Limited sampling strategies for monitoring tacrolimus in paediatric liver transplant recipients

Ce travail de recherche a été réalisé dans le laboratoire de pharmacologie clinique, au Centre Hospitalier Universitaire Sainte-Justine, à Montréal. C'est une étude rétrospective basée sur le suivi thérapeutique du Tacrolimus prescrit chez les enfants après transplantation hépatique. Ce suivi est nécessaire car le Tacrolimus possède une importante variabilité pharmacocinétique inter et intra-individuelle ainsi qu'un index thérapeutique très étroit. Actuellement, l'individualisation des doses prescrites est basée sur la mesure de la concentration de base - du médicament dans le sang (C0), mais des études récentes montrent que cette mesure ne reflète pas précisément l'exposition du Tacrolimus dans l'organisme chez les enfants. Le meilleur reflet de cette exposition est la mesure de l'aire sous la courbe (AUC). Cependant, cette dernière implique la mesure de multiples concentrations tout au long de l'intervalle entre 2 doses de médicament (Tacrolimus: 12 heures) ce qui est long, cher et impraticable en ambulatoire. De nouvelles méthodes utilisant un nombre limité de prélèvements ont donc été développées pour prédire au mieux cette AUC. Ce sont les "Limited sampling strategies" ou LSS. La plupart de ces LSS pour le Tacrolimus ont été développées et validées chez des patients transplantés adultes et leur application directe chez les transplantés pédiatriques n'est pas possible en raison de différences importantes au niveau des paramètres pharmacocinétiques du médicament entre ces deux populations. Aussi, le but de ce travail était de développer et valider, pour la première fois, des LSS chez les enfants transplantés hépatiques. Pour cela, une analyse de 36 profils pharmacocinétiques de 28 patients transplantés hépatiques âgés de 0.4- 18.5 ans a été effectuée. Tous les profils ont été réalisés au Centre Hospitalier Universitaire Sainte-Justine entre janvier 2007 et janvier 2009. Les LSS comportant au maximum 4 mesures de concentration ont été développées en utilisant une analyse de régression multiple. Parmi tous les modèles obtenus, cinq ont été sélectionnés sur la base de critères précis puis validés selon la méthode décrite par Sheiner et Beal.¦Les résultats montrent que ces cinq modèles peuvent prédire l'AUC du Tacrolimus avec une précision cliniquement acceptable de ± 15% alors que la C0 présente la plus faible corrélation avec l'AUC.¦En conclusion, cette étude confirme que la C0 ne permet pas de prédire de manière efficace l'exposition du Tacrolimus dans l'organisme dans notre population de patients pédiatriques contrairement aux LSS analysées qui offrent une méthode pratique et fiable. Par ailleurs, en permettant d'obtenir une estimation précise et simplifiée de l'AUC complète du Tacrolimus chez les patients, ces LSS ouvrent la porte à de futures études prospectives visant à mieux définir l'AUC cible du médicament et à déterminer si le suivi basé sur la mesure de l'AUC est plus efficace et plus sûr que celui basé sur la mesure de la C0.

Antibody-fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice.

To improve the detectability of tumors by light-induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein-MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein-MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I-MoAb-(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti-CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 micrograms of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 micrograms of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser-induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Long-term follow-up of colorectal carcinoma patients by repeated CEA radioimmunoassay.

One of the major practical applications of carcinoembryonic antigen (CEA) assay is the monitoring of colorectal carcinoma patients after complete tumor resection. During the last 5 years, we have followed by repeated CEA assays 66 patients with histologically confirmed colon or rectum adenocarcinoma. Among 19 patients who developed a tumor recurrence, 17 had increased CEA levels preceding the clinical diagnosis by 2 to 26 months. Among the 47 patients who did not show any clinical evidence of tumor recurrence, 35 had CEA values remaining below the limit of 5 ng/ml, whereas 12 had moderate elevations of CEA level fluctuating around this limit. The majority of patients in this last group were heavy smokers or had liver enlargement, but in a few of them we did not find a satisfactory explanation for their moderately increased CEA levels. While our results confirm that repeated CEA assays can predict tumor recurrence with a lead time of several months over clinical diagnosis, they also give a word of warning concerning the interpretation of moderate elevations of CEA level. A moderate increase of CEA level can be the result of early distant metastases, local recurrence or exacerbation of an inflammatory disease. We feel that the decision of second look operations based on CEA results should be made only if increasing CEA values have been observed on three different blood samples taken within a period of 3 months and if no nonmalignant diseases known to increase CEA level are present. Ultimately only randomized clinical studies will determine if second look operations motivated by elevated CEA levels can improve the quality and length of survival of patients with colorectal carcinoma.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Survival after lung metastasectomy in colorectal cancer patients with previously resected liver metastases

