The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.Mitochondria play a central role in apoptosis. Mitochondrial outer membrane permeabilization (MOMP) leads to release of proapoptotic factors such as cytochrome c and AIF (1). Furthermore, loss of mitochondrial membrane potential (Δψ m ) is thought to contribute to cell death by disruption of normal mitochondrial function (2, 3). Interaction of members of the Bcl-2 family of proteins regulates MOMP, the key event of cytochrome c release into the cytoplasm (3, 4). What is less clear, however, is the precise role of caspase proteases in mitochondrial events of apoptosis. Although upstream caspases, such as caspase 2 and caspase 8, affect mitochondrial events in both death-receptor and mitochondrial pathways of apoptosis, either directly or through interaction with Bcl-2 family members, the role of presumed downstream "effector" caspases in this process is less clear (5, 6). Therefore, we studied the two highly related effectors, caspase 3 and caspase 7, to elucidate their functions in apoptosis.* To whom correspondence should be addressed. richard.flavell@yale.edu. We generated caspase 7 −/− mice ( fig. S1), which were born in ratios consistent with Mendelian inheritance. They had normal appearance, organ morphology, and lymphoid development. When caspase 7 −/− mouse embryonic fibroblasts (MEFs) were treated with inducers of apoptosis, they exhibited a slight survival advantage as compared with wild-type MEFs. Apoptosis caused by a range of insults in other caspase 7 −/− cells proceeded normally, however, including the death of activated T cells following stimulation of the T cell receptor, thymocyte apoptosis, Fas-mediated death of B cells, and Fas-mediated death of hepatocytes ( fig. S2). Supporting Online MaterialCaspase 3, which is structurally similar to caspase 7, might compensate for the lack of caspase 7, which would lead to this relatively mild antiapoptotic phenotype (7,8). Thus, we bred caspase 7 −/− mice to caspase 3 −/− mice previously described by our laboratory (9). The embryonic stem cells containing the mutation were from the 129/SvJ genetic background. Mice derived from these embryonic stem cells were backcrossed six generations onto the C57BL/6 background. We obtained no live caspase 3 −/− /caspase 7 −/− double-knockout (DKO) mice when progeny were genotyped at an age of 10 to 14 days. DKO mic...

The immune response to foreign antigens in the liver is often suboptimal and this is clinically relevant in chronic persistence of hepatotropic viruses. In chronic infection with the hepatitis C virus, activated CD8؉ T cells specific for viral epitopes are present in the peripheral blood and the liver, yet viral clearance is unusual. To define the fate of activated CD8؉ entering the liver, we developed a mouse model of portal vein injection of activated CD8؉ T cells in vivo. Activated CD8؉ T cells are retained very efficiently by the liver and undergo an approximately 8-fold expansion in the first 48 hours. This expansion is followed by apoptosis and a decline in numbers of the retained cells over the next 4 days. The presence of high affinity (HA) antigen does not affect the initial retention by the liver but greatly limits the expansion in the first 48 hours by increasing apoptosis of the retained cells. In the absence of Kupffer cells, the initial retention and expansion are unchanged, but HA antigen does not limit the expansion of the liver CD8؉ T cell pool. In conclusion, these data identify a previously unknown phase of CD8؉ T cell expansion after entering the liver, demonstrate that HA antigen limits the hepatic CD8؉ T cell pool by inducing apoptosis, and that this effect requires Kupffer cells. Interfering with antigen presentation by Kupffer cells may be a strategy to limit HA antigen-induced deletion of activated CD8؉ T cells entering the liver.

The reduction of portal pressure in patients with early compensated cirrhosis may be more responsive to drugs increasing intrahepatic vasodilatation than those reducing portal venous inflow. The PDE-V inhibitor sildenafil can potentially reduce portal pressure by decreasing intrahepatic resistance, but its systemic vasodilatory effects may be deleterious. Aim:Evaluate the effect of sildenafil on systemic and portal hemodynamics in an open-label pilot study. Methods:Twelve patients with compensated cirrhosis and baseline hepatic venous pressure gradient (HVPG) >5 mmHg received 25mg of oral sildenafil. Mean arterial pressure(MAP), heart rate (HR) and HVPG were repeated after 30 and 60 minutes and in 9/12 patients at 90 minutes (after an additional 25mg of sildenafil). HVPG tracings were read by 3 blinded observers.Results: All 12 patients were Child A with median MAP 92 mm Hg(84-95) and median HVPG 10.4 mmHg(6.6-13.0). While MAP decreased significantly at all time points, sildenafil had no effect on HVPG. Conclusion:As shown with other vasodilators, in compensated cirrhotic patients, sildenafil at therapeutic doses for erectile dysfunction reduces MAP without reducing portal pressure. The search should continue for specific intrahepatic vasodilators. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. KeywordsDisclosures: Dr. Garcia-Tsao is consultant for Dong Pharmaceuticals that makes udenafil (a long-acting PDE-V inhibitor) Portal hypertension is the major cause of morbidity and mortality in patients with cirrhosis. Portal hypertension is initially due to increased intrahepatic resistance and subsequently maintained by an increase in portal blood inflow. A reduction in portal pressure results in a lower rate of complications of cirrhosis(1-3).Non-selective beta-blockers (BB) reduce portal pressure by reducing portal venous inflow. Although BB prevent variceal hemorrhage in patients with varices they do not prevent the development of varices(4), perhaps because portal hypertension in patients without varices (early disease) is more dependent on increased intrahepatic resistance than increased portal inflow. Experimental studies have demonstrated that cirrhotic rats without ascites already have impaired intrahepatic vasorelaxation(5).Since portal pressure reduction in patients with early (compensated) cirrhosis is related to a decrease in outcomes(4), pharmacological therapy should be targeted at reducing intrahepatic resistance by increasing intrahepatic nitric oxide (NO). Available vasodilators not only reduce intrahepatic resistance but also have a systemic effect t...

Background & Aims Large-volume paracentesis (LVP) is the treatment of choice for patients with cirrhosis and refractory ascites. However, LVP can lead to post-paracentesis circulatory dysfunction (PCD), which is associated with faster ascites recurrence and renal failure. PCD results from vasodilatation, which reduces effective blood volume, and is prevented by intravenous administration of albumin. Vasoconstrictors could be used instead of albumin and, with longer use, prevent PCD and delay ascites recurrence. Methods We performed a multicenter, randomized, double-blind, placebo controlled trial to compare albumin with the vasoconstrictor combination of octreotide and midodrine in patients with refractory ascites who underwent LVP. Patients in the albumin group received a single intravenous dose of albumin at the time of LVP plus placebos for midodrine and octreotide (n=13). Patients in the vasoconstrictor group received saline solution (as a placebo for albumin), 10 mg of oral midodrine (3 times daily), and a monthly, 20 mcg intra-muscular injection of long-acting octreotide (n=12). Patients were followed until recurrence of ascites. Results The median times to recurrence of ascites were 10 days in the albumin group and 8 days in the vasoconstrictor group (P=.318). There were no significant differences in PCD between the albumin group (18%) and the vasoconstrictor group (25%, P=.574). When ascites recurred, serum levels of creatinine were higher in the vasoconstrictor group (1.2 vs 0.9 mg/dL in the albumin group, P=.051). Conclusions The combination of midodrine and octreotide after LVP is not superior to albumin in delaying recurrence of ascites or preventing PCD in patients with cirrhosis. Outcomes appear to be worse in patients given octreotide and midodrine.