dysfunction correlating with the degree of jaundice. After internal biliary drainage, there is a measurable improvement of cardiac function.

Pancreatic intraductal sampling during ERCP in patients with chronic pancreatitis and pancreatic cancer: cytologic studies and k-ras-2 codon 12 molecular analysis in 47 cases.

BACKGROUND: A preoperative tissue diagnosis of pancreatic cancer is desirable but difficult to obtain. METHODS: Pancreatic brush cytology, salvage cytology, and collection of pancreatic juice were attempted prospectively during ERCP in 34 patients with pancreatic cancer and 11 with chronic pancreatitis. K-ras-2 codon 12 was analyzed for presence and type of point mutations. RESULTS: Brush cytology coupled with salvage cytology had a sensitivity of 74%. The addition of cytologic analysis of pancreatic juice did not substantially improve sensitivity (76%). K-ras-2 was mutated in both cancer (87%) and pancreatitis (40%). The specificity for cytology was 100% and for K-ras-2 mutations 60%. Combining cytology with mutation analysis increased sensitivity to 93% but reduced the positive predictive value. The negative predictive value never exceeded 75%. None of the patients with chronic pancreatitis had cancer develop (median follow-up 60 months). CONCLUSIONS: Pancreatic ductal brushing with salvage cytology is useful in the diagnosis of cancer, whereas cytologic analysis of pancreatic juice can be abandoned. At present, K-ras-2 mutation is not useful for differentiating pancreatic cancer from chronic pancreatitis or the identification of patients with chronic pancreatitis at risk for malignant transformation.

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones.

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.

Melatonin versus vitamin E as protective treatment against oxidative stress after extra-hepatic bile duct ligation in rats.

The aims of the present study were first to compare the effects of melatonin and vitamin E on the cholestasis syndrome and their protective effect on liver injury, and second, to evaluate the activity of antioxidant enzymes after treatment with these antioxidant drugs. Cholestasis was achieved in adult male Wistar rats by double ligature and section of the extra-hepatic biliary duct. Hepatic and plasma oxidative stress markers were evaluated by changes in the amount of lipid peroxides, measured as malondialdehyde (MDA) and reduced glutathione (GSH) in plasma and homogenates of hepatic tissue. Serum bilirubin, alkaline phosphatase (AP), and gamma-glutamyl-transpeptidase (GGT) were used to evaluate the severity of cholestasis, and serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to evaluate the hepatic injury. Both vitamin E and melatonin were administrated 1 day before and 7 days after bile duct ligation. Acute ligation of the bile duct was accompanied by a significant increased in MDA and a decrease in GSH levels in both plasma and liver, as well as a significant reduction in antioxidant enzymes activities. The overall analysis of both treatments showed that melatonin (500 microg/kg daily) offered significantly better protection against cholestasis and a superior protective effect on hepatic injury than did vitamin E (15 mg/kg daily). Although vitamin E treatment resulted in a reduction of parameters of oxidative stress, the results were significantly better after a much lower daily dose of melatonin. Moreover, melatonin treatment was associated with a significant recovery of antioxidative enzymes. In conclusion, the present paper demonstrates a correlation between the intensity of biliary tract obstruction and increased free radical generation, as well as a direct correlation between the level of oxidative stress and the biochemical markers of liver injury. Melatonin (at a much lower dose than vitamin E) was much more efficient than vitamin E in reducing the negative parameters of cholestasis, the degree of oxidative stress and provided a significantly greater hepatoprotective effect against the liver injury secondary to the acute ligation of the biliary duct.

Anorexia and the effect of internal biliary drainage on food intake in patients with obstructive jaundice.

