Trends in indications, complications and outcomes for venous resection during pancreatoduodenectomy.

BACKGROUND: Pancreatoduodenectomy with superior mesenteric-portal vein resection has become a common procedure in pancreatic surgery. The aim of this study was to compare standard pancreatoduodenectomy with pancreatoduodenectomy plus venous resection at a high-volume centre, and to examine trends in management and outcome over a decade for the latter procedure. METHODS: This retrospective observational study included all patients undergoing pancreatoduodenectomy with or without venous resection at Oslo University Hospital between January 2006 and December 2015. Trends were evaluated by assessing preoperative clinical and radiological characteristics, as well as perioperative outcomes in three time intervals (early, intermediate and late). RESULTS: A total of 784 patients had a pancreatoduodenectomy, of whom 127 (16·2 per cent) underwent venous resection. Venous resection resulted in a longer operating time (median 422 versus 312 min; P = 0·001) and greater estimated blood loss (EBL) (median 700 versus 500 ml; P = 0·004) than standard pancreatoduodenectomy. The rate of severe complications was significantly higher for pancreatoduodenectomy with venous resection (37·0 versus 26·3 per cent; P = 0·014). The overall burden of complications, evaluated using the Comprehensive Complication Index (CCI), did not differ (median score 8·7 versus 8·7; P = 0·175). Trends in venous resection over time showed a significant reduction in EBL (median 1050 versus 375 ml; P = 0·001) and duration of hospital stay (median 14 versus 9 days; P = 0·011) between the early and late periods. However, despite an improvement in the intermediate period, severe complication rates returned to baseline in the late period (18 of 43 versus 9 of 42 versus 20 of 42 patients in early, intermediate and late periods respectively; P = 0·032), as did CCI scores (median 20·9 versus 0 versus 20·9; P = 0·041). CONCLUSION: Despite an initial improvement in severe complications for venous resection during pancreatoduodenectomy, this was not maintained over time. Every fourth patient with venous resection needed relaparotomy, most frequently for bleeding.

Resection margin involvement and tumour origin in pancreatic head cancer.

BACKGROUND: Assessment of the origin of adenocarcinoma in pancreatoduodenectomy specimens (pancreatic, ampullary or biliary) and resection margin status is not performed in a consistent manner in different centres. The aim of this review was to identify the impact of such variations on patient outcome. METHODS: A systematic literature search for articles on pancreatic, ampullary, distal bile duct and periampullary cancer was performed, with special attention to data on resection margin status, pathological examination and outcome. RESULTS: The frequent reclassification of tumour origin following slide review, and the wide variation in published incidence of pancreatic (33-89 per cent), ampullary (5-42 per cent) and distal bile duct (5-38 per cent) cancers indicate that the histopathological distinction between the three cancer groups is less accurate than generally believed. Recent studies have shown that the wide range of rates of microscopic margin involvement (R1) in pancreatoduodenectomy specimens (18-85, 0-27 and 0-72 per cent respectively for pancreatic, ampullary and distal bile duct cancers) is mainly caused by differences in pathological assessment rather than surgical practice and patient selection. As a consequence of the existing inconsistency in reporting of these data items, the clinical significance of microscopic margin involvement in each of the three cancer groups remains unclear. CONCLUSION: Inaccurate and inconsistent distinction between pancreatic, ampullary and distal bile duct cancer, combined with inaccuracies in resection margin assessment, results in obfuscation of key clinicopathological data. Specimen dissection technique plays a key role in the quality of the assessment of both tumour origin and margin status. Unless the pathological examination is meticulous and standardized, comparison of results between centres and observations in multicentre trials will remain of limited value.

Hepatic radiofrequency ablation using perfusion electrodes in a pig model: effect of the Pringle manoeuvre.

