Rate constant for the H˙ + H2O → ˙OH + H2 reaction at elevated temperatures measured by pulse radiolysis.

Maintaining the structural integrity of materials in nuclear power plants is an essential issue associated with safe operation. Hydrogen (H2) addition or injection to coolants is a powerful technique that has been widely applied such that the reducing conditions in the coolant water avoid corrosion and stress corrosion cracking (SCC). Because the radiation-induced reaction of ˙OH + H2 → H˙ + H2O plays a crucial role in these systems, the rate constant has been measured at operation temperatures of the reactors (285-300 °C) by pulse radiolysis, generating sufficient data for analysis. The reverse reaction H˙ + H2O → ˙OH + H2 is negligibly slow at ambient temperature; however, it accelerates considerably quickly at elevated temperatures. Although the reverse reaction reduces the effectiveness of H2 addition, reliable rate constants have not yet been measured. In this study, the rate constants have been determined in a temperature range of 250-350 °C by pulse radiolysis in an aqueous I- solution.

Ultra-high dose-rate (FLASH) radiotherapy: Generation of early, transient, strongly acidic spikes in the irradiated tumor environment.

Monte Carlo simulations of γ/fast electron-radiolysis of water show that the in situ formation of H3O+ temporarily renders each "native" isolated spur/track region very acidic. For pulsed (FLASH) irradiation with high dose rate, this early time, transient "acid-spike" response is shown to extend evenly across the entire irradiated volume. Since pH controls many cellular processes, this study highlights the need to consider these spikes of acidity in understanding the fundamental mechanisms underlying FLASH radiotherapy.

Mitogen-induced replication of woodchuck hepatitis virus in cultured peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) isolated from woodchucks chronically infected with the woodchuck hepatitis virus (WHV) carry low levels of nonreplicating WHV DNA. When PBLs from chronic carrier woodchucks were activated in culture with the generalized mitogen lipopolysaccharide (LPS), WHV DNA replication was initiated in cells obtained from one of three animals examined. Intracellular WHV core particles, containing WHV DNA replication intermediates, RNA/DNA hybrid molecules, and an active endogenous DNA polymerase, appeared 3 days after the start of LPS stimulation. After 5 to 7 days of LPS stimulation, WHV DNA-containing particles, which displayed the properties of intact, mature virions, were released into the culture medium. These studies provide evidence for reactivation of a latent WHV infection of circulating lymphoid cells and indicate that the presence of nonreplicating hepadnaviral DNA in lymphoid cells represents a potentially active infection following cellular activation.

An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus.

The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.

Cloned fragment of the hepatitis delta virus RNA genome: sequence and diagnostic application.

Hepatitis delta virus (HDV) is a replication-defective etiological agent of hepatitis that requires hepatitis B virus (HBV) as a helper. A complementary DNA (cDNA) fragment of the RNA genome of HDV was cloned into the plasmid vector pBR322, and the primary nucleotide sequence and predicted protein products of the cDNA fragment were determined. This cloned cDNA fragment has been used as a sensitive radioactive probe for the detection of HDV RNA in the serum of patients with either acute or chronic HDV infections.

Treatment of chronic delta hepatitis with lamivudine vs lamivudine + interferon vs interferon.

Chronic delta hepatitis is the most severe form of chronic viral hepatitis for which interferon (IFN) is the only available treatment. In 39 patients (25 were treatment-naïve, 14 had previously used IFN), efficacy of 1-year treatment with IFN (9 MU, t.i.w.) or lamivudine (LAM; 100 mg, q.d.) alone was compared with IFN and LAM combination (2 months of LAM to be followed by combination treatment). IFN monotherapy was given only to treatment-naïve patients. In both treatment-naïve and previous IFN users, end of treatment virological and biochemical responses were similar with IFN-LAM combination and superior to LAM monotherapy (P < 0.05). Improvement in liver histology occurred more often with IFN +/- LAM than with LAM alone (P < 0.05). In treatment-naïve patients, combination treatment was not superior to IFN monotherapy. After treatment discontinuation, virological and biochemical response rates decreased in LAM and IFN combination and IFN monotherapy. On treatment virological response at month 6 of treatment predicted sustained virological response. The results of this study suggest that addition of LAM to IFN for the treatment of delta hepatitis is of no additional value and that both treatment modalities are superior to LAM monotherapy.

In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent.

The woodchuck model of HDV infection.