BACKGROUND: Anorexia is a frequent finding in patients with biliary obstruction (BO). This study investigates the role of biochemical and hormonal factors in the pathogenesis of reduced food intake in BO and the effects of internal biliary drainage. STUDY DESIGN: Sixty-two patients with BO were prospectively investigated. Transaminases, amylase, cholecystokinin, secretin, bile acids, tumor necrosis factor-alpha, and endotoxin were determined at admission. Caloric intake was quantified by a controlled diet. In a subset of 27 patients, studies were repeated after internal biliary drainage. RESULTS: Sixty-six percent of patients had spontaneous food intakes below the estimated caloric requirements. Serum bilirubin, alkaline phosphatase, and cholecystokinin plasma levels were independent predictor factors for calorie intake (p = 0.0001). After internal biliary drainage, cholestasis parameters and cholecystokinin concentrations decreased significantly; this was associated with an improvement of spontaneous food intake in both benign and malignant biliary obstruction (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: Decreased food intake in BO was associated with the degree of obstruction and with increased cholecystokinin plasma levels. Biliary drainage improved biochemical and food intake derangements.

Investigation of HLA class I downregulation in breast cancer by RT-PCR.

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.

Results of liver transplantation in a Spanish group: a report from the Cordoba unit.

The liver transplantation unit from Córdoba, Spain, performed its first liver transplantation in 1989. Since then, 500 liver transplants have been performed in our institution up to November 2001. Our one-year recipient and graft survival rates are 77% and 76%, respectively. These recipient and graft survival rates are lower when compared with other results in the English language literature, and we present evidence here that the disparity results from 4 especially problematic aspects of our program: 1) the expanded use of donors, 2) the policy of allocation and prioritization in our institution, 3) the recurrence of primary liver disease and 4) de-novo neoplasms. Liver transplantation with unstable, hypernatremic donors or donors with a lengthy hospitalization and grafts with a prolonged cold ischemia time leads to diminished graft survival. When several marginal criteria accumulate in a donor, the results are even poorer. Consequently, the delayed non-function rate is especially high in our series. Attention to severe liver preservation injury as a primary mechanism of graft losses with marginal donors is given. The impact of the policy of the "sickest-first" principle in our center with a long waiting list seems to benefit urgent but not elective patients. Survival in elective patients is poorer than expected considering their clinical condition. The use of the sickest-first or urgency principle in our unit has not been efficient or equitable and may partially explain the poorer survival of our patients. Finally, the recurrence of primary disease and incidence of de-novo tumors after transplantation in our unit are similar those reported in English language literature.

A proposal for scoring marginal liver grafts.

The aim of this study is to assess the effect of accumulation of marginal liver graft criteria on the immediate outcome of liver transplantation (LT). The last 325 consecutive LT performed in 293 patients were analyzed retrospectively with respect to donor acceptance criteria. A marginal liver score was elaborated on the basis of the following features: donor > 60 years, ICU stay > 4 days, cold ischemia times > 13 h, hypotensive episodes < 60 mmHg > 1 h, bilirubin > 2.0 mg/dl, ALT > 170 U/l, and AST > 140 U/l were scored with the value 1. The use of dopamine doses > 10 microg/kg per min and peak serum sodium > 155 mEq/l were labeled with value 2. The cut-off point at 6 months after LT revealed 42 deaths (14%), with 65 graft losses (20%) and 32 (9%) retransplants. Recipient survival was not affected by the combined effect of marginal criteria. However, recipients transplanted with marginal livers with score 3 or more showed a decrease in graft survival (log-rank 6.21; P = 0.045) and an increase in delayed non-function rate (10 out of 33 vs 4 out of 156; P = 0.03). The use of marginal liver donors with more than three risk factors must be carefully reviewed or refused because of the cumulative dysfunction of these grafts.