AIM: To assess the influence of the Pringle manoeuvre on volume and geometry of coagulations close to the portal vein using an impedance-controlled radiofrequency ablation system with perfusion electrodes. METHODS: Twelve pigs were randomly assigned to a control group (n = 6) and a group where the Pringle manoeuvre was applied during ablation (n = 6). One coagulation was made in each animal close to the portal vein. All animals were sacrificed 4 days after ablation, and the livers were removed for gross and histopathologic analysis. RESULTS: Effective coagulation volume in the Pringle group (10.8 +/- 5.0 cm(3)) was significantly increased (p = 0.03) compared to the control group (4.1 +/- 4.1 cm(3)). The efficacy ratio, defined as the effective coagulation volume divided by the coagulation volume, was not significantly different in the Pringle group (0.47 +/- 0.27) compared to the control group (0.33 +/- 0.22). The geometrical centre of the effective coagulation volume did not correspond to the position of the ablation electrode. Thermal damage of the gallbladder was found in three animals, all belonging to the Pringle group. CONCLUSIONS: The Pringle manoeuvre was associated with increased effective coagulation volume, but did not significantly influence the predictability of coagulation volume or geometry.

Effects of butyrate on epidermal growth factor receptor binding, morphology, and DNA synthesis in cultured rat hepatocytes.

We have examined the effect of butyrate on morphology, DNA synthesis, and epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. Butyrate added 2 h after plating retarded the flattening and maintained the polyhedral shape of the hepatocytes in culture. Both insulin- and EGF-stimulated DNA syntheses were slightly stimulated by butyrate at 1 mM but strongly inhibited at 5 mM. EGF receptor binding was also strongly affected by butyrate treatment of the hepatocytes. The freshly isolated hepatocytes (prior to plating) and the early-stage cultures (2 h) exhibited two classes of surface EGF receptors with high and low affinity (Kd approximately 0.05 and approximately 0.7 nM, respectively). With increasing time in culture there was a decrease in the total EGF receptor number and a corresponding reduction in the capacity for receptor-mediated EGF internalization. The high-affinity receptor class was more strongly reduced than the low-affinity class and was almost absent after 40 h in culture. Butyrate dose-dependently counteracted the decrease in the number of surface EGF receptors during culturing and preserved the high-affinity binding component. Thus, after 40 h, the cells cultured in the presence of butyrate (5 mM) had an approximately 50% elevation in the total number of receptors and the capacity to endocytose EGF compared to control cells, whereas the binding at low ligand concentration (0.02 nM) was increased 4-fold. The results suggest that butyrate, in addition to affecting morphology and DNA synthesis, also has marked effects on the hepatocyte EGF receptor status.

n-butyrate and dexamethasone synergistically modulate the surface expression of epidermal growth factor receptors in cultured rat hepatocytes.

n-Butyrate was previously found to increase the epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. We show here that butyrate and dexamethasone synergistically modulate the surface expression of the EGF receptors. The butyrate-induced enhancement of high-affinity EGF binding was only slight in the absence of glucocorticoid, but was strongly and dose-dependently amplified by dexamethasone. Butyrate counteracted the inhibition by insulin of the dexamethasone-induced increase in EGF binding. The results indicate that the glucocorticoid has a permissive effect on a butyrate-sensitive process that determines the surface expression of the high-affinity class of EGF receptors.

p53 accumulation confers prognostic information in resectable adenocarcinomas with ductal but not with intestinal differentiation in the pancreatic head.

The aim of the study was to examine the relation between p53 protein accumulation, clinicopathological variables and prognosis in resectable adenocarcinomas of the pancreatic head. The clinical records and tissue specimens of 82 consecutive patients resected for adenocarcinomas located in the head of the pancreas were reviewed retrospectively. Formalin-fixed and paraffin-embedded specimens from each tumour were stained with the monoclonal antibody DO7, and the nuclear p53 positivity within each tumour was assessed. Histopathological reclassification showed that 60 tumours exhibited ductal differentiation and 22 tumours intestinal differentiation. Twenty-five percent (15/60) of the ductal tumours and 50% (11/22) of the intestinal tumours were positive for p53 accumulation. p53 immunoreactivity was significantly correlated to a worse prognosis in the tumours of ductal differentiation, with median survival 0.76 years for p53 positive and 1.44 years for p53 negative patients. The p53 positivity of tumours with intestinal differentiation showed no such correlation. No correlation was found between p53 accumulation and other known prognostic factors in either the ductal or the intestinal type of tumours. Our results indicate that the tumour biology of ductal adenocarcinomas differs significantly from that of adenocarcinomas of the intestinal type located in the pancreatic head, and that p53 accumulation confers a worse prognosis only of ductal tumours. Subclassification of these tumours based on type of differentiation is therefore suggested since periampullary tumours include ductally as well as intestinally differentiated adenocarcinomas.

