We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B-and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4؉ -T-cell responses. To better characterize the CD8 ؉ -T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8؉ -T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8؉ -T-cell responses after vaccination. The most frequently recognized peptides were

Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The application of these conclusions to human gene therapy with recombinant adenovirus should lead to the development of strategies to overcome the formation of such neutralization antibodies, which have been shown to limit the efficacy of gene transfer in humans.

The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2-specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. Conclusion: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs. (HEPATOLOGY 2012;56:861-872) S tudies on the mechanisms of hepatitis C virus (HCV) infection have been hampered by limited in vivo and ex vivo models 1-4 as the consequence of poor replication of primary viral isolates and restriction of susceptible hosts (i.e., humans and chimpanzees). Recent advances have yielded cell-culture replication-competent chimeric viruses that have the capacity to achieve the complete infectious cycle. Of note, the infectious clone of genotype 2a HCV, Japanese fulminant hepatitis type 1 (JFH-1), 5,6 allowed us to develop chimeric viruses with exceptionally efficient replication, 7 because so-called cell-culture-grown hepatitis C virus (HCVcc) can be propagated, with infectivity titers in the range of 10 5 focus-forming units (ffu)/mL in the highly permissive Huh-7 subline, Huh-7.5.1. 8,9 However, this system has several limitations that include the inability to study the effects of pharmacologic inhibitors targeting the nonstructural proteins of the most prevalent, problematic viral strains (e.g., genotypes 1a and 1b). Moreover, the study of virus/host cell interactions is limited, because the permissive cell lines are transformed and poorly differentiated. Attempts to infect primary hepatocytes using primary HCV isolates resulted in limited poor viral replication and low production of de novo infectious virus particles. 10,11 Other models using primary human HCVcc-infected hepatocytes 12 or HCV-transfected hepatocytes cocultured with stellate cell lines 13 have been developed, which allowed virus replication of the entire viral life cycle with relatively high viral titers.

This study examines in detail the capsid-specific humoral immune response of BALB/c mice after one single injection of a replication-defective adenovirus. Two routes of immunization, intravenous (i.v.) and intraperitoneal (i.p.), were compared for the response induced against the adenovirus particle and the three major components of the viral capsid, hexon, penton base, and fiber. A single immunization with the replication-defective adenovirus induces a long and persistent humoral response specific for the virus. However, the molecular components of the viral capsid are differentially recognized depending on the route of immunization. The sera from mice immunized i.p. recognized only the hexon protein and a preferential switch to the IgG2a subclass was obtained which remained stable 100 days post-immunization. The sera obtained from mice immunized i.v. gave a more complex response. At the beginning of the response, an isotype bias toward the IgG2a subclass was observed, but the isotype distribution changed during the whole period of the response. Neutralizing activity was maximum 45 days after immunization by both routes, and no activity was detectable after 3 months. However, the i.v. serum displayed a higher neutralizing activity than the i.p. serum. The IgM antiviral antibodies appeared to be an important component of the neutralizing activity, and the two routes of immunization do not induce the same IgG isotypes to neutralize viral infectivity. Extension of these findings to human gene therapy using recombinant adenoviruses may help to characterize the precise viral protein targets of neutralizing antibodies.

An efficient vaccine against human immunodeficiency virus (HIV) must induce good cellular immune responses. To do this, it must be processed and presented by dendritic cells, which are required for primary T-lymphocyte stimulation. We have previously shown that a model lipopeptide containing a short epitopic peptide from HIV-1 was endocytosed and presented in association with major histocompatibility complex class I molecules by human dendritic cells to specific CD8 ؉ T lymphocytes, but the cross-presentation pathway needed to be precisely determined. We have studied a longer lipopeptide (Pol 461-484 ) and another lipopeptide (Nef 66-97 ) currently being used in vaccine trials. Like the shorter lipopeptide, the rhodamine-labeled Pol 461-484 lipopeptide was internalized by endocytosis, as assessed by confocal microscopy. The lipopeptides were processed by dendritic cells and presented to CD8 ؉ T cells specific for the HLA-A*0201-restricted Pol 476-484 and the HLA-A*0301-restricted Nef 73-82 epitope, respectively. Presentation of both lipopeptides was inhibited by brefeldin A. Presentation of the Pol lipopeptide was inhibited by epoxomycin, a proteasome-specific inhibitor, but not by monensin. This shows that it gained access to the cytosol to be digested by the proteasome. In contrast, presentation of the Nef lipopeptide was not inhibited by epoxomycin but was inhibited by monensin, a classical inhibitor of acid-dependent endosomal enzyme activity, indicating an endocytic processing pathway yielding to major histocompatibility complex class I-restricted presentation. Therefore, the two lipopeptides followed different cross-presentation pathways, both resulting in efficient presentation to CD8 ؉ T lymphocytes.

.-In the livers of humans and many other mammalian species, ␤ 2-adrenergic receptors (␤2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic ␤ 2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (André C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: [417][418][419][420][421][422][423][424] 1996), which carry the human ␤ 2-AR gene under the control of its own promoter. In these mice, hepatic ␤ 2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to ␤-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased ␤ 2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and L-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of ␤-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the ␤ 2-AR agonist clenbuterol and increased by the ␤-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic ␤2-AR system physiopathology and for testing the effects of ␤-AR ligands on liver metabolism.␤-adrenergic receptor; glycogen; glucose; phosphoenolpyruvate carboxykinase; L-pyruvate kinase THE INTERACTION OF CIRCULATING CATECHOLAMINES with adrenergic receptors plays an important role in the regulation of lipid, protein, and carbohydrate metabolism. Epinephrine enhances lipolysis in adipose tissue, glycogenolysis in muscle, and glucose production in liver that result from both direct stimulation of glycogenolysis and indirect stimulation of gluconeogenesis (3,21,26).Both ␣ 1 -and ␤ 2 -adrenergic receptors (␣ 1 -ARs and ␤ 2 -ARs) are expressed in the liver and are involved in the direct effect of catecholamines. Whereas ␣ 1 -ARs are linked to the phosphoinositide turnover/calcium mobilization, ␤ 2 -ARs mediate their effects via the adenylyl cyclase/cAMP-signaling pathway. The relative contribution of the two adrenergic receptor subtypes to catecholamine-induced glycogenolysis depends on animal species, sex, and physiological state (16,19).In the livers of most species, including humans, the number of both ␣ 1 -and ␤ 2 -ARs is high, and catecholamine-stimulated glycogenolysis occurs primarily via ␤ 2 -ARs in normal physiological conditions (3,17,21,26,28,34). In male rats and mice, however, there is a reversal in predominance from ␤ 2 -ARs to ␣ 1 -ARs during development. The ␤ 2 -AR density, ␤ 2 -AR-induced cAMP responses, and ␤ 2 -AR-dependent glycogen...

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Plasma diazepam concentrations were measured by gas-liquid chromatography in samples of blood from adult female patients follwing diazepam 10 mg orally, alone or in combination with metoclopramide, morphine, pethidine or atropine. Patients receiving metoclopramide had higher plasma diazepam concentrations than those in the control group, while the addition of morphine, pethidine or atropine resulted in lower plasma diazepam concentrations throughout the 90-min period of the study. In the control goup peak plasma concentrations were reached by 60 min. The addition of metoclopramide increased the rate of diazepam absorption and peak concentrations were reached by 30 min, while morphine, pethidine and atropine reduced the rate of absorption with no apparent peak being reached by 90 min.