Papel del trasplante ortotópico de hígado en el tratamiento familiar homozigota / Role of orthotopic liver transplant in the treatment of homozygous familial hypercholesterolemia

La hipercolesterolemia familiar homocigota es un trastorno metabólico hereditario que condiciona una disminución del catabolismo del colesterol unido a las lipoproteínas de baja densidad, provocando cuadros de isquemia miocárdica en la primera-segunda década de la vida. Dado que el hígado presenta de un 50 a 75 por ciento de los receptores de las lipoproteínas de baja densidad, el trasplante de hígado se ha introducido como una opción terapéutica en esta enfermedad. OBJETIVO: aportar nuestra experiencia en el tratamiento de la hipercolesterolemia familiar homocigota mediante trasplante ortotópico de hígado, evaluando los resultados metabólicos obtenidos y la supervivencia de los pacientes. PACIENTES: presentamos el tratamiento de dos hermanos afectados, en el que en uno de ellos fue precisa, además, la realización de un trasplante cardíaco previo dada la grave afectación coronaría que presentaba. RESULTADOS: en ambos pacientes la evolución tras la intervención fue favorable, existiendo una disminución brusca de los niveles de colesterol en los primeros días con un descenso progresivo posterior hasta alcanzar niveles normales. A los 4 años de ambas intervenciones la función de los injertos era adecuada, los niveles de colesterol plasmático estaban dentro de los límites normales sin necesidad de fármacos hipolipemiantes y existía ausencia de progresión de la enfermedad. CONCLUSIONES: apoyándonos en nuestros resultados y en lo publicado en la literatura, consideramos que el trasplante hepático permanece actualmente como el tratamiento de elección en la hipercolesterolemia familiar homocigota hasta que la terapia génica se convierta en una opción viable. El trasplante hepático debe realizarse preferentemente antes del desarrollo de complicaciones cardiovasculares (AU)

Identification of a new DRB3*02 allelic variant (DRB3*0209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Levels of HBV-DNA and HBsAg after acute liver allograft rejection treatment by corticoids and OKT3.

The aim of this work was to analyze whether the treatment of acute rejection of orthotopic liver transplants (OLT), either with corticoids or OKT3, has any effect on the levels of hepatitis B virus (HBV)-DNA and HBsAg in individuals which were originally affected by cirrhosis or fulminant hepatic failure as a result of B virus. We have found that HBV-DNA is present in macrophages, B cells and both CD4+ and CD8+ T cells after OLT in all cases studied. Interestingly, the levels of HBV-DNA and HBsAg in the serum analyzed were increased extremely rapidly in the patients treated with OKT3 in an acute rejection episode. However, the serum levels of HBV-DNA and HBsAg found were lower when the patients were treated with steroids, and were not found in non-treated patients. As the serum levels of HBV-DNA increase, the process of liver reinfection could be accelerated; therefore, these results may help to understand how OKT3 and corticoids immunosuppressive therapy may accelerate the reinfection of OLT by HBV. In conclusion, our results suggest that special care must be taken in the use of OKT3 in the treatment of acute liver rejection episodes in chronic or fulminant HBV transplanted patients.

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the northeast of Italy: absence of sequence variability.

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Novel associations among HLA-DQA1 and -DQB1 alleles, revealed by high-resolution sequence-based typing (SBT).

Althought it is a valuable tool for the identification of HLA alleles, sequence-based typing (SBT) presents difficulties when used to determine HLA-DQA1 and -DQB1 alleles. Specifically, some HLA-DQA1 alleles have a three-base deletion at codon 56 of exon 2 that interferes with the sequencing read. Moreover, the frequently used primers for HLA-DQB1 may co-amplify the HLA-DQB2 pseudogene. To overcome these problems, we amplified DQA1 exon 2 using five group-specific polymerase chain reactions (PCRs) which allowed separation of deleted from non-deleted DQA1 alleles. DQB1 exon 2 was amplified using two group-specific amplifications. To increase typing resolution, we also analyzed DQA1 exons 1, 3 and 4 and DQB1 exon 3 by PCR using sequence-specific primers (PCR-SSP) or SBT analysis. Using this method we found some important associations between DQA1 and DQB1 alleles: DQA1*05011 and DQB1*0201, DQA1*0505 and DQB1*03011, DQA1*01021 and DQB1*06, DQA1*01022 and DQB1*0502.