Regulation of hepatocyte epidermal growth factor receptors by n-butyrate and dimethyl sulfoxide: sensitivity to modulation by the tumor promoter TPA.

n-Butyrate and dimethyl sulfoxide (DMSO) are known to promote differentiated characteristics in certain cells, including hepatocytes. We have previously reported that butyrate up-regulates the surface expression of hepatocyte epidermal growth factor (EGF) receptors and preserves a high-affinity receptor subpopulation. In the present study, culturing of hepatocytes with DMSO dose-dependently (0.5-2%) increased EGF binding and maintained a high-affinity binding component which was otherwise down-regulated during culturing. Although butyrate was more effective than DMSO in most experiments, the two agents caused qualitatively the same alteration in hepatocyte EGF receptor status. The high-affinity component of the EGF binding present in cells treated with butyrate or DMSO was reduced by treatment (10 nM-1 microM, 1 h) with the phorbol ester tumor promoter TPA, an activator of protein kinase C. Butyrate- or DMSO-treated hepatocytes were more susceptible to this response to TPA than were untreated hepatocytes. The present data indicate that in hepatocytes both butyrate and DMSO preserve a high-affinity EGF receptor subpopulation which is otherwise down-regulated during hepatocyte culture, and that this effect particularly comprises receptors that are sensitive to modulation by the tumor promoter TPA.

The regulation of cyclic AMP levels in cultured MH1C1 rat hepatoma cells and in solid tumours derived from MH1C1 cell inoculates.

Several studies have found high cAMP content in hepatomas in vivo, while hepatoma cells in vitro have very low levels. To explore this discrepancy and the regulation of cAMP in hepatomas, we have examined the cell line MH1C1 from Morris hepatoma 7795. These cells in culture contained low intracellular cAMP concentrations (approximately 0.5 pmol/mg protein at confluency), and were unresponsive to glucagon and prostaglandins (PG) E1 and E2. In contrast, solid hepatomas in rats developed from inoculates of MH1C1 had a 40-fold higher basal cAMP concentration and were stimulated by PGE1 and PGE2. Fibroblasts cultured from these tumours also contained high cAMP levels and responded strongly to PGE1. This may suggest that the difference in cAMP regulation between hepatomas in vivo and hepatoma cells in vitro results from the presence of other cells in the solid tumour rather than from selection of low-cAMP cells during the cloning procedure. Low-Km and intermediate-Km cAMP phosphodiesterase activity was high in MH1C1, compared to normal hepatocytes. This might contribute to the low cAMP level. The ability of MH1C1 to form cAMP was not defective, as the level could be increased more than 200-fold by beta-adrenergic activation in the presence of the phosphodiesterase inhibitor methylisobutylxanthine.

Reclassification of tumour origin in resected periampullary adenocarcinomas reveals underestimation of distal bile duct cancer.

BACKGROUND: Primary adenocarcinomas removed by pancreatoduodenectomy originate from the duodenum (DC), ampulla (AC), distal bile duct (DBC), or pancreas (PC). Pathobiology, staging, survival, and adjuvant chemotherapy vary among these cancers. The proximity of the structures of possible origin renders it difficult to obtain a correct diagnosis, which might lead to inconsistencies in reported data and inappropriate adjuvant treatment. METHODS: Records of 207 patients undergoing pancreatoduodenectomy (1998-2009) for periampullary adenocarcinoma were reviewed. Routine histopathology reports of tumour origin performed by multiple pathologists were independently re-evaluated based on predetermined criteria by two experienced pancreatic pathologists. RESULTS: Slide review changed the diagnosis in 55 (27%) patients. After reclassification, final distribution was 29 (14%) DC, 52 (25%) AC, 57 (28%) DBC, and 69 (33%) PC. The diagnosis was revised in 4 (14%) DC, 7 (17%) AC, 30 (53%) DBC and 14 (19%) PC. The underestimation of DBC during routine histopathology was caused by misinterpretation of DBC either PC or AC. Misclassification of PC was mainly due to erroneous diagnosis of AC. Reassignment of tumour origin caused no significant changes in survival within cancer type, but resulted in a significant difference in survival between DBC and PC (p = 0.004). CONCLUSION: Specialist slide review resulted in reassignment of tumour origin in 27% of periampullary adenocarcinomas. Distal bile duct cancer was found to be most frequently misdiagnosed (53%). Correct diagnosis of tumour origin is crucial for data quality, appropriate adjuvant therapy, and patient inclusion in clinical trials.