The Eastern woodchuck, Marmota monax, has been a useful model system for the study of the natural history of hepadnavirus infection and for the development and preclinical testing of antiviral therapies. The model has also been used for hepatitis delta virus (HDV). In this chapter several new applications of the woodchuck model of HDV infection are presented and discussed. The development of a woodchuck HDV inoculum derived from a molecular clone has facilitated the analysis of viral genetic changes occurring during acute and chronic infection. This analysis has provided insights into one of the more important aspects of the natural history of HDV infection-whether a superinfection becomes chronic. These results could renew interest in further vaccine development. An effective therapy for chronic HDV infection remains an important clinical goal for this agent, particularly because of the severity of the disease and the inability of current hepadnaviral therapies to ameliorate it. The recent application of the woodchuck model of chronic HDV infection to therapeutic development has yielded promising results which indicate that targeting the hepadnavirus surface protein may be a successful therapeutic strategy for HDV.

Alpha-fetoprotein in the woodchuck model of hepadnavirus infection and disease: normal physiological patterns and responses to woodchuck hepatitis virus infection and hepatocellular carcinoma.

Persistent infection of the eastern woodchuck (Marmota monax) with the woodchuck hepatitis virus (WHV) produces disease sequelae similar to those observed in humans with persistent hepatitis B virus infection, including hepatocellular carcinoma (HCC). To further characterize serological markers of HCC in the woodchuck, serum alpha-fetoprotein (AFP) was measured under normal physiological conditions and following infection with WHV. Serum AFP was elevated in association with WHV-induced hepatitis and HCC and was a useful indicator of hepatic responses in individual animals throughout the course of experimental WHV infection. The frequent occurrence of normal elevations in serum AFP during the fall and winter, however, limits the use of AFP as a marker for early detection of HCC. The present temporal studies of AFP responses in WHV-infected woodchucks have identified several stages of infection where virological and cellular interactions can be investigated at the molecular level. Studies of AFP in the woodchuck model should provide opportunities to further elucidate the physiological and immunological functions of AFP and to understand virus-host cell interactions during the course of experimental hepadnavirus infection leading to HCC.

Expression of hepatitis B virus surface and e antigen genes cloned in bovine papillomavirus vectors.

Sequences of the hepatitis B virus (HBV) genome coding for the surface antigen (HBsAg), but lacking the regulatory (preS) sequences, were cloned into a bovine papillomavirus (BPV) vector consisting of the transforming 69% of the BPV genome (BPV 69T), and transformed into mouse c127 cells. Clones carrying HBV and BPV sequences in the same transcriptional orientation did not produce immunologically active HBsAg, while those with the opposite orientation produced and secreted HBsAg. RNA species of an HBsAg producer were 3400, 9600, 11000 and 18000 nucleotides long and hybridized with both HBV and BPV probes. Mouse cells (c127) transformed with the whole BPV genome (BPV-1) carrying both the HBsAg-coding sequences and the regulatory preS sequences of HBV DNA were stable and produced and secreted HBsAg 22 nm particles. These yielded 11500 and 12500 nucleotide RNA transcripts which also hybridized with both BPV and HBV DNA probes. BPV-1 carrying whole HBV DNA monomer or dimer genomes yielded transformants which initially produced HBsAg as well as HBV e antigen (HBeAg), but these were not stable. The hybrid genomes, with the exception of those carrying the HBV dimers, existed as multicopy plasmids (50-100 copies per cell) and often acquired new sequences.

Age-related increased susceptibility of high-density lipoproteins (HDL) to in vitro oxidation induced by gamma-radiolysis of water.

In the present study, we investigated the age-related susceptibility of high-density lipoproteins (HDL) to oxidation. HDL were obtained from healthy, normolipidemic young, middle-aged and elderly subjects. Oxidation of HDL was induced in vitro by oxygen free radicals generated by water gamma-radiolysis, and followed by the decrease of endogenous vitamin E and the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the alterations of apolipoproteins A-I/A-II. The resistance of HDL to oxidation, evaluated by the length of the lag phase, decreased with aging. This increased oxidizability of HDL with aging could have a dramatic impact on the development of atherosclerosis in the elderly population.

Enhanced antiviral benefit of combination therapy with lamivudine and alpha interferon against WHV replication in chronic carrier woodchucks.

Cell culture studies in our laboratory previously demonstrated synergistic antiviral activity for the combinations of lamivudine and a novel recombinant hybrid human alpha B/D interferon (rHu alpha B/D IFN) against hepatitis B virus (HBV) replication. Based on these results, a study was designed to determine if an enhanced antiviral effect with this drug combination could be demonstrated in vivo using the woodchuck hepatitis virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers during previous studies by our laboratories. Two combination treatment regimens were compared to matched monotherapies in a placebo-controlled trial. The first used simultaneous administration of rHu alpha B/D IFN and lamivudine for 24 weeks. The other combination treatment regimen used a staggered dosing schedule