Objective: Resection of hepatic metastases is indicated in selected Stage IV colorectal cancer (CRC) patients. A minority will eventually develop pulmonary metastases and may be candidates for lung surgery. The aim of this study was to assess clinical outcome, and identify parameters predicting survival after pulmonary metastasectomy, in patients who underwent prior resection of hepatic CRC metastases.Methods: We performed a retrospective analysis of 27 consecutive patients (median age 62 [range 33-75] years) who underwent resection of pulmonary metastases from CRC in two institutions from 1996 to 2009. All patients considered in the analysis had previously undergone colorectal and hepatic surgery with curative intent, and were considered cured in both locations (R0).Results: Median follow-up was 32 (range 3-69) months after resection of lung metastases and 65 (range 19-146) months after resection of primary CRC. Eleven (40?7%) patients had their primary tumors located in the rectum, and 12 (44%) patients presented initially with synchronous liver metastases (stage IV). Median disease-free interval between primary colorectal tumor and development of first metastasis was 6 (range 0-50) months. At the time of last follow-up, seven patients only (26%) were alive without evidence of recurrence. Three- and 5-year overall survival rates after lung surgery were 56% and 39%, respectively. Median survival after pulmonary metastasectomy was 46 months. In multivariate analysis, the number of pulmonary lesions was significantly correlated with survival (log-rank test, p = 0?035).Conclusion: Resection of lung metastases from CRC patients is compatible with prolonged (median = 4 years) survival, even when those patients had undergone prior resection of liver metastases. While prolonged, disease-free survival remains the exception, patients presentin

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.

Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.

Late-onset cytomegalovirus (CMV) disease commonly occurs after discontinuation of antiviral prophylaxis. We determined the utility of testing CD8+ T-cell response against CMV as a predictor of late-onset CMV disease after a standard course of antiviral prophylaxis. Transplant patients at high-risk for CMV disease were enrolled. CD8+ T-cell-mediated immunity (CMI) was tested using the QuantiFERON-CMV assay at baseline, 1, 2 and 3 months posttransplant by measurement of interferon-gamma response to whole blood stimulation with a 21-peptide pool. The primary outcome was the ability of CMI testing to predict CMV disease in the first 6 months posttransplant. There were 108 evaluable patients (D+/R+ n = 39; D-/R+ n = 34; D+/R- n = 35) of whom 18 (16.7%) developed symptomatic CMV disease. At the end of prophylaxis, CMI was detectable in 38/108 (35.2%) patients (cutoff 0.1 IU/mL interferon-gamma). CMV disease occurred in 2/38 (5.3%) patients with a detectable interferon-gamma response versus 16/70 (22.9%) patients with a negative response; p = 0.038. In the subgroup of D+/R- patients, CMV disease occurred in 1/10 (10.0%) patients with a detectable interferon-gamma response (cutoff 0.1 IU/mL) versus 10/25 (40.0%) patients with a negative CMI, p = 0.12. Monitoring of CMI may be useful for predicting late-onset CMV disease.

Hemobilia, a rare cause of acute pancreatitis after percutaneous liver biopsy: diagnosis and treatment by endoscopic retrograde cholangiopancreatography.

We here report the case history of a 75-yr-old woman who developed pancreatitis and recurrent symptomatic, cholestasis-induced hemobilia after percutaneous liver biopsy. An endoscopic sphincterotomy with clot extraction led to relief of symptoms. The risk of hemobilia after percutaneous liver biopsy is less than one per 1000 procedures, and only two cases of acute pancreatitis after percutaneous liver biopsy have previously been reported. To our knowledge, this is the first case in which endoscopic retrograde cholangiopancreatography was used to both diagnostic and therapeutic ends.

Diagnostic immunologique du cancer

Two different types of immunological reaction are of assistance in the diagnosis of cancer: The first is detection of a weak immunological response of the patient toward his own tumor cells. Unfortunately the currently available techniques for the demonstration of humoral or cellular immunological reaction against autologous tumor cells are not reproducible enough to be recommended as routine clinical tests. Secondly, it is possible to use antisera, obtained by immunization of animals with human tumor extracts, for the detection of substances released into the blood by the tumor cells. The two major antigens associated with human cancer that can be measured in the blood by very sensitive immunological methods are the alphafetoprotein (AFP) and the carcinoembryonic antigen (CEA). It is very important for the physician to be fully alive to the usefulness and limitations of such tests in order to interpret them correctly. Clinical situations in which the measurement of AFP and CEA can provide useful information are reviewed.

Changes in biomarkers of liver disease during successful combination antiretroviral therapy in HIV-HCV-coinfected individuals.

