Irving S. Benjamin

Changes in E-cadherin immunoreactivity in the adenoma-carcinoma sequence of the large bowel

We have used an avidin-biotin immunoperoxidase technique to localise epithelial cadherin (E-cadherin), a calcium-dependent cell-cell adhesion molecule, in 107 paraffin-embedded sections from 93 patients consisting of 24 with colorectal adenoma, 55 with rectal carcinoma and 14 with liver metastases. The corresponding primary colorectal tumours were also studied in these cases. E-cadherin was expressed by normal colorectal epithelial cells with typical membranous staining at the intercellular junctions. Loss of normal membranous E-cadherin expression and presence of cytoplasmic staining were found frequently in adenomas larger than 1 cm (P<0.01), with high grade dysplasia and villous histology (P<0.01). In primary rectal cancers, loss of membranous expression correlated with high tumour grade. No correlation was seen with Dukes and Jass stage, local extramural spread and 5-year recurrence rate. Complete loss of membranous E-cadherin immunoreactivity was seen in 7/14 (50%) liver metastases in which 6/7 (86%) showed intense membranous E-cadherin immunoreactivity in the corresponding primary tumour. Our data indicate that changes in E-cadherin immunoreactivity and cellular localisation correlate with size, severe dysplasia in adenomas and tumour grade in carcinomas. However, there seems to be no correlation between loss of membranous E-cadherin immunoreactivity and the invasive and metastatic potential of the carcinomas.

A simple and accurate mathematical method for calculation of the EC50

A simple, accurate, and speedy noncomputational technique for the calculation of the EC50 or any other concentration-related parameter of concentration-effect curves is presented. It avoids the necessity for graph construction or computational curve-fitting programs and allows accurate calculation of the EC50, where the value falls between two known concentrations The technique has been applied to a concentration-response curve constructed to hepatic arterial (HA) vasoconstrictor responses to HA injections of noradrenaline in an isolated dual-perfused rat liver preparation. EC50 values calculated by the new technique were compared to those calculated by conventional, established, noncomputational techniques. The new technique is faster, more accurate, and simpler to perform than other established noncomputational techniques used for the calculation of the EC50 and can be widely applied to many other pharmacological investigations.

The transhepatic action of ATP on the hepatic arterial and portal venous vascular beds of the rabbit: the role of nitric oxide

1 The effect of bolus administration of adenosine 5′‐triphosphate (ATP) into the portal vein on hepatic arterial pressure (the transhepatic action of ATP) and portal venous pressure, and the contribution of nitric oxide towards these responses, was studied in the in vitro dual‐perfused rabbit liver. 2 At basal tone, hepatic arterial and portal venous vasoconstriction followed ATP injection, while at a tone raised with methoxamine (10−6‐10−5m) ATP caused hepatic arterial vasodilatation, and a phasic vasodilatation followed by vasoconstriction in the portal venous vascular bed. 3 To determine whether the transhepatic arterial dilatation was due to the diffusion of nitric oxide (NO) from the portal venous vasculature, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μm), an inhibitor of NO synthesis, was infused selectively into the portal vein. L‐NAME infusion potentiated portal venous vasoconstriction to ATP (‐log M ED50 5.32 ± 0.31 to 6.51 ± 0.43, P < 0.05, Students paired / test) indicating the possible inhibition of a NO‐mediated vasodilator component of the portal venous response to ATP. There was, however, no demonstrable difference in the transhepatic arterial vasodilatation induced by ATP during this infusion. 4 Simultaneous perfusion of both the hepatic arterial and portal venous inflows with L‐NAME (100 μm) resulted in a significant decrease in the amplitude of hepatic arterial responses to ATP demonstrating that these responses were ultimately mediated by an NO‐dependent mechanism. 5 This study has thus demonstrated a vasodilator component of the portal venous response to ATP that is NO‐mediated. It also provides evidence that it is not portally‐derived NO, but NO released from the hepatic arterial vascular bed, that accounts for the hepatic arterial vasodilatation to intra‐portal administration of ATP. This implies that ATP itself, and not a second messenger, diffuses from the portal venous to hepatic arterial vascular bed to elicit the hepatic arterial response.

