Intraoperative radiation therapy as adjuvant treatment in locally advanced stage tumours involving the middle ear: a hypothesis-generating retrospective study.

The objective of this study was to evaluate the safety, effectiveness and functional outcomes of intraoperative radiotherapy (IORT) followed by intensity-modulated radiation therapy (IMRT) in locally advanced stage tumours involving the middle ear. Data on 13 consecutive patients treated for malignant tumor of external auditory canal involving the middle ear were retrospectively reviewed. Median follow-up was 33 months (range 6-133). Five (38%) patients were stage III and 8 (62%) were Stage IV according to the University of Pittsburgh staging system. Lateral temporal bone resection (LTBR) was performed in all cases. LTBR was associated with parotidectomy in 5 (38%) cases, and with neck dissection and parotidectomy in 6 (46%) cases. No patients had gross residual tumour. Surgical treatment was followed by IORT (12 Gy) and IMRT (50 Gy). Adjuvant chemotherapy was used in 4 (30%) cases. Preoperative and postoperative audiometric tests were performed to assess hearing loss. 5-year local-control (LC), 5-year distant-metastasis (DM), 5-year disease-free-survival (DFS) and 5-year overall-survival (OS) were calculated with Kaplan-Meyer method. Significant changes in bone conduction were reported after treatment. Partial flap necrosis was the only early complication observed in three (23%) cases, while meningeal fistula was seen in one (7.6%) case as a late complication. The 5-year LC-rate was 68%. The 5-year DM-rate was 90%. The 5-year DFS-rate was 61%. The 5-year OS-rate was 69%. IORT followed by IMRT for the treatment of advanced external auditory canal and middle ear tumours seems to be safe. No intraoperative death was reported. IORT may reduce the postoperative irradiation of remnant tissue obtaining the same full dose on the tumour bed. No complications of the residual external ear were observed. Detriment of neurosensory hearing may be expected. Future studies are required to confirm the benefit of this procedure in the ear.

Activity and safety of bevacizumab plus fotemustine for recurrent malignant gliomas.

BACKGROUND: No established chemotherapeutic regimen exists for the treatment of recurrent malignant gliomas (rMGs). Herein, we report the activity and safety results of the bevacizumab (B) plus fotemustine (FTM) combination for the treatment of rMGs. PATIENTS AND METHODS: An induction phase consisted of B 10 mg/kg days 1, 15 plus FTM 65 mg/m(2) days 1, 8, and 15. Nonprogressive patients entered the maintenance phase with B 10 mg/kg plus FTM 75 mg/m(2) every 3 weeks. The primary endpoint was response rate; secondary endpoints included safety, progression free survival (PFS), and overall survival (OS). RESULTS: Twenty-six patients affected by recurrent MGs (50% glioblastoma) were enrolled. Eight partial responses (31%) were observed. Median PFS and OS were 4 (95% C.I.: 2.8-5.1) and 6 months (95% C.I.: 4.2-7.8), respectively. Responses were significantly associated with both improved PFS and OS (P = 0.002 and P = 0.001, resp.). Treatment adverse events were mostly mild to moderate in intensity. Bevacizumab-related adverse events included grade 3 venous thromboembolic event (8%), grade 2 epistaxis (4%), hypertension (8%), and gastrointestinal perforation (4%). CONCLUSIONS: Bevacizumab plus FTM showed activity and good tolerability in pretreated MGs. Further investigations are needed in order to verify the benefits deriving from the addition of B to a cytotoxic in this clinical setting of patients.

Anatomical and dose changes of gross tumour volume and parotid glands for head and neck cancer patients during intensity-modulated radiotherapy: effect on the probability of xerostomia incidence.

AIMS: To quantify the changes in dose as well as in the prediction of parotid gland toxicity due to anatomical changes during therapy of head and neck cancer patients. MATERIALS AND METHODS: Fifteen patients with advanced locoregional head and neck cancer, with no evidence of distant metastasis, were enrolled in a prospective study. All patients were treated with intensity-modulated radiotherapy. Multiple computed tomography scans were repeated at the end of each treatment week. The original treatment plans were copied to the per-treatment scans to create hybrid plans. The normal tissue complication probability (NTCP) was calculated assuming the end point to be grade ≥3 xerostomia according to the Radiation Therapy Oncology Group late toxicity scale. RESULTS: The gross tumour volume dose coverage was slightly affected by the anatomical changes, whereas the mean dose (D(mean)) to the parotids changed from 26.1 ± 6.0 to 27.4 ± 7.4 Gy, with a mean increase of 0.22 Gy/treatment week. Consequently, the mean NTCP increased from 0.15 ± 0.06 to 0.18 ± 0.10, primarily due to a few patients exhibiting a marked increase. The absolute gross tumour volume shrinkage and the percentage parotids shrinkage were the best independent predictors for the NTCP variations. CONCLUSIONS: On average, the increase in the parotids D(mean) as well as in NTCP during treatment is limited, and the observed variations were strongly patient-dependent.

