[Suppurated hepatic metastasis revealing a colonic stromal tumor].
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Colonic Neoplasms↗
Biomedical subjects
D Franco
Publications and source records attributed to D Franco.
[Should surgeons get involved in research?].
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Animals↗
[Allotransplantation in utero and immortalization of primate fetal hepatocytes].
We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.
Animals↗
[Gallstones and their complications].
Gallbladder stones are present in more than 10% of the population in Europe, particularly in women. Most patients are and will remain asymptomatic. No treatment is required. Patients with gallstones and biliary colics should have elective laparoscopic cholecystectomy which can be performed in almost every case. Acute gall-stone cholecystitis is the most severe complication. It should prompt urgent cholecystecomy that can be done by laparoscopy. Other complications such as biliobiliary fistulas have become unfrequent.
Cholecystectomy↗
Multiple transcriptional domains, with distinct left and right components, in the atrial chambers of the developing heart.
During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.
Aging↗
Chamber formation and morphogenesis in the developing mammalian heart.
In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.
Animals↗
Suppression of atrial myosin gene expression occurs independently in the left and right ventricles of the developing mouse heart.
Many cardiac genes are broadly expressed in the early heart and become restricted to the atria or ventricles as development proceeds. Additional transcriptional differences between left and right compartments of the embryonic heart have been described recently, in particular for a number of transgenes containing cardiac regulatory elements. We now demonstrate that three myosin genes which become transcriptionally restricted to the atria between embryonic day (E) 12.5 and birth, alpha-myosin heavy chain (MHC), myosin light chain (MLC) 1A and MLC2A, are coordinately downregulated in the compact myocardium of the left ventricle before that of the right ventricle. alpha-MHC protein also accumulates in the right, but not left, compact ventricular myocardium during this period, suggesting that this transient regionalization contributes to fktal heart function. dHAND and eHAND, basic helix-loop-helix transcription factors known to be expressed in the right and left ventricles respectively at E10. 5, remain regionalized between E12.5 and E14.5. Downregulation of alpha-MHC, MLC1A, and MLC2A in iv/iv embryos, which have defective left/right patterning, initiates in the morphological left (systemic) ventricle regardless of its anatomical position on the right or left hand side of the heart. This points to the importance of left/right ventricular differences in sarcomeric gene expression patterns during fktal cardiogenesis and indicates that these differences originate in the embryo in response to anterior-posterior patterning of the heart tube rather than as a result of cardiac looping. Dev Dyn 2000;217:75-85.
Animals↗
An atrioventricular canal domain defined by cardiac troponin I transgene expression in the embryonic myocardium.
During early cardiac development the atrial myocardium is continuous with the ventricular myocardium throughout the atrioventricular canal. The atrioventricular canal undergoes complex remodelling involving septation, formation of atrioventricular valves and insulation between atria and ventricles except at the level of the atrioventricular node. Understanding of these processes has been hampered by the lack of markers specific for this heart region. We have generated transgenic mice expressing beta-galactosidase under the control of the cardiac troponin I gene that show transgene expression mainly confined to the atrioventricular canal myocardium during early embryonic development. With further development beta-galactosidase positive cells are observed in the atrioventricular node and in the lower rim of both right and left atria, supporting the view that atrioventricular canal myocardium contributes to the atrioventricular node and is in part incorporated into the lower rim of the atria. These results identify the atrioventricular canal myocardium as a distinct transcriptional domain.
Animals↗
[Effect of portal branch ligation on liver regeneration in the rat].
GOAL: The aim of this study was to assess liver regeneration after partial portal ligation. METHODS: 70% partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry. RESULTS: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximum at 28 hours with a peak at 12.5% and was prolonged beyond the 48th hour. CONCLUSIONS: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.
Animals↗
Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells.
We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.
Carcinoma, Hepatocellular↗
Evaluation of extracts from Gevuina avellana hulls as antioxidants.
The antioxidant activity of the extracts from Gevuina avellana hulls was evaluated and compared with that of BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole), using the beta-carotene bleaching assay, the accelerated oxidation of crude soybean oil, and the 2,2-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging method. Solvents of different polarity were used to obtain the extracts. Both the extraction yield and the antioxidant activity were strongly dependent on the solvent. The ethanol and diethyl ether soluble fractions were the most active with the beta-carotene assay. Ethanol and methanol extracts were the most active in hydrogen radical scavenging activity. Water and methanol inhibited more efficiently the oxidation of soybean oil at 70 and 80 degrees C, respectively. As a general trend, increased antioxidant activity was observed for increased extract concentration. Except the acetone extracts, all were stable after 6 months storage at 4 degrees C. The ethanol solubles from G. avellana hulls present antioxidant activity similar to that of synthetic antioxidants and to other reported residual agroindustrial materials.
Antioxidants↗
Analgesic effect of low-dose intrathecal morphine and bupivacaine in laparoscopic cholecystectomy.