Hepatic function during prolonged isolated rat liver perfusion using a new miniaturized perfusion circuit

Previous designs of isolated rat liver perfusion circuits used for toxicological investigations are often expensive, cumbersome, or traumatic in relation to hepatic biocompatibility following extended perfusion times. A new, miniaturized circuit that incorporates a novel design of organ bath, to maintain a buoyant preparation, and a high-efficiency miniaturized membrane tubing oxygenator is described. Livers from male Sprague-Dawley rats were perfused continuously for 6 hr in vitro using rat blood diluted to a perfusate hematocrit of 9.75 +/- 0.35% with Krebs-Henseleit buffer (KHB). Hepatic function was evaluated by measurement of perfusion pressure, flow rate, bile volume production, bile bilirubin content, hepatic oxygen uptake (HOU), bromosulphthalein (BSP) removal, hepatic enzyme activities, and electrolyte concentrations, and finally by histological examination. Perfusion pressure and flow rate remained stable at 8.7 +/- 1.7 mmHg and 1.92 +/- 0.06 mL min-1 g-1 liver, respectively. Bile volume production and HOU were maximal at 784 +/- 84 microL h-1 and 0.99 micromol/L min-1 g-1 respectively. Erythrocyte damage in the perfusate was evaluated by measurement of reduction in perfusate hematocrit from 9.75 +/- 0.35% to 9.27 +/- 0.24%, and increases in plasma free hemoglobin, which rose from 85.8 +/- 12.3 mg% to 650.1 +/- 53.3 mg% over the 6-hr perfusion period studied. Using bile volume production, hepatic oxygen uptake, and the liberation of plasma free hemoglobin as the most sensitive indices of adverse conditions, the new circuit was capable of supporting an isolated perfused rat liver for periods of up to 6 hr under close-to-physiological conditions.

Cold‐storage of Rabbit Thoracic Aorta in University of Wisconsin Solution Reduces Endothelium‐independent Vasodilation

Optimum preservation conditions for storage of donor livers and blood vessels are essential for successful transplantation. The blood vessels are used as vascular conduits to facilitate anastomosis of the liver to the recipients systemic vasculature. Failure of some transplants has been ascribed to thrombosis of these vascular conduits possibly because of alterations in vascular reactivity owing to inadequate storage techniques. To restrict data variability previously associated with studies using a heterogeneous sample of vessels from man, this study investigated changes in vascular reactivity in segments of rabbit thoracic aorta from male, age‐matched, New Zealand White rabbits stored at 4°C in either University of Wisconsin solution (UW; Du Pont Pharmaceuticals, UK) or Krebs‐Bülbring buffer (KB).

The action of ATP on the hepatic arterial and portal venous vascular networks of the rabbit liver: the role of adenosine

ATP is released from blood vessels during periods of hypoxia and may be responsible for hepatic arterial vasodilatation during instances of reduced hepatic portal venous flow. The role of adenosine in ATP-induced vasodilator and vasoconstrictor responses of the hepatic arterial and portal venous vascular networks respectively was studied in the isolated dual-perfused rabbit liver in vitro to ascertain whether ATP could be catabolised to adenosine during transit through the hepatic parenchyma. Intra-arterial and intra-portal injections of ATP (-10 to -4 log mol/100 g liver) resulted in dose-dependent vasodilatation in the hepatic artery and vasoconstriction in the portal vein. Addition of 8-phenyltheophylline (10 microM), a non-selective P1-purinoceptor antagonist, to the hepatic arterial and portal venous perfusate significantly inhibited the hepatic arterial ED50 for responses to intra-arterial injected ATP from -8.70 +/- 0.22 to -7.63 +/- 0.28 log mol/100 g liver (P < 0.001); it also inhibited hepatic arterial responses to, mid-range, portal venous injections of ATP. The data suggest that the hepatic arterial vasodliatation to ATP is partly mediated via catabolism to adenosine and may be an important mechanism during periods of relative hepatic hypoxia associated with portal flow reduction.