Morphological features and modulation of the intrahepatic biliary epithelium.

Much has been learned in the past few years concerning the morphology and function of the intrahepatic biliary epithelium. Immunohistochemistry, together with ultrastructural studies has allowed a better identification or the smallest branches of the biliary tree and of subcellular components (cytoskeleton, specializations of the cell membrane, specific receptors). Modulation of the biliary epithelium in response to physiological or pathological stimuli has renewed the interest concerning the existence of facultative stem cells in the liver. More information is needed however concerning the mechanisms of cell loss in vanishing bile duct syndromes.

Primary biliary cirrhosis: modalities of injury and death in biliary epithelium.

BACKGROUND/AIM: Despite the number of studies on primary biliary cirrhosis, contrasting data remain concerning modalities of cholangiocyte death. Liver biopsies obtained from 40 patients with anti mitochondrial antibody-positive primary biliary cirrhosis, at various stages of the disease, were examined, and special attention was paid to the expression of subcellular damage and evidence of apoptosis. METHODS: Liver sections were stained with haematoxylin/eosin or Sirius red. Ductular mass was evaluated on sections after cytokeratin 7 staining. Apoptosis was evaluated on haematoxylin/eosin stained material or after processing for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling assay. In 16 patients, part of the biopsy was processed for electron microscopy. Twenty histologically normal liver biopsies were used for control purposes. RESULTS: According to Scheuer's classification, 29 patients were classified as stage I-II, and 11 as stage III-IV. A strong staining of bile ducts was evident after immunohistochemistry for cytokeratin 7, often associated with ductular metaplasia in lobular zone 1. Cytokeratin 7-positive cells occupied 3.0+/-1.3% of liver mass as compared to 0.25+/-0.03% in controls. Ductular metaplasia accounted for 1.4+/-0.07% of all cytokeratin 7-positive cells. Regardless of staging, apoptotic bodies were seen only exceptionally in epithelial wall of bile ducts, whereas cholangiocyte damage leading to extensive lytic necrosis appeared responsible for most of the bile duct mass loss, as also confirmed by ultrastructural studies. A few terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling-positive nuclei were occasionally associated with the inflammatory infiltrate and evidence of apoptosis in hepatocytes was frequent, especially in zone 1. CONCLUSION: Regardless of staging, lytic necrosis and not apoptosis accounts for most of the bile duct loss in primary biliary cirrhosis. Furthermore, ductular metaplasia appears as a late event with highly variable pattern being observed between patients.

The function of alkaline phosphatase in the liver: regulation of intrahepatic biliary epithelium secretory activities in the rat.

We studied the effects of alkaline phosphatase (AP) on the secretory processes of the rat intrahepatic biliary epithelium as well as the role of the intrahepatic biliary epithelium in the uptake and biliary secretion of exogenous AP. The effects of acute and chronic administration of AP on bile secretory parameters were investigated in vivo in normal and bile duct ligated (BDL) rats and in vitro in isolated rat bile duct units (IBDU). In vivo, acute AP administration decreased bile flow and biliary bicarbonate excretion and abolished secretin choleresis in BDL rats but not in normal rats. On the contrary, the AP inhibitor, levamisole, increased in BDL rat bile flow and biliary bicarbonate excretion. In vitro, basal and secretin-stimulated Cl(-)/HCO(3)(-) exchanger activity in IBDU was immediately inhibited by AP intraluminal microinjection (apical exposure) but only after a prolonged exposure to the basolateral pole. Levamisole increased the Cl(-)/HCO(3)(-) exchanger activity of IBDU. A significant basolateral uptake of AP occurs in IBDU with a progressive transport to the apical domain. AP chronic treatment increased AP and gamma-glutamyltranspeptidase (gamma-GT) activities in the intrahepatic bile ducts and hepatocyte canalicular pole, promoted enlargement of bile canaliculi, and decreased bile flow and biliary bicarbonate excretion. In conclusion, the intrahepatic biliary epithelium plays a role in the uptake and biliary secretion of serum AP. AP inhibits the secretory processes of the intrahepatic biliary epithelium and induces features of intrahepatic cholestasis after chronic administration. These findings indicate that AP plays an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium.

