07 vs 0.76 log IU/mL, P = 0.038), 2 weeks (2.73

vs 1.01, P = 0.009), 4 weeks (2.72 vs 1.55, P = 0.059), and 12 weeks (4.56 vs 3.24, P = 0.104). The sustained virological response rates in the IL28B major genotype were similar between IFN-β group (47.1%, 8/17) and PEG group (53.3%, 8/15). Selleck BVD-523 In contrast, the sustained virological response rates in the IL28B minor genotype were numerically higher in IFN-β group (50.0%, 3/6) than in PEG group (12.5%, 1/8), although not statistically significant. It was suggested that lead-in twice-daily IFN-β/ribavirin treatment followed by PEG-IFN/ribavirin combination therapy may modify the HCV-RNA dynamics compared with that by PEG-IFN/ribavirin therapy, and it is particularly useful for the IL28B minor genotype. “
“Hepatic ischemia and reperfusion injury (IRI), 5-Fluoracil concentration an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate

cyclase-activating polypeptides (PACAP) in a murine model of partial liver “warm” ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the

expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily click here restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. Conclusion: Our novel findings document the importance of PACAP-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to manage liver inflammation and IRI in transplant patients.

6 times more likely to develop cholestatic/mixed type of DILI. This may not come as a surprise, considering that 83% of the mitochondria hazardous drugs in our cohort also fall into the quinone/epoxide-forming category, reemphasizing the importance of the chemical structure and corresponding reactive intermediates of the drug. Our findings suggest that the SOD2 polymorphism studied is related to cholestatic/mixed type of DILI.

Subsequently, oxidative stress may have a bigger influence on cholestatic/mixed than hepatocellular type of liver injury. Indeed, retention of hydrophobic bile acids is known to enhance mitochondrial oxidative stress.26 The fact that cholestatic patients are generally older than hepatocellular patients supports a role for SOD2 PI3K Inhibitor Library cell line in cholestatic injury, as the antioxidant defence system is known to decline with age.8, 27 In line with this theory was the finding that our cholestatic/mixed DILI cases homozygous for the SOD2 Ala allele had a higher mean age than the corresponding heterozygous DILI cases. In an earlier study, we found that the presence of either the GSTM1 or GSTT1 null allele was associated with low or no increased risk of developing DILI, respectively, but that carriers of both null alleles had a 2.7-fold increased risk for DILI.17 This suggests that the two GST genes have

a synergistic role in Cobimetinib DILI development. Similarly, the SOD2 and GPX1 polymorphisms synergistically enhance the risk of DILI, irrespective of the type of liver injury as DILI patients were more likely to contain two or more risk alleles (SOD2 Ala and GPX1 Leu allele). Combined sources of oxidative stress are therefore more likely to lead to DILI development, probably because of cumulative mitochondrial dysfunction, and have the potential to overcome the disease threshold faster, as seen in the shorter time to onset in cases with two or more risk alleles. Diplotypes abundant in pro-oxidant alleles are consequently disadvantageous to the cell. The number of pro-oxidant alleles is therefore limited, and none of our cases or controls was found to be homozygous for see more the SOD2 Ala and GPX1 Leu alleles as well as

GSTM1/T1 double nulls. In conclusion, carriers of the SOD2 Ala/Ala and GPX1 Leu/Leu genotype are more susceptible to developing cholestatic/mixed type of DILI. The risk of DILI is augmented with drugs forming reactive intermediates during bioactivation. The SOD2 Ala and GPX1 Leu risk alleles have a synergistic effect on the risk of DILI, and increased number of risk alleles appear to shorten the time to disease onset. The authors thank D. Ramón Hidalgo of the Servicio Central de Informática de la Universidad de Málaga for his invaluable help in the statistical analyses. The funding source had no involvement in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

