Jean-Pierre Campion

High yield preparation of isolated human adult hepatocytes by enzymatic perfusion of the liver

Abstract We have developed experimental conditions allowing the isolation of as many as 1–10 × 10 8 human adult hepatocytes by perfusing the left lobe of the liver. About 70–85% cells were viable with the trypan blue exclusion test; they attached to substrates and formed monolayers which survived for up to 8 days in culture.

Primary liver cancer in genetic hemochromatosis: A clinical, pathological, and pathogenetic study of 54 cases

BACKGROUND Although liver cancer arises frequently in the course of genetic hemochromatosis (GH), it has not been previously studied in a large series of patients with well-defined GH. METHODS The bioclinical and pathological data from 1 cholangiocarcinoma and 53 hepatocellular carcinomas (HCCs) complicating GH in 32 untreated and 22 de-ironed patients are reported. RESULTS This study (1) adds three new well-documented cases of HCC in noncirrhotic but only fibrotic hemochromatotic liver, (2) shows the high prevalence (83%) of proliferative and often dysplastic (70%) iron-free foci in the nontumorous liver of untreated patients, and (3) emphasizes the significant increase of cirrhosis (81% vs. 28%) and of associated noniron-related risk factors, mainly chronic alcoholism (48% vs. 25%) and tobacco smoking (50% vs. 18%) in patients with HCC compared with matched hemochromatotic patients without HCC. CONCLUSIONS These data (1) suggest that iron-free foci may be markers of an early stage of HCC in GH and (2) supply the basis for defining a cost-effective policy for the screening of HCC in GH patients.

The Effects of Small-Dose Ketamine on Morphine Consumption in Surgical Intensive Care Unit Patients After Major Abdominal Surgery

In a randomized, double-blinded study, we evaluated the analgesic effect of ketamine in the management of pain in a surgical intensive care unit after major abdominal surgery. Patients received morphine patient-controlled analgesia with either placebo (Group M) or ketamine (Group K). Morphine was administered with initial loading doses of 2 mg until the visual analog scale (VAS) score was <30 and thereafter with bolus doses of 1 mg and a lockout time of 7 min. Ketamine was administered with an initial bolus of 0.5 mg/kg followed by a perfusion of 2 &mgr;g · kg−1 · min−1 during the first 24 h and 1 &mgr;g · kg−1 · min−1 during the following 24 h. The 4-h cumulative morphine doses were measured over 48 h. The VAS scores at rest and at mobilization were measured every 4 h during 48 h. A total of 101 patients were enrolled, and 93 were analyzed (41 in Group K and 52 in Group M). VAS scores at rest and at mobilization were similar. The cumulative consumption of morphine was significantly smaller in Group K (P < 0.05). We concluded that small doses of ketamine were a valuable adjunct to opioids in surgical intensive care unit patients after major abdominal surgery.

Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads : A step toward a reliable bioartificial liver

Orthotopic liver transplantation is the most effective treatment for fulminant hepatic failure. As an alternative treatment, an efficient extracorporeal bioartificial liver should contain a large yield of functional hepatocytes with an immunoprotective barrier, for providing temporary adequate metabolic support to allow spontaneous liver regeneration or for acting as a bridge toward transplantation. Survival, proliferation, and functions of porcine hepatocytes were evaluated in primary cultures and after embedding in alginate beads, which were subsequently coated with a membrane made by a transacylation reaction between propylene glycol alginate and human serum albumin. Disruption of total pig livers by collagenase perfusion/recirculation allowed the obtention of up to 10(11) hepatocytes with a viability greater than 95%. Hepatocytes in conventional cultures or embedded in coated alginate beads survived for about 10 days, secreted proteins, particularly albumin, and maintained several phase I and II enzymatic activities, namely ethoxyresorufin-O-deethylase, oxidation of nifedipine to pyridine, phenacetin deethylation to paracetamol, glucuroconjugation of paracetamol, and N-acetylation of procainamide. Typical features of mitosis and [3H]thymidine incorporation indicated that porcine hepatocytes proliferated in both conventional cultures and alginate beads. The efficacy of the membrane surrounding alginate beads for protecting cells from immunoglobulins was tested by embedding HLA-typed human lymphocytes, which were subsequently incubated with specific anti-HLA immunoglobulin G and complement. These data show that large yields of porcine hepatocytes that are embedded in coated alginate beads remain functional and are isolated from large molecular weight molecules, such as immunoglobulins. This system represents a promising tool for the design of an extracorporeal bioartificial liver, containing xenogeneic hepatocytes, to treat acute liver disease in humans.

