Sébastien Lepreux

Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.

UNLABELLED BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis. METHODS Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration. RESULTS Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors. CONCLUSIONS The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.

Detection of C3d-Binding Donor-Specific Anti-HLA Antibodies at Diagnosis of Humoral Rejection Predicts Renal Graft Loss

Antibody-mediated rejection (AMR) is a major cause of kidney graft loss, yet assessment of individual risk at diagnosis is impeded by the lack of a reliable prognosis assay. Here, we tested whether the capacity of anti-HLA antibodies to bind complement components allows accurate risk stratification at the time of AMR diagnosis. Among 938 kidney transplant recipients for whom a graft biopsy was performed between 2004 and 2012 at the Lyon University Hospitals, 69 fulfilled the diagnosis criteria for AMR and were enrolled. Sera banked at the time of the biopsy were screened for the presence of donor-specific anti-HLA antibodies (DSAs) and their ability to bind C1q and C3d using flow bead assays. In contrast with C4d graft deposition, the presence of C3d-binding DSA was associated with a higher risk of graft loss (P<0.001). Despite similar trend, the difference did not reach significance with a C1q-binding assay (P=0.06). The prognostic value of a C3d-binding assay was further confirmed in an independent cohort of 39 patients with AMR (P=0.04). Patients with C3d-binding antibodies had worse eGFR and higher DSA mean fluorescence intensity. In a multivariate analysis, only eGFR <30 ml/min per 1.73 m(2) (hazard ratio [HR], 3.56; 95% confidence interval [CI], 1.46 to 8.70; P=0.005) and the presence of circulating C3d-binding DSA (HR, 2.80; 95% CI, 1.12 to 6.95; P=0.03) were independent predictors for allograft loss at AMR diagnosis. We conclude that assessment of the C3d-binding capacity of DSA at the time of AMR diagnosis allows for identification of patients at risk for allograft loss.

Non-Complement–Binding De Novo Donor-Specific Anti-HLA Antibodies and Kidney Allograft Survival

C1q-binding ability may indicate the clinical relevance of de novo donor-specific anti-HLA antibodies (DSA). This study investigated the incidence and risk factors for the appearance of C1q-binding de novo DSA and their long-term impact. Using Luminex Single Antigen Flow Bead assays, 346 pretransplant nonsensitized kidney recipients were screened at 2 and 5 years after transplantation for de novo DSA, which was followed when positive by a C1q Luminex assay. At 2 and 5 years, 12 (3.5%) and eight (2.5%) patients, respectively, had C1q-binding de novo DSA. De novo DSA mean fluorescence intensity >6237 and >10,000 at 2 and 5 years, respectively, predicted C1q binding. HLA mismatches and cyclosporine A were independently associated with increased risk of C1q-binding de novo DSA. When de novo DSA were analyzed at 2 years, the 5-year death-censored graft survival was similar between patients with C1q-nonbinding de novo DSA and those without de novo DSA, but was lower for patients with C1q-binding de novo DSA (P=0.003). When de novo DSA were analyzed at 2 and 5 years, the 10-year death-censored graft survival was lower for patients with C1q-nonbinding de novo DSA detected at both 2 and 5 years (P<0.001) and for patients with C1q-binding de novo DSA (P=0.002) than for patients without de novo DSA. These results were partially confirmed in two validation cohorts. In conclusion, C1q-binding de novo DSA are associated with graft loss occurring quickly after their appearance. However, the long-term persistence of C1q-nonbinding de novo DSA could lead to lower graft survival.

Expression and cellular localization of fibrillin-1 in normal and pathological human liver.

BACKGROUND The expression and the distribution of fibrillin-1 and elastin were studied in normal and pathological human liver samples. METHODS As controls, histologically normal/subnormal liver samples (n = 24) were used. Pathological samples corresponded to seven cirrhosis and eight hepatocellular carcinomas (HCC) developed on cirrhotic (four) or noncirrhotic (four) liver. RESULTS In normal liver, fibrillin-1 and elastin co-localized in vessel walls and portal tract connective tissue. Fibrillin-1 alone was detected along sinusoids and in portal spaces at the interface with the limiting hepatocytic plates and close to the basement membrane of bile ducts. By transmission electron microscopy, typical bundles of microfibrils were detected both in Disse space and in portal zones. Cirrhotic nodules were usually rich in fibrillin-1 along sinusoids; fibrillin-1 and elastin were co-localized in fibrotic septa surrounding nodules. In HCC, fibrillin-1 was present between tumoral hepatocytes; stromal reaction around the tumors contained both fibrillin-1 and elastin. CONCLUSIONS Fibrillin-1 was associated with elastin in portal mesenchyme and vessel walls of normal liver, in fibrotic septa around cirrhotic nodules and stromal reaction around HCC, but was expressed alone in the perisinusoidal space. The functional roles for fibrillin-1 in non-elastic tissues, such as the liver, remain to be elucidated.