MO-CTL treatment did not affect the level of c-FLIP, whereas MO1 injection down-regulated c-FLIPS to 36% of the PD-0332991 chemical structure control level (Fig. 6A). More important, we found that coinjection of mRNA encoding human c-FLIPS (75 pg/embryo) could

rescue the MO1-induced liver defect in 75% of the injected embryos (Fig. 6B; N = 44). Taken together, these results suggested that knockdown of SNX7 induced the degradation of c-FLIPS, which led to the activation of the caspase 8–dependent pathway and subsequent cell death. Many molecules involved in hepatogenesis have been identified from various model systems. The majority of them can be grouped into one of the following categories: (1) cell-signaling molecules, such as FGF, BMP, Wnt, Hedgehog, or RA pathway-related genes; (2) transcriptional factors, such as Gata and HNF family members,

Hhex, Prox1, and so on; and (3) epigenetics-related molecules, such as Dnmt1/2, Hdac1/3, and Uhrf1. We report here that SNX7, a SNX family member supposed to be involved in vesicular trafficking and protein sorting, is crucial for embryonic liver development in zebrafish. SNX7 is an early endosome and multivesicular-body–distributed protein (data not shown). Interestingly, a recent study reveals that tomm22, selleck compound a regulator of protein traffic from cytoplasm into the mitochondria, is required for liver development in zebrafish.24 Disruption of this gene induces extensive apoptosis of hepatocytes, which is similar to what we observed in SNX7 morphants. On the other hand, mutation in vacuolar protein sorting protein 18 (vps18), a class C vacuolar protein-sorting gene, causes hepatomegaly (i.e., large liver) in zebrafish.47 Vps18 is involved in the regulation of vesicles from late endosome to lysosome, and mutation in vps18 causes the accumulation of cytoplasmic vacuoles, which eventually leads to the hepatomegaly phenotype. These observations suggest that different subcellular protein-traffic pathways could affect different aspects of liver development. Thus, SNX7 could provide us with new opportunities to study the molecular

mechanism of liver development. The specification of hepatoblasts was normal in SNX7 morphants; however, these cells underwent apoptosis see more during the budding stage. Knockdown of SNX7 by siRNAs in Hela or HepG2 cells induced apoptosis as well. We revealed that SNX7 regulated the death-receptor–mediated caspase 8 pathway, but not the mitochondrion-related caspase 9 pathway. c-FLIP is a catalytically inactive homolog of caspase 8 and is able to interfere with the activation of caspase 8. We demonstrated that down-regulation of SNX7 decreased the intracellular level of c-FLIPS, and this regulation appeared to be proteasome dependent (data not shown). Proteasomes are large protein complexes involved in ubiquitin-dependent protein degradation.

42, 95% CI 1.37-8.54; P = 0.004) and having drawn the blood sample for vitamin D measurement during the winter or spring months (OR 2.79, 95% CI 1.35-5.74; P = 0.005) were the only independent predictors of low vitamin D serum levels. When taking into account all HCV genotypes, a direct relationship was observed between higher 25-OH vitamin D serum levels and a higher rate of treatment response (Table 5). In particular, 25-OH vitamin D serum levels were strongly associated with the rates of RVR and cEVR. In multivariate analysis, 25-OH vitamin D serum levels >20 ng/mL predicted an SVR independent of the other well-known predictors of viral response reported in Table 3. Considering all genotypes,

the final model of viral response prediction included HCV genotype, the IL-28B Nutlin-3a concentration rs12979860 C/T polymorphism, GGT, HCV RNA, cholesterol, and 25-OH vitamin D, with an area under the receiver operating characteristic (ROC) curve of 0.827 (Table 6). When difficult-to-treat genotypes were analyzed separately, even stronger associations were detected between serum vitamin D levels and the rate of viral response (Table 5). Moreover, the serum vitamin D level was confirmed to be an independent predictor of an SVR. In difficult-to-treat genotypes, the final model of viral response prediction included the IL-28B rs12979860