Correction of Bilirubin Conjugation in the Gunn Rat Using Hepatocytes Immobilized in Alginate Gel Beads as an Extracorporeal Bioartificial Liver

A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat. Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca++-alginate beads. The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures. The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group). Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals. Both bilirubin monoconjugales and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography. Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%). In the test group, the concentration of bilirubin conjugates increased rapidly, attaining median values of 72.26 pM and 92.59 pM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation. In the control group, the levels of either conjugate did not exceed 0.87 μM throughout the experiments. Statistical analysis showed a significant difference between the two groups (p < 0.0023). These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rats genetic defect. Such a system might be of therapeutic interest in acute liver failure.

Detoxifying activity in pig livers and hepatocytes intended for xenotherapy.

BACKGROUND Both livers and hepatocytes from pigs have been proposed for the treatment of end-stage liver diseases, as an alternative to allogeneic liver transplants. However, little is known of the capability of porcine hepatocytes to fulfill the biotransformation pathways of toxic compounds, including those released from livers in acute failure. We have studied the activity and expression of detoxifying enzymes in porcine livers and in cultured hepatocytes and their induction by phenobarbital. METHODS Cytochromes P450 (CYP) 1A, 2B, and 3A and GST-like activities were tested with the following specific substrates: 7-ethoxyresorufin, 7-pentoxyresorufin, nifedipine, testosterone, 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrinic acid. CYP 1A1/2-, 2B1/2-, 2E1- and 3A4-related and GSTalpha proteins were analyzed by Western blotting and CYP 1A1/2, 2B1/2, 2C6, 2E1, and 3A4, aldehyde dehydrogenase, epoxide hydrolase, and GSTalpha-like RNA by Northern blotting. RESULTS Enzymatic activities reflecting the expression of CYP 1A-, CYP 2B-, CYP 2E1-, and CYP 3A-like genes, that is, ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase, nifedipine oxidase and testosterone 6beta-hydroxylase, and chlorzoxazone 6-hydroxylase, were identified in pig livers. CYP 1A and CYP 2E1, GSTalpha-like proteins, CYP 1A, 2C, and 2E, epoxide hydrolase, aldehyde dehydrogenase, and GST like RNA were expressed in vivo and in vitro. CYP 2B and CYP 3A RNA and proteins, and their associated activities were induced by phenobarbital. CONCLUSIONS Porcine hepatocytes express the most important biotransformation enzymes and their corresponding activities and RNA. Thus, livers and hepatocytes from pigs can detoxify a large spectrum of exogenous and endogenous compounds, which makes them a convenient substitute for allogeneic transplants for patients with liver failure.

Interleukin 4 inhibits the production of some acute-phase proteins by human hepatocytes in primary culture.

Interleukin 4 (IL4) has been shown to exhibit anti‐inflammatory effects by inhibiting the secretion by monocytes of proinflammatory cytokines such as interieukin 1 (IL1), interleukin 6 (IL6), and tumor necrosis factor (TNF) and by inducing the secretion of the IL1 receptor antagonist. We investigated the role of this cytokine on the production of acute‐phase proteins in primary human hepatocyte cultures. Cells were exposed to either IL4 and/or IL6, the most potent mediator of hepatic acute phase proteins. IL4 led to decreased production of haptoglobin, C‐reactive protein and albumin while α1‐antitrypsin and fibrinogen remained unaffected. These inhibitory effects of IL4 were also observed at the mRNA level. In addition, IL4 inhibited the IL6‐induced production of haptoglobin although it had no effect on the induced C‐reactive protein and fibrinogen. Our results demonstrate that IL4 can affect the production of a subset of acute‐phase proteins by human hepatocytes and can antagonize some of the effects of IL6. These observations reinforce the notion that IL4 can be considered as an anti‐inflammatory cytokine.