The role of proton therapy in the treatment of large irradiation volumes: a comparative planning study of pancreatic and biliary tumors.

PURPOSE: The purpose of this study was to examine the potential benefit of proton therapy for abdominal tumors. Extensive comparative planning was conducted investigating the most up-to-date photon and proton irradiation technologies. METHODS AND MATERIALS: A number of rival plans were generated for four patients: two inoperable pancreatic tumors, one inoperable and one postoperative biliary duct tumor. The dose prescription goal for these large targets was 50 Gy, followed by a boost dose up to 20 Gy to a smaller planning target volume (PTV). Photon plans were developed using "forward" planning of coplanar and noncoplanar conformal fields and "inverse" planning of intensity-modulated (IM) fields. Proton planning was simulated as administered using the so called spot-scanning technique. Plans were evaluated on the basis of normal tissues' dose-volume constraints (Emami B, Lyman J, Brown A, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys 1990;21:109-122) and coverage of treatment volumes with prescribed doses. RESULTS: For all cases, none of the forward calculated photon plans was able to deliver 50 Gy to large PTVs at the same time respecting the dose-volume constraints on all critical organs. Nine evenly spaced IM fields achieved or nearly achieved all maximum dose constraints to critical structures for two out of three inoperable patients. IM plans also obtained good results for the postoperative patient, even though the dose to the liver was very close to the maximum allowed. In all cases, photon irradiation of large PTV1s to 50 Gy followed by a 20 Gy boost entailed a risk very close to or higher than 5% for serious complications to the kidneys, liver, or bowel. Simple arrangements of 2, 3, and 4 proton fields obtained better dose conformation to the target, allowing the delivery of planned doses including the boost to all patients, without excessive risk of morbidity. Dose homogeneity inside the targets was also superior with protons. CONCLUSION: For the irradiation of large PTVs located in the abdominal cavity, where multiple, parallel structured organs surround the target volumes, proton therapy, delivered with a sophisticated isocentric technique, has the potential to achieve superior dose distributions compared with state-of-the-art photon irradiation techniques. IM photon plans obtain better results in the postoperative case, because the reduced volume lessens the effect of the unavoidable increase of integral dose to surrounding tissues.

Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.

We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on D-myo-Inositol 1,4,5-triphosphate (IP(3)) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3', 5'-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca(2+) chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKCalpha, we evaluated (1) for the presence of PKCalpha in cholangiocytes and (2) the effect of gastrin on the PKCalpha protein expression in a triton-soluble (containing cytoplasm + membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP(3) levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKCalpha from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP(3), Ca(2+), and PKCalpha.

Subtype specific regulation of human vascular alpha(1)-adrenergic receptors by vessel bed and age.

BACKGROUND: alpha(1)-adrenergic receptors (alpha(1)ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (alpha(1a), alpha(1b), alpha(1 d)) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human alpha(1)AR subtype expression differs with vascular bed, and (2) age influences human vascular alpha(1)AR subtype expression. METHODS AND RESULTS: Five hundred vessels from 384 patients were examined for alpha(1)AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel alpha(1)AR density is 16+/-2.3fmol/mg total protein. alpha(1a)AR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, alpha(1)AR mRNA subtype expression varies with vessel bed (alpha(1a) higher in splanchnic versus central arteries, P<0.05); competition analysis (selected vessels) and functional assays demonstrate alpha(1a) and alpha(1b)-mediated mammary artery contraction. Overall alpha(1)AR expression doubles with age (<55 versus > or = 65 years) in mammary artery (no change in saphenous vein), accompanied by increased alpha(1b)>alpha(1a) expression (P< = 0.001). CONCLUSIONS: Human vascular alpha(1)AR subtype distribution differs from animal models, varies with vessel bed, correlates with contraction in mammary artery, and is modulated by aging. These findings provide potential novel targets for therapeutic intervention in many clinical settings.

Effect of pharmacological modulation of liver P-glycoproteins on cyclosporin A biliary excretion and cholestasis: a study in isolated perfused rat liver.