Localisation of hepatic vascular resistance sites in the isolated dual-perfused rat liver

The locations of the vascular resistance sites which regulate vascular tone in the hepatic arterial and portal venous vasculatures of the rat liver were identified using a new, in vitro, dual-perfused liver preparation. Twelve livers of male Wistar rats were perfused via the hepatic artery and portal vein at fixed flow and at physiological pressure. Dose-related vasoconstriction to injections or infusions of noradrenaline was measured as transient or sustained increases in perfusion pressure, respectively, in the hepatic arterial and portal venous vasculatures. Direct injections/infusions of noradrenaline refer to those administered into the vasculature from which pressure was recorded, e.g., the effects of hepatic arterial (direct) injections/infusions of noradrenaline upon hepatic arterial perfusion pressure. Indirect injections/infusions of noradrenaline were those administered to the adjacent afferent vasculature, e.g., the effects of portal venous (indirect) injections of noradrenaline upon hepatic arterial perfusion pressure. The converse applies for recordings of portal venous perfusion pressure. The -log(M) ED50 values to direct (hepatic arterial) and indirect (portal venous) injections in the hepatic artery were 4.25+/-0.20 and 3.40+/-0.10, respectively, and were significantly different (P < 0.01, Students unpaired t-test); the -log(M) ED50 values to direct (portal venous) and indirect (hepatic arterial) injections in the portal vein were 3.91+/-0.08 and 3.85+/-0.11, respectively, and were not significantly different (P > 0.05, Students unpaired t-test). Similarly, the -log(M) ED50 values to direct (hepatic arterial) and indirect (portal venous) infusions in the hepatic artery were 5.28+/-0.11 and 3.75+/-0.12, respectively, and were significantly different (P < 0.01, Students unpaired t-test); the -log(M) ED50 values to direct (portal venous) and indirect (hepatic arterial) infusions in the portal vein were 5.31+/-0.19 and 5.70+/-0.16, respectively, and were not significantly different (P > 0.05, Students unpaired t-test). These results demonstrated that there is little transfer of noradrenaline from the portal venous to the hepatic arterial resistance sites, but significant transfer from the hepatic artery to the portal venous suggesting that; (a) the portal venous resistance sites are located at the sinusoidal or post-sinusoidal level; and (b) the hepatic arterial resistance sites are located at the pre-sinusoidal level.

Differentiation Between the Effects of Unprocessed Portal Blood and Reduced Liver Function on Brain Indole Amine Metabolism in the Portacaval Shunted Rat

Changes in brain 5-HT turnover which have been associated with portal-systemic encephalopathy (PSE) in man were studied in rats with experimental PSE for intervals up to 15 weeks following the surgical construction of end-to-side portacaval shunts (PCS). These were compared to changes measured in portacaval transposed rats (PCT) which, show little hepatic dysfunction or cerebral abnormalities but, in common with the PCS rat, sustain total portal-systemic diversion. Thus any differences between these two groups were indicative of hepatic dysfunction and not the systemic diversion of portal blood. After 15 weeks, sustained increases were measured in brainstem and cerebral concentrations of the catabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), from 0.25±0.01 to 0.68±0.01*** μg g−1 brain and from 0.18±0.01 to 0.31±0.03*** μg g−1 brain respectively in PCS rats and were statistically greater to those measured in the brainstem and cerebrum of PCT and control rats. Sustained increases in cerebral concentrations alone of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, from 0.17±0.01 to 0.23±0.02 μg g−1 brain were measured in PCS rats and were significantly*** greater than in PCT control rats after 15 weeks. Some early increases in 5-HTP were measured in PCS above control rats but these were not significant after 15 weeks. No sustained significant differences between the 3 groups were measured in 5-HT after 15 weeks. These data confirm previous evidence that the elevations in 5-HTP and 5-HIAA concentrations observed in experimental chronic liver failure and PSE are due to liver dysfunction and not portal-systemic diversion and may contribute additional information regarding the role of derangements in central 5-HT turnover as one of the causes of PSE. *** p<0.001, Newman-Keuls ANOVAR followed by Students unpaired t-test for individual comparisons, (data shown are mean ± SEM).

The Transhepatic Response to Noradrenaline in the Rabbit Liver: the Influence of Arterioportal Pressure Gradient

The dose‐related responses of the hepatic arterial and portal venous vascular beds to bolus administration of noradrenaline (10−10−10−4mol), injected into the hepatic artery and portal vein, were studied in the isolated dual‐perfused rabbit liver at both basal and raised tone. The transhepatic ratio, defined as the ratio between the intra‐arterial molar ED50 dose and the intraportal dose required to give the same arterial response, was calculated for arterial and venous responses to noradrenaline.