We assessed the peri-operative analgesic efficiency of low-dose intrathecal morphine combined with a low dose of bupivacaine after elective laparoscopic cholecystectomy since postoperative pain in such procedures, although less than after a conventional open technique, may be significant, particularly during the first 12-24 h. After informed consent, 34 ASA I or II patients were randomly allocated to one of two groups to receive either a lumbar intrathecal injection of morphine (75 or 100 microg) combined with 5 mg of isobaric bupivacaine (spinal group) or a subcutaneous injection of a saline solution (control group). Intra-operatively, opioid requirements, blood pressure response and heart rate changes after insufflation were recorded. Postoperatively, morphine requirements, pain scores and opioid-related side-effects were assessed by a physician blinded to the randomisation. Intra-operative opioid requirements did not differ significantly between groups. Mean (SD) postoperative morphine requirements were significantly lower in the spinal group [13 (10) vs. 23 (10) mg; p = 0.04] as were postoperative pain scores (p < 0.001). Side-effects were of comparable incidence and severity between groups. Low-dose intrathecal morphine combined with low-dose bupivacaine provided effective postoperative analgesia for elective laparoscopic cholecystectomy.
Adult↗
Activation of V(D)J recombination induces the formation of interlocus joints and hybrid joints in scid pre-B-cell lines.
V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/+) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/+ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.
Abelson murine leukemia virus↗
Radio-isotopic in situ hybridization on tissue sections. Practical aspects and quantification.
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Animals↗
Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.
To determine the frequency of Wnt/Wingless beta catenin pathway alteration in human hepatocellular carcinoma, a beta catenin and APC gene mutation screening was performed in a series of 119 tumors. An activating beta catenin mutation in exon 3 was found in 18% of the cases. Among tumors lacking beta catenin mutation, no APC mutation has been evidenced in a subset of 30 cases tested. The correlation between beta catenin mutation status and chromosome segment deletions was studied on a set of 48 hyperploid tumors. Chromosome 1p, 4q and 16p deletions were significantly associated with the absence of beta catenin mutation (P<0.05). Furthermore the Fractional Allelic Loss was significantly smaller in the beta catenin mutated tumors than in the non-mutated tumors (0.12 versus 022). Taken together, these results suggest, the existence of two carcinogenesis mechanisms. The first mechanism implies a beta catenin activating mutation associated with a low rate of loss of heterozygosity. The second mechanism, operating in a context of chromosomal instability, would involve tumor suppressor genes.
Aneuploidy↗
Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas.
To explore the role of hepatitis B virus (HBV) X protein in liver carcinogenesis, independently from its role in viral replication, we have analyzed X gene structure and expression in tumorous and non-tumorous tissues obtained from 9 hepatitis B surface antigen (HBsAg)-negative, HBV DNA-positive patients. HBV replication was undetectable in tumorous tissues. HBV X gene was truncated at its 3' end in 5 of 9 tumorous tissues and 1 of 8 non-tumorous livers. Sequence analysis performed on uninterrupted X genes from 3 tumors and 3 surrounding non-tumorous tissues showed a high rate of mutations, selectively in the tumorous livers. In 1 of the 3 tumors, a frameshift mutation induced a new stop at codon 129. HBV RNAs were tested by reverse transcriptase-polymerase chain reaction (RT-PCR) with surface (S), core (C) and X specific primers. X, but not S and C, RNA expression was found in 6 of 8 tumors and in 6 of 7 non-tumorous tissues. This finding was consistent with immunohistochemical detection of X, but not S and C, antigens in all tumors also expressing X RNA. Our results provide evidence for selective expression of HBV X, but not S and C, RNA and protein in the tumorous and non-tumorous tissue of HBsAg-negative, HBV DNA-positive patients. It also shows that the structure of the X gene is modified (interrupted or highly mutated) in the majority of tumorous livers. Taken together, our findings are consistent with a potential role of mutated X proteins in HBV-related liver oncogenesis.
Adolescent↗
Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart.
The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium.
Animals↗
Liver resection and needle liver biopsy cause hematogenous dissemination of liver cells.
We have investigated whether liver resection and needle liver biopsy cause dissemination of liver cells into peripheral blood circulation, using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay targeted against alpha-fetoprotein (AFP) mRNA. Twelve patients with and 16 without primary liver cancer (PLC) undergoing liver resection were tested before skin incision, after liver mobilization, after hepatic parenchyma transection, after abdominal wall suture, and 4 days after surgery. Two patients with and 20 without PLC were tested before, 20 minutes after, and 24 hours after needle liver biopsy. Six of 14 patients with and 0 of 36 patients without PLC scored positive before intervention (P <.001). Liver cell spreading was induced at different times after surgery and liver biopsy in 14 of 14 patients with but also 23 of 36 without PLC (P <.05). We conclude that liver resection and needle liver biopsy induce release of cells from the liver, which are not necessarily liver tumor cells, into the peripheral blood circulation. This may be, however, an important mechanism of liver cancer cell dissemination deserving further investigations.
Adult↗