In different cell types P-glycoproteins (P-gp) are involved in the transport of cyclosporin A (CyA). The aim of this study was to evaluate the effect of the pharmacological modulation of the hepatic P-gp on biliary secretion of CyA and on cholestasis induced by acute administration of CyA in the isolated perfused rat liver (IPRL). Verapamil was used as a P-gp specific inhibitor and acetylaminofluorene (AAF) as a P-gp inducer. CyA biliary excretion was determined by administering in the IPRL a tracer dose of [3H]CyA with or without verapamil or AAF. The effect on bile flow was evaluated by administering increasing doses of CyA (2.8, 8, and 20 mg/kg body wt) in the IPRL. Morphological evidence of damage was evaluated by optical and electron microscopy in the liver as well as in primary culture of rat hepatocytes exposed to CyA +/- verapamil. Verapamil significantly inhibited the biliary excretion of a tracer dose of [3H]CyA (0.15+/-0.04 vs 0.33+/-0.07%; P < 0.05). In contrast, pretreatment with AAF significantly increased the biliary excretion of [3H]CyA, (0.61+/-0.10 vs 0.33+/-0.07%; P < 0.05). CyA induced a dose-dependent inhibition of bile flow with a maximal effect at 20 mg/kg CyA (-49.3+/-4.5% decrease of basal bile flow). CyA cholestasis was significantly worsened by the P-gp inhibitor, verapamil (-75.5+/-7.5%; P < 0.05), but it was unaffected by induction of P-gp via AAF pretreatment (-44.9+/-1.7%). During CyA cholestasis, the cumulative biliary excretion of [3H]CyA was lower than in the absence of cholestasis (0.22+/-0.05 vs 0.33+/-0.07%; P < 0.05), was inhibited by verapamil (0.08+/-0.01%; P < 0.05), but was unaffected by AAF (0.23+/-0.05%). No morphological evidence of damage was observed in the liver, and no evidence of cytoskeleton derangement was seen in primary cultures of rat hepatocytes exposed to CyA +/- verapamil. We demonstrated that pharmacological modulation of P-gp may influence the biliary excretion of CyA. The acute cholestatic effect of CyA is worsened by P-gp inhibitors, while it is unaffected by P-gp inducers. This indicates CyA should not be given with other P-gp substrates or inhibitors.

Hepatic stellate cell activation and liver fibrosis are associated with necroinflammatory injury and Th1-like response in chronic hepatitis C.

BACKGROUND/AIMS: The involvement of a direct viral cytopathic effect or an immune-mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA-positive untreated patients with chronic hepatitis. METHODS: Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, interferon gamma (IFNgamma)-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry. RESULTS: HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The alphaSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNgamma-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load. CONCLUSIONS: HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1-like response, a plausible target for the treatment of chronic hepatitis C.

Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats.

BACKGROUND & AIMS: To investigate the role of the cholinergic system in regulation of cholangiocyte functions, we evaluated the effects of vagotomy on cholangiocyte proliferation and secretion in rats that underwent bile duct ligation (BDL rats). METHODS: After bile duct ligation (BDL), the vagus nerve was resected; 7 days later, expression of M3 acetylcholine receptor was evaluated. Cholangiocyte proliferation was assessed by morphometry and measurement of DNA synthesis. Apoptosis was evaluated by light microscopy and annexin-V staining. Ductal secretion was evaluated by measurement of secretin-induced choleresis, secretin receptor (SR) gene expression, and cyclic adenosine 3',5'-monophosphate (cAMP) levels. RESULTS: Vagotomy decreased the expression of M3 acetylcholine receptors in cholangiocytes. DNA synthesis and ductal mass were markedly decreased, whereas cholangiocyte apoptosis was increased by vagotomy. Vagotomy decreased ductal secretion. Forskolin treatment prevented the decrease in cAMP levels induced by vagotomy, maintained cholangiocyte proliferation, and decreased cholangiocyte apoptosis caused by vagotomy in BDL rats. Cholangiocyte secretion was also maintained by forskolin. CONCLUSIONS: Vagotomy impairs cholangiocyte proliferation and enhances apoptosis, leading to decreased ductal mass in response to BDL. Secretin-induced choleresis of BDL rats was virtually eliminated by vagotomy in association with decreased cholangiocyte cAMP levels. Maintenance of cAMP levels by forskolin administration prevents the effects of vagotomy on cholangiocyte proliferation, apoptosis, and secretion.

Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.

Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4 damages only large ducts. CCl4 or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4 administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4 converts to its radicals) only in large cholangiocytes. CCl4 induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver.