Rapid constitutive internalization and externalization of epidermal growth factor receptors in isolated rat hepatocytes. Monensin inhibits receptor externalization and reduces the capacity for continued endocytosis of epidermal growth factor.

It was previously demonstrated that freshly isolated rat hepatocytes can internalize severalfold more epidermal growth factor (EGF) molecules than the number of surface EGF receptors, suggesting extensive reutilization of receptors during endocytosis (Gladhaug, I. P. & Christoffersen, T. (1987) Eur. J. Biochem. 164, 267-275). The present report attempts to explore the pathways involved in the externalization of EGF receptors. Incubation of hepatocytes at 37 degrees C in the absence of ligand increased the surface receptor pool by 50-100% within 45 min. Pretreatment with monensin inhibited the turnover of the surface EGF receptor pool by 50-60% within 10 min and blocked the temperature-dependent externalization of receptors. Cycloheximide caused a slower attenuation of the surface receptor pool, whereas tunicamycin and chloroquine did not significantly affect the exchange of receptor pools. Monensin reduced the surface receptor pool and the endocytic uptake in corresponding proportions, without affecting the internalization of prebound EGF. Endocytic uptake was unaffected by chloroquine and slightly reduced by cycloheximide. The internalization of unoccupied receptors and the endocytosis of prebound EGF followed similar kinetics (t1/2 approximately 5 min), suggesting that unoccupied receptors are internalized at a rate comparable to that of occupied receptors. The results suggest that there is a rapid turnover of the surface pool of EGF receptors with constitutive internalization of unoccupied surface receptors and externalization of internal receptors. This is consistent with, but does not prove, a true recycling of the EGF receptors in the hepatocytes. The monensin-sensitive externalization pathway determines the capacity for continued endocytosis of EGF.

Kinetics of epidermal growth factor binding and processing in isolated intact rat hepatocytes. Dynamic externalization of receptors during ligand internalization.

The kinetics of binding and processing of epidermal growth factor (EGF) was studied in freshly isolated rat hepatocytes. After isolation the hepatocytes had a nonhomogeneous population of surface EGF receptors consisting of approximately 9000 high-affinity sites (Kd 21 pM) and 165,000 low-affinity sites (Kd 0.62 nM). Incubation at 37 degrees C (45 min) increased the number of surface receptors per cell to about 260,000. This increase was selective for the low-affinity receptors and was cycloheximide-sensitive. During 5 h of incubation at 37 degrees C the hepatocytes internalized 6-7-times more EGF molecules than the number of cell surface receptors, based on clearance measurements. The uptake was unaffected by cycloheximide. Concomitant estimation, using acid/salt elution, of surface-bound EGF and internalized EGF showed that the number of internalized EGF molecules exceeded the decrease in surface-binding 6 times. The ratio between internalized EGF and the decrease in surface binding was temperature-dependent, being reduced to a one-to-one stoichiometry at 10 degrees C. After down-regulation (approximately equal to 75%) induced by 5 nM unlabeled EGF the surface EGF receptors did not recover during subsequent incubation (2 h) at 37 degrees C. However, the remaining surface receptors internalized EGF in ninefold excess of their number. The large discrepancy between internalization capacity and cell surface binding capacity was also found in the presence of cycloheximide. The results support the idea that internalized EGF receptors are partly replaced by externalization of preformed intracellular receptors during EGF uptake in isolated hepatocytes, involving recycling of a small population of EGF receptors and/or recruitment of unexposed, pre-existing receptors.

Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes.

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.

Regulation of surface expression of high-affinity receptors for epidermal growth factor (EGF) in hepatocytes by hormones, differentiating agents, and phorbol ester.