BACKGROUND: We investigated changes in biomarkers of liver disease in HIV-HCV-coinfected individuals during successful combination antiretroviral therapy (cART) compared to changes in biomarker levels during untreated HIV infection and to HIV-monoinfected individuals. METHODS: Non-invasive biomarkers of liver disease (hyaluronic acid [HYA], aspartate aminotransferase-to-platelet ratio index [APRI], Fibrosis-4 [FIB-4] index and cytokeratin-18 [CK-18]) were correlated with liver histology in 49 HIV-HCV-coinfected patients. Changes in biomarkers over time were then assessed longitudinally in HIV-HCV-coinfected patients during successful cART (n=58), during untreated HIV-infection (n=59), and in HIV-monoinfected individuals (n=17). The median follow-up time was 3.4 years on cART. All analyses were conducted before starting HCV treatment. RESULTS: Non-invasive biomarkers of liver disease correlated significantly with the histological METAVIR stage (P<0.002 for all comparisons). The mean ±sd area under the receiver operating characteristic (AUROC) curve values for advanced fibrosis (≥F3 METAVIR) for HYA, APRI, FIB-4 and CK-18 were 0.86 ±0.05, 0.84 ±0.08, 0.80 ±0.09 and 0.81 ±0.07, respectively. HYA, APRI and CK-18 levels were higher in HIV-HCV-coinfected compared to HIV-monoinfected patients (P<0.01). In the first year on cART, APRI and FIB-4 scores decreased (-35% and -33%, respectively; P=0.1), mainly due to the reversion of HIV-induced thrombocytopaenia, whereas HYA and CK-18 levels remained unchanged. During long-term cART, there were only small changes (<5%) in median biomarker levels. Median biomarker levels changed <3% during untreated HIV-infection. Overall, 3 patients died from end-stage liver disease, and 10 from other causes. CONCLUSIONS: Biomarkers of liver disease highly correlated with fibrosis in HIV-HCV-coinfected individuals and did not change significantly during successful cART. These findings suggest a slower than expected liver disease progression in many HIV-HCV-coinfected individuals, at least during successful cART.

Impact of donor and recipient IL28B rs12979860 genotypes on hepatitis C virus liver graft reinfection.

BACKGROUND & AIMS: Recent studies have described a major impact of genetic variations near the IL28B gene on the natural course and outcome of antiviral therapy in chronic hepatitis C. We therefore, aimed to explore the impact of donor and recipient genotypes of these polymorphisms on hepatitis C virus (HCV) liver graft reinfection. METHODS: Donor and recipient genotypes of IL28B rs12979860C>T single nucleotide polymorphism were determined in 91 patients with HCV liver graft reinfection, 47 of whom were treated with pegylated interferon-α (PEG-IFN-α) and ribavirin. IL28B genetic polymorphisms were correlated with the natural course and treatment outcome of recurrent hepatitis C. RESULTS: Patients requiring liver transplantation due to end-stage chronic hepatitis C appeared to be selected toward the adverse genotypes rs12979860 CT/TT compared to non-transplanted HCV-infected patients (p=0.046). Patients with the donor genotype rs12979860 CC had higher peak ALT and HCV RNA serum concentrations than those with CT/TT (p=0.04 and 0.06, respectively). No association was observed between ALT/HCV RNA serum concentrations and recipient genotypes (p>0.3). More important, donor IL28B rs12979860 CC vs. CT/TT genotypes were associated with rapid, complete early, and sustained virologic response (RVR, cEVR, SVR) to treatment with PEG-IFN-α and ribavirin (p=0.003, 0.0012, 0.008, respectively), but weaker associations of recipient genotypes with RVR, cEVR, and SVR were observed as well (p=0.0046, 0.115, 0.118, respectively). CONCLUSIONS: We provide evidence for a dominant, but not exclusive impact of the donor rather than the recipient IL28B genetic background on the natural course and treatment outcome of HCV liver graft reinfection.

Screening of the inhibitory effect of xenobiotics on alcohol metabolism using S9 rat liver fractions.

The purpose of this work was to develop and optimize a simple and suitable method to detect the potential inhibitory effect of drugs and medicines on alcohol dehydrogenase (ADH) activity in order to evaluate the possible interactions between medicines and alcohol metabolism. Commonly used medicines that are often involved in court litigations related with driving under the influence of alcohol were selected. Alprazolam, flunitrazepam and tramadol were tested as drugs with no known effect on ADH activity. Cimetidine, reported previously as having inhibitory effect on ADH, and 4-methylpyrazole (4-MP), a well known ADH inhibitor, were tested as positive controls. Apart from 4-MP, tramadol was identified as having the higher inhibitory effect with an IC50 of 44.7×10(-3)mM, followed by cimetidine (IC50 of 122.9×10(-3)mM). Alprazolam and flunitrazepam also reduced liver ADH activity but to a smaller extent (inhibition of 11.8±5.0% for alprazolam 1.0mM and 34.5±7.1% for flunitrazepam 0.04mM). Apart from cimetidine, this is the first report describing the inhibitory effect of these drugs on ethanol metabolism. The results also show the suitability of the method to screen for inhibitory effect of drugs on ethanol metabolism helping to identify drugs for which further study is justified.