A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis

Ductal plate malformations (DPMs) are developmental anomalies considered to result from lack of ductal plate remodeling during bile duct morphogenesis. In mice, bile duct development is initiated by the formation of primitive ductal structures lined by two cell types, namely ductal plate cells and hepatoblasts. During ductal plate remodeling, the primitive ductal structures mature to ducts as a result from differentiation of the ductal plate cells and hepatoblasts to cholangiocytes. Here, we report this process is conserved in human fetal liver. These findings prompted us to evaluate how DPMs develop in three mouse models, namely mice with livers deficient in hepatocyte nuclear factor 6 (HNF6), HNF1β, or cystin‐1 (cpk [congenital polycystic kidney] mice). Human liver from a patient with a HNF1B/TCF2 mutation, and from fetuses affected with autosomal recessive polycystic kidney disease (ARPKD) were also analyzed. Despite the epistatic relationship between HNF6, HNF1β, and cystin‐1, the three mouse models displayed distinct morphogenic mechanisms of DPM. They all developed biliary cysts lined by cells with abnormal apicobasal polarity. However, the absence of HNF6 led to an early defect in ductal plate cell differentiation. In HNF1β‐deficient liver, maturation of the primitive ductal structures was impaired. Normal differentiation and maturation but abnormal duct expansion was apparent in cpk mouse livers and in human fetal ARPKD. Conclusion: DPM is the common endpoint of distinct defects initiated at distinct stages of bile duct morphogenesis. Our observations provide a new pathogenic classification of DPM. (HEPATOLOGY 2011;)

Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: A novel organization for these secreted proteins

OBJECTIVE Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets. MATERIALS AND METHODS TIMP expression in MKs derived from blood CD34(+) progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting. RESULTS Biosynthesis of TIMPs 1-4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other alpha-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established alpha-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an alpha-granule location. Western blotting confirmed the presence of TIMPs 1-4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in alpha-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial alpha-granules; however, localization of the TIMPs was unchanged. CONCLUSIONS TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in alpha-granule pools.

The identification of small nodules in liver adenomatosis.

BACKGROUND/AIMS Liver adenomatosis is characterized by the presence of multiple adenomas of various sizes in the liver. The aim of this study was to characterize the morphology of small nodules which can be difficult to identify. METHODS Seven patients included in this study underwent surgery for the removal of one or several nodules. All, but one, were females. Three out of seven presented with acute bleeding. Five had six or more nodules at presentation, and two only one, who developed nodules later on during follow-up. RESULTS All of the large nodules that were >2 cm, except one, were typical adenomas with or without hemorrhagic areas. Smaller nodules (1-2 cm), some of which discovered on the resected specimen were either typical adenomas or non-typical nodules. These non-typical nodules were characterized by a polylobulated aspect with steatotic zones, and in between, bands of non-steatotic hepatocytes with portal tracts-like structures containing occasional cytokeratin 7 and less often cytokeratin 19 positive biliary cells. Numerous steatotic foci were also seen in four cases. They were isolated or grouped forming microadenomas or non-typical micronodules (<1 cm) containing biliary elements. Our findings lead to the following hypothesis: adenomatosis is characterized by the simultaneous occurrence of multiple adenomas; if several adenomatous foci expand at the same time in the same area, they will form one polylobulated nodule containing non-adenomatous tissue with portal tracts in between areas of adenomatous tissue (non-typical micronodule). Such a small micronodule may in turn expand and join another micronodule to form a bigger one by the same process (non-typical nodule). As nodules grow, their non-adenomatous components including hepatocytic plates and portal tracts constituents will progressively disappear to end up with the classical aspect of an adenoma. CONCLUSIONS This hypothesis supports the concept that non-typical nodules/micronodules are adenomas precursors. However, they can be difficult to classify since they resemble focal nodular hyperplasia precursor.

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis

OBJECTIVES Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease. METHODS A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied. RESULTS Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation. CONCLUSIONS Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.

Differential expression of the anterior gradient protein-2 is a conserved feature during morphogenesis and carcinogenesis of the biliary tree

Background: The anterior gradient protein‐2 (AGR2) is overexpressed in numerous tumours such as breast, prostate or pancreas carcinomas and recently reported in fibrolamellar hepatocellular carcinoma (HCC).

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices

Background: The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open.