Freshly isolated adult rat hepatocytes exhibit a nonhomogeneous population of epidermal growth factor (EGF) receptors with about 10,000 high-affinity binding sites (Kd 20 pM) and about 200,000 low-affinity sites (Kd 600 pM) per cell. With culturing as primary monolayers under conditions where the cells show a marked increase in the sensitivity to the growth-stimulatory effect of EGF, a gradual reduction in the number of EGF receptors and an almost complete loss of high-affinity EGF receptors is seen. Insulin, which promotes growth of hepatocytes in concert with EGF, enhances the down-regulation of these high-affinity receptors. The differentiating (and growth-inhibitory) agent n-butyrate counteracts this down-regulation and preserves the high-affinity receptors. This effect of butyrate is synergistic with the glucocorticoid agent dexamethasone. Another differentiating agent, dimethylsulfoxide (DMSO), also counteracts the down-regulation of high-affinity EGF receptors. Moreover, the tumor promoter, tetradecanoylphorbol acetate (TPA), down-regulates the EGF receptor. This effect is particularly evident when studying the high-affinity receptors up-regulated by prior treatment with butyrate plus dexamethasone. Taken together these results provide strong support for the notion that an inverse relationship exists between expression of high-affinity EGF binding and responsiveness to growth activation by EGF.

Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes.

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.

DNA synthesis in cultured adult rat hepatocytes: effect of serum and calcium.

We have investigated the influence of serum and of varying Ca2+ concentrations on the DNA synthesis in primary monolayer cultures of adult rat hepatocytes, using a defined basic medium. Supplementation of the medium with 10% horse serum did not significantly affect the time course of the DNA synthesis induced by insulin and epidermal growth factor. Dose effect curves showed that serum moderately sensitized the cells to low concentrations of insulin and slightly sensitized them to epidermal growth factor, but was not required for full responses to maximal concentrations of the hormones. In the serum-free cultures a wide range of Ca2+ concentrations (0.4 - 1.8 mmol/l) yielded maximal DNA synthesis, suggesting a broad Ca2+ optimum of the S phase entry in the hepatocyte monolayers.

Studies of glucocorticoid enhancement of the capacity of hepatocytes to accumulate cyclic AMP.

Pretreatment of cultured rat hepatocytes with dexamethasone markedly enhanced the acute cAMP response to glucagon, isoproterenol or forskolin. The effect of dexamethasone was apparent within 3-6 hr and was maximal after 20-30 hr. The amplification of the cAMP response to glucagon could also be produced by other glucocorticoids, with relative potency dexamethasone much greater than methylprednisolone greater than hydrocortisone. The increased cAMP response was associated with a reduced cAMP phosphodiesterase activity in cell lysates and a reduced effect of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in intact cells, indicating that the glucocorticoid pretreatment reduced cAMP degradation. However, the increase in response to glucagon in glucocorticoid-treated cells was relatively larger than the increase in forskolin response and also larger than the decrease in phosphodiesterase activity, suggesting that other factors in addition to down-regulation of phosphodiesterases was responsible for the effect. Cycloheximide abolished the difference in phosphodiesterase activity and cAMP response between dexamethasone-treated and control cells. The results suggest that the glucocorticoids increase the ability of hepatocytes to accumulate cAMP due to protein synthesis-dependent processes which at least in part involve reduced degradation of cAMP.

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase.

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Immunoglobulin prolongs survival of pig kidneys perfused ex vivo with human blood.

Intravenous immunoglobulin (IVIG) (Octagam), was used to determine the effect on hyperacute rejection in an ex vivo xenograft model. Six pig kidneys were perfused with IVIG and fresh human AB blood, and six control pig kidneys were simultaneously perfused with albumin and blood from the same donation. The survival of the IVIG-perfused xenografts (median, 6.5 h) was significantly (P = 0.03) longer than the albumin-perfused xenografts (median, 3.5 h). Complement was activated in both groups. The administration of IVIG to the perfused blood resulted in immediate and significantly higher complement activation in the fluid phase as compared with the albumin group. At rejection the fluid phase complement activation was higher in the IVIG group than in the albumin group for C1rs/C1inh complexes, C4bc, Bb and TCC. At the time of rejection both the albumin and the IVIG group demonstrated interstitial tubular haemorrhage, vasculitis or necrosis of glomerular capillaries and glomerular microthrombi. IgM, C1q, C3c, C4 and fibrin were located in arteries and glomeruli and IgG in the interstitium in both groups at rejection. The fluid phase findings are consistent with a modulatory effect of IVIG on complement activation by deviating the classical pathway activation towards the fluid phase. The prolonged survival of the IVIG-perfused kidneys suggests that IVIG may be useful to dampen hyperacute rejection.

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry.

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.