The aim of this study was to develop a model of selective duct damage restricted to hormone-responsive segments corresponding to the ducts damaged in primary biliary cirrhosis (PBC). Carbon tetrachloride (CCl4) was fed by gavage to rats, and 2, 7, 14, and 28 days later, small and large cholangiocytes were isolated. Apoptosis was determined in situ by morphology and in purified cholangiocytes by assessment of nuclear fragmentation by 4, 6-diamidino-2-phenylindole (DAPI) staining. Cholangiocyte proliferation was evaluated in situ by morphometry of liver sections stained for cytokeratin-19 (CK-19) and by proliferating cellular nuclear antigen (PCNA) staining in liver sections and in purified cholangiocytes by PCNA gene expression. Ductal secretion was assessed by measurement of secretin receptor (SR) gene expression and secretin-induced cyclic adenosine 3',5'-monophosphate (cAMP) synthesis and secretin-induced choleresis. Two days after CCl4 administration, there was an increased number of small ducts, but a reduction of large ducts. Apoptosis, observed only in large ducts, was associated with decreased DNA synthesis and ductal secretion. Conversely, small cholangiocytes expressed de novo the SR gene and secretin-stimulated cAMP synthesis 2 days after CCl4 treatment. Proliferation of large cholangiocytes was delayed until 7 days, which was associated with a transient increase in ductal secretion in vivo. CCl4 effects on cholangiocytes were reversed by day 28. CCl4 treatment causes a decrease in large duct mass as a result of a higher rate of apoptosis and absence of initial proliferation in large cholangiocytes. These processes were concomitant with a decrease of ductal secretion in large cholangiocytes. Small cholangiocytes appear resistant to CCl4-induced apoptosis, and proliferate and transiently compensate for loss of proliferative and secretory activity of large cholangiocytes.

The significance of apoptosis in the liver.

Apoptosis is a form of cell death which occurs in normal as well as in pathological tissues. We provide a description of the morphological changes during apoptosis and an overview of the role of apoptosis dysregulation in the pathogenesis of non-neoplastic liver diseases.

Radiotherapy, local control and survival in brain tumors.

High grade glial brain tumors and brain metastases are a complex subject with still unsatisfactory therapeutic results for the frequent absence of early and precise diagnosis as well as for the limited therapeutic interval between the tumor and presumed healthy tissues. The therapeutic problems of cellular hypoxia, the rapid recovery of sublethal damage for neoplastic cells, the rapid regrowth, have led to a number of efforts to deliver the dose of radiotherapy in various associations. The constant technological trend to increase the high dose gradient to the peripheral tumor, is reported.

Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice.

Hepatocellular carcinoma (HCC) frequently overexpresses the MDR1 gene and is resistant to drugs transported by the multidrug-resistance efflux pump. A xanthine analog, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (CT-2584,CTI), is cytotoxic to many tumors in culture and was four times more effective than verapamil in inhibiting Rhodamine 123 secretion in MDR1-overexpressing Chinese hamster ovary cells. However, studies using PRF/PLC/5 (Alexander) cells revealed that CT-2584 is cytotoxic by another mechanism not involving inhibition of MDR1 function. Alexander cells have integrated the hepatitis B surface antigen (HBsAg) gene and quantitatively secrete HBsAg. The parent cell line, Alex 0, has low MDR1 expression and is drug-sensitive, whereas a derived line, Alex 0.5, is drug-resistant and overexpresses MDR1 100 times. Both cell lines were similarly killed within 24 or 48 hours by CT-2584. Freshly isolated rat and human hepatocytes were considerably more resistant to killing by CT-2584. In vivo, CT-2584 significantly reduced tumor growth in SCID mice bearing Alex 0 or 0.5 xenografts as determined by serial measurements of HBsAg. Hepatic parenchyma was normal, whereas apoptosis and cellular loss were observed in xenografts. The xenograft model is useful for testing pharmacological therapy of HCC.

Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.

UNLABELLED: We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 microM) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)- ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (50 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM). CONCLUSIONS: M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.

Organ preservation in the management of pancreatic cancer.

Over a 5-year period, 6 patients with potentially resectable (cT2) pancreatic adenocarcinoma underwent exclusive radiotherapy. Surgery was ruled out because of the patients' age (> 75 years) in 5 cases and for contraindication for anesthesia in the remaining one. Four patients underwent intraluminal brachytherapy (50 Gy) with linear 192Ir sources; the remaining two underwent concomitant radiochemotherapy (39.6 and 50.4 Gy respectively) followed by a boost dose (50 and 20 Gy respectively) of intraluminal brachytherapy. All patients completed the treatment without relevant acute side-effects. One patient undergoing combined treatment showed gastric ulcer 8 months after treatment. Two patients showed local progression of the disease and two distant metastases. In one of the two patients with local progression, digestive by-pass was required. All patients died. Median survival was of 14 months. Actuarial survival at 1, 2 and 3 years was 66%, 33% and 16% respectively. No patient showed pain during follow-up. The fairly good quality of life observed in these patients undergoing conservative treatment and the rate of survival, comparable with that of surgical series, suggest that irradiation should be considered in those patients in whom radical surgery in contraindicated.