A new mutation of E-cadherin gene in familial gastric linitis plastica cancer with extra-digestive dissemination

Over a 12-month period, we diagnosed poorly differentiated infiltrative independent-cell gastric adenocarcinoma in two brothers and one sister aged 41 to 47 years. Their father had died from antral cancer at the age of 34 years. These cancers had two characteristic clinical features: rapid course and distant malignant dissemination. In all three patients, polymerase chain reaction-sequencing of the E-cadherin (CDH1) gene of white blood cells identified a heterozygous nonsense mutation of exon 3, producing a stop codon at position 95 (Q95X), resulting in a truncated protein. The alteration of this protein, which plays a crucial role in epithelial cell adhesion, probably explains the clinical expression in this type of familial diffuse gastric cancer.

Laparoscopic rectopexy for full-thickness rectal prolapse: a single-institution retrospective study evaluating surgical outcome

BackgroundThe laparoscopic approach promises to become the gold standard for the transabdominal management of full-thickness rectal prolapse. The aim of this study was to review our experience and to highlight the functional results achieved with this new technique.MethodsForty-eight patients with full-thickness external prolapse underwent laparoscopic repair between February 1997 and February 2003. All patients underwent preoperative evaluation of their rectal function. Patients with isolated rectal ulcer without prolapse or with internal prolapse and patients deemed by the anesthesiologist to be unfit for general anesthesia were excluded from the study. The laparoscopic technique was either a mesh rectopexy without resection (n = 35) or a suture rectopexy with sigmoid resection (n = 13). Patients with intractable constipation preceding the development of the rectal prolapse were advised to have a resection–rectopexy. In the postoperative follow-up, attention was paid to mortality, morbidity, recurrent prolapse, incontinence, and constipation. Follow-up was done by clinical review and postal questionnaire.ResultsThere were no deaths and no septic or anastomotic complications. The postoperative morbidity rate was 5%. Oral intake was started on postoperative day 1. Discharge from the hospital was on postoperative day 4 in patients without sigmoid resection and on postoperative day 7 in patients with sigmoid resection. Two patients (4%) developed recurrent total prolapse during a median follow-up period of 36 ± 15 months (range, 7–77). The functional results were good or excellent in 72% of the cases, without digitations or dyschesia. Continence was improved in 31% of the patients and remains unchanged in 64% of them. In 11 patients (23%), constipation was worsened by the procedure.ConclusionLaparoscopic rectopexy with or without resection is both safe and effective. Advantages include low-morbidity, improved cosmesis, the rapid return of intestinal function, early discharge from hospital, and a low recurrence rate. The fecal continence score is improved; however, constipation is frequently worsened.

Neoadjuvant chemotherapy and hyperfractionated radiotherapy with concurrent low-dose chemotherapy for squamous cell esophageal carcinoma

PURPOSE We conducted a prospective study of neoadjuvant treatment for squamous cell carcinoma of the esophagus, modifying the chemotherapy protocol by adding l-folinic acid and giving bifractionated radiotherapy with a cis-diaminedichloroplatinum (CDDP) injection before each fraction. METHODS AND MATERIALS Thirty-two patients, 30 men, 2 women, mean age 56.2+/-8.9 years, with resectable squamous cell carcinoma of the esophagus (TNM stage I=4, IIA=4, IIB=13, III=11) were included. Chemotherapy, CDDP (80 mg/m2 D2), 5-fluorouracil (5-FU; 600 mg/m2, D1-4), and l-folinic acid (200 mg/m2, D1-4), was given in two sessions with a 3-week interval during which the patients received radiotherapy (45 Gy), two fractions per day (150 cGy/fraction). A 3-mg injection of CDDP was given prior to each fraction. Patients underwent surgery 4 to 7 weeks after neoadjuvant therapy. RESULTS No severe side effects were observed in 12 patients. Grade 3 effects (WBC, platelets, mucositis) occurred in 16 patients and grade 4 effects (platelets, mucositis) in four including 1 death due to septicemia with an infected catheter. Surgery was performed in 29 patients; 26 had resectable tumors (81%). Operative mortality was 10%. The 26 surgical specimens showed complete response (n=18), persistent microscopic residues (n=4), or not significant modification (n=4). Survival at 1, 2, and 3 years was 81, 61, and 51.6% and disease-free survival was 75, 59, and 54% respectively. CONCLUSIONS This new therapeutic combination is aggressive and associated with a high postoperative mortality but has a remarkable histological effect since complete response was achieved in 56% (95% CI: 39-73%) of the patients and 3-year survival reached 52%, a very high rate in our experience.