C/T polymorphism, viral Antiinfection Compound Library load, and serum vitamin D level, with an area under the ROC curve of 0.836 (Table 6). The following allelic and genotypic frequencies for the IL-28B rs12979860 C/T polymorphism were detected: C, 0.623; T, 0.377; C/C, 76 (36.0%); C/T, 111 (52.6%); selleck screening library and T/T, 24 (11.4%). Genotypic frequencies did not differ from what was expected based on the Hardy-Weinberg equilibrium equation (P > 0.05). Considering all of the patients, the SVR rate occurred as follows: C/C, 60/76 (78.9%); C/T, 60/111 (54.1%); and T/T, 14/24 (58.3%) (P = 0.004). In difficult-to-treat HCV genotypes, the SVR rates were: C/C, 24/32 (75.0%); C/T, 16/61 (26.2%); and T/T, 7/17 (41.2%) (P = 0.002). To verify the usefulness of the combined assessment of the IL-28B rs12979860 C/T polymorphism and the serum

vitamin D level, we identified four groups of patients: C/C homozygotes with vitamin D levels of >20 ng/mL (group A), C/C homozygotes with vitamin D levels of ≤20 ng/mL (group B), C/T heterozygotes and T/T homozygotes with vitamin D levels of >20 ng/mL (group C), and C/T heterozygotes and T/T homozygotes with vitamin D levels of ≤20 ng/mL (group D). Considering the whole cohort of treated patients, a highly significant linear trend was found in the decrease of the SVR rate: group A, 35/43 (81.4%); group B, 25/33 (75.8%); group C, 43/70 (61.4%); and group D, 31/65 (47.7%) (P = 0.0001). Taking into account only difficult-to-treat HCV genotypes (1, 4, and 5), an even more pronounced stratification of the data occurred: group A, 18/21 (85.7%); group B, 6/11 (54.5%); group C, 14/38 (36.8%); and group D, 9/40 (22.5%) (P < 0.0001).

Disclosures: Olivier Chazouillères – Consulting: APTALIS, MAYOLY-SPINDLER The following people have nothing to disclose: Véronique D. Barbu, Isabelle Jéru, Christophe Corpechot, Eric Fernandez, Laure Muller, Fabienne Dufernez, Yannick Marie, Zhenyu Xu, Chantal Housset Background and Aim: Fenofibrate is a novel therapy for Primary Biliary Cirrhosis (PBC). We sought to perform a systematic review and a meta-analysis of studies that assessed

the efficacy of fenofibrate in the treatment of PBC patients. Methods: electronic database search was performed for relevant studies. Database searched included KU-57788 chemical structure PubMed, Scopus, and ScienceDirect. In addition, search of abstracts presented in the main scientific meetings in the field and articles in press was performed. Random effect model was used to pool the effect size across studies for changes in means of alkaline phosphatase, GGT, bilirubin and IgM levels before and after treatment and the overall rate of having complete response to fenofibrate therapy. Publication bias, heterogeneity testing and sensitivity analysis were also performed.

Results: Six studies with 102 patients (90% female) met the inclusion criteria. All studies were case JNK inhibitor crossover where patients who had no or incomplete response to UDCA had fenofibrate added at a dose of 100-200 mg daily. Treatment duration ranged from selleck compound 8-100 weeks. Treatment with fenofibrate was associated with a significant decrease in the pooled mean alkaline phosphatase (−114 IU/L, 95% CI:−152 to −76, p<0.0001); a significant decrease in GGT level (−92 IU/L, 95%CI:−149 to −43; p=0.0004); significant decrease in total bilirubin (−0.11mg/dl;95%CI:−0.18 to −0.08; p=0.0008), and a significant decrease in IgM level (−88mg/dl; 95%CI:−119 to −58; p<0.0001);. The pooled complete response rate was 69% (95% CI: 53-82%; p=0.024). The odds ratio of achieving complete response while on fenofi-brate was 2.43 (95% CI: 1.44-4.1, p=0.0009). Conclusions: Fenofibrate therapy at doses of 100-200 mg daily appears to be an effective

adjunctive therapy in PBC patients who had no or incomplete response to UDCA. There is a critical need for larger scale randomized trial to confirm its efficacy and define its position in the treatment paradigm of PBC. Disclosures: Cynthia Levy – Consulting: Lumena, Gilead, Evidera The following people have nothing to disclose: Alla Grigorian, Houssam E. Mardini, Christophe Corpechot, Raoul Poupon Background: Primary biliary cirrhosis (PBC) is a chronic, cholestatic liver disease that can lead to cirrhosis & liver failure. Ursodeoxycholic acid (UDCA) improves transplant-free survival, but up to 40% may not achieve adequate biochemical response. Fibrates may decrease alkaline phosphatase (ALP), but no study has examined their impact on transplant-free survival.

Treatment-related AEs were consistent with the known tolerability profile of onabotulinumtoxinA when injected into the head and neck muscles, and no newly emerged safety findings were observed. There were significantly more treatment-related AEs in the onabotulinumtoxinA group than in the placebo group. Individual Ulixertinib mouse AEs occurred in fewer than 10% of patients,

were mild to moderate in severity, and were generally transient. Although the precise mechanism of onabotulinumtoxinA as headache prophylaxis in CM is not fully elucidated, human and animal studies have shown that onabotulinumtoxinA blocks release of neurotransmitters associated with the genesis of pain.40-43 The presumed mechanism for headache prophylaxis is that, DAPT by blocking release of neurotransmitters, such as substance P, calcitonin gene-related peptide, and glutamate, from the peripheral termini of primary

afferents,40,41,44 onabotulinumtoxinA inhibits peripheral signals to the central nervous system and thus indirectly inhibits central sensitization. Central sensitization results from ongoing input from C-fiber nociceptors. Central sensitization may lead to cutaneous allodynia, which manifests as pain after ordinary nonnociceptive stimulation of skin. Bigal et al45 reported that in a population-based study, persons with migraine who experienced headache on ≥15 days per month reported significantly higher prevalence as well as significantly more severe cutaneous allodynia during headache attacks than did persons with migraine who experienced

headache on <15 days per month. These results suggest that persons with higher migraine headache day frequency are more susceptible selleck chemical to the adverse consequences of central sensitization and that a treatment directed at blocking this aspect of disease manifestation may be helpful. Immunogenicity manifested as antibody formation has been reported as an uncommon occurrence with chronic use of onabotulinumtoxinA in other therapeutic indications; such toxin neutralizing antibodies (TNA) can specifically inhibit the clinical effectiveness of treatment.46-48 Long-term management of CM may involve the administration of onabotulinumtoxinA injections to patients repeatedly over several months or years. Samples collected in phase 2 studies that evaluated up to 3 repeated treatments (every 12 weeks) of onabotulinumtoxinA doses as high as 260 U10,11,28 were evaluated for TNA using the validated mouse protection bioassay (MPA). The MPA is the gold standard for detection of TNA to onabotulinumtoxinA.49,50 The TNA analysis included 505 onabotulinumtoxinA-treated patients, of whom 496 had analyzable samples. There were no positive TNA, and 1 patient of 496 (0.2%) had inconclusive results.

Several researchers reported that when darts hit at a perpendicular angle to the animal, the largest samples (includes blubber and skin) are excised and retained, and minimal behavioral reactions are observed. Fourth, experienced vessel operators are paramount to the success of safely collecting biopsy samples and to minimizing disturbance. Several studies reported that slow approaches appear to minimize disturbance during biopsy sampling. Cetaceans also demonstrate less evasion when approached RG-7388 molecular weight slowly, increasing the probability of sampling success. Fifth, researchers should make a concerted effort to monitor and record the physiological and behavioral responses of cetaceans to

biopsy sampling. Norman et al. (2004) discuss several physiological parameters that should be monitored during the capture-release, handling, and tagging of odontocetes; and these are also applicable during surgical biopsy techniques. For remote biopsy techniques, however, other methods need to be utilized. For example, Mesnick, Wenzel, and their colleagues recommended specific data to be collected during each biopsy attempt and provided examples of sampling forms in their publications (Mesnick et al. 1999, Wenzel et al. 2010). The use of video cameras, particularly those

affixed to biopsy dart firing devices, allows researchers to more accurately quantify animals’ reactions to sampling events. Similarly, documenting the healing process with digital photographs of biopsy sites is important for assessing

long-term impacts and providing information on the time period required for healing, which is still unknown for most cetacean species. The VX-770 research buy standardization and systematic collection of data on factors that influence the success of acquiring samples and factors that influence behavioral and physiological this website responses are also critical to more easily compare results across studies and to better assess the impacts of cetacean biopsy techniques so that methods can be improved to yield the best samples with minimal disturbance. It is equally important to conduct studies that assess potential long-term impacts of biopsy sampling. Finally, in order to properly assess both short- and long-term effects of biopsy sampling, it is imperative that properly designed controls be implemented into research regimes. We thank L. Jones for her support and encouraging us to write this manuscript. We are indebted to S. Kromann from the National Marine Mammal Laboratory Library at the NOAA Alaska Fisheries Science Center for locating many of the references required for this review. D. Janiger, Curatorial Assistant (Mammals) from the Natural History Museum of Los Angeles County, California also provided PDFs of many papers that were included in this review. Finally, we greatly appreciate T. McCosh’s assistance with formatting and editing the text and tables and B. Diehl’s assistance with preparing figures. This manuscript was greatly improved by comments from P. Best, C. Emmons, M.

In contrast, measurements of protein induced by vitamin K absence or antagonist II (PIVKA-II) and AFP-lectin fraction (AFP-L3) show a characteristically

high specificity (∼95%) and are thus widely used in Japan. In hepatocellular carcinoma surveillance, tumor markers are used as a supplement to imaging tests. In such a situation, when tumor marker levels are elevated beyond their thresholds, even if abdominal ultrasonography fails to detect a lesion, there may be a case for performing high-sensitivity examinations such as dynamic CT. Under this circumstance, tumor marker levels with a high positive likelihood ratio (a ratio to increase post-test probability when it is positive) must be established. EX 527 concentration The absolute values of tumor markers can be viewed as a substitute for the total tumor mass

in the liver or body. Measurements of tumor markers before and after treatment enable one to objectively assess the effect of the therapy in reducing the tumor mass. In particular, they are considered to be highly useful for TACE. For tumor markers having high specificity, an evaluation of negativization may allow one to review radical cure by resection or local therapy. Japan is the only country where measurements of all the three types of tumor markers mentioned above are covered by the National Health Insurance. Therefore, Japan is making a substantial contribution in this field, and the majority of evidence has been collected from Pexidartinib manufacturer this country. CQ7 Is it useful to measure two or more tumor markers for the diagnosis of hepatocellular carcinoma? For the diagnosis of small hepatocellular carcinoma, measurement of two or more tumor markers is recommended. (grade A) In Japan, measurements of AFP, protein induced by vitamin K absence or antagonist II (PIVKA-II) and AFP-L3 are covered by the National Health Insurance, as tumor makers for hepatocellular carcinoma. α-Fetoprotein is the tumor marker that has been used for the longest time.

In the past, 500 ng/mL or more was a widely accepted level for making a definitive diagnosis of hepatocellular carcinoma. However, high AFP levels are rare in small hepatocellular carcinomas that can be detected by regular screening. Therefore, with the progress of diagnostic imaging, the position of AFP in the diagnosis of hepatocellular carcinoma has declined. PIVKA-II, also referred to as des-γ-carboxy prothrombin, see more is an abnormal prothrombin that has no coagulation activity and is synthesized in the liver. It has also been commonly employed in Japan as a hepatocellular carcinoma-specific tumor marker. As with AFP, PIVKA-II has a low positive rate in patients with small hepatocellular carcinomas. The AFP fraction with affinity to the Lens culinaris agglutinin (AFP-L3) is characterized by higher specificity for hepatocellular carcinoma than AFP. The sensitivity of AFP measurement for the diagnosis of hepatocellular carcinomas that were 3 cm or less in diameter was 23.

The effect of antiviral therapy on incidence of HCC has not been well established. The aim of this analysis

was to examine HCC incidence using a prediction model. Methods: The incidence of HCC in patients treated with TDF was obtained from the 6-year follow-up data of the registration trials for HBeAg-positive (GS-US-174-0103) and HBeAg-negative (GS-US-174-0102) patients. The predicted risk of HCC in individual patients was estimated using a model validated in cirrhotics and non-cirrhotics (the REACH-B model: Yang et al., Lancet Oncology, 2011). Standardized incidence ratios [SIR] were calculated Trametinib purchase between the observed and predicted numbers of HCC in the study cohort. Results: In the two studies, 641 patients received TDF for 6 years (375 subjects

in study 102 and 266 in 103). During this time, 14 patients with newly diagnosed HCC were reported. Nine were in study 102HBeAg positive; Epacadostat order among them 3 were cirrhotic. Five were in study 103HBeAg negative; among them 3 were cirrhotic. From the 14 HCC cases, 4 were genotype (gt) C, 5 gt-D, 2 gt-B, 1 gt-E, 1 gt-F and 1 unable to genotype. The 10th HCC case occurred at 3.3 years, at which

time the REACH-B model predicted 11.2 cases. Beyond that time, there was a progressive divergence between the predicted and observed number of HCC cases. In non cirrhotic patients, the effect of TDF became significant (55% reduction) at 6 years of therapy and the SIR was 0.45 (95% confidence interval [CI] = 0.227–0.909) for the last case selleckchem reported near week 336. Conclusion: Based on the REACH-B risk calculator, after long term therapy with TDF, the incidence of HCC decreased compared to the predicted risk. However, despite the small number of patients who developed HCC, continued surveillance is needed for CHB patients receiving long term oral antiviral treatment. AJ WIGG,1 R WUNDKE,1 R MCCORMICK,1 RJ WOODMAN2 1Hepatology and Liver Transplant Medicine Unit, Flinders Medical Centre, Adelaide. 2Division of General Practice, Flinders University, Adelaide.

HeLa, HEK293T, and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum (Invitrogen) GDC-0068 chemical structure containing penicillin and streptomycin. The Tac backbone construct was kindly provided by Dr. John Marshall

(Brown University, Providence, RI). The schematic structure of the TacCterm chimeras, consisting of the extracellular and transmembrane domains of Tac and the C-terminal tail of BSEP, is shown in Fig. 1. The Tac coding sequence was amplified by polymerase chain reaction (PCR) insertion of EcoRV and XbaI sites for subcloning into pcDNA3 (Invitrogen). TacCterm chimeras were constructed by a two-stage PCR method, using two sets of overlapping primers and ligating into the Tac construct using EcoRV and the XbaI site. The C-terminal tail of BSEP encoding residues D1284 to S1321 was amplified using human BSEP (kindly provided by beta-catenin tumor Dr. Bruno Stieger, University Hospital, Zurich, Switzerland). Deletion mutants of TacCterm (del 1298-1316; del1308-1316) and alanine substitutions in the following mutants were generated by site-directed mutagenesis: YY (Y1310A, Y1311A); LM (L1303A, M1304A); and

LMYY (L1303A, M1303A, Y1310A, Y1311A). A shorter version of the C-terminal BSEP, Tac8A-YY, was also generated by inserting eight alanines and the corresponding two residues G1308AYYKLV1314). All constructs were confirmed by DNA sequencing. Human full-length BSEP was amplified by PCR and inserted into the EcoRV site of the pWAY21-EGFP expression vector provided by Dr. Anton Bennett (Yale University, New Haven, CT). Mutant GFP-BSEP (Y1310A/Y1311A) was selleck compound generated by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). HEK293T cells were plated on poly-L-lysine coverslips and transiently transfected using LipofectAMINE 2000 reagent (Invitrogen) for 18 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS; with Mg and Ca) and labeled with mouse anti-Tac antibody (IL-2Rα, 0.5 μg/mL, 30 minutes, 4°C; BD Transduction Laboratories, San Jose, CA) in labeling buffer (PBS/Mg/Ca/0.2% bovine serum albumin [BSA]).

Internalization was initiated by warming to 37°C, carried out for the indicated time, and then stopped by washing repeatedly with ice-cold labeling buffer. Cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. TacCterm–anti-Tac complexes were detected with Alexa 488 or Alexa 568 anti-mouse secondary antibody (1:500; 1 hour), and fluorescent images were acquired on an LSM 510 confocal microscope (Carl Zeiss Inc., Thornwood, NY). Internalization of Tac chimeras was examined after cotransfection with the dominant-negative construct K44A dynamin (provided by Dr. Pietro de Camilli, Yale University) and with wild-type Rab5a-DsRed and dominant-negative N133I Rab5a-DsRed, kindly provided by Dr. Richard Pagano (plasmids 13050, 13051, Addgene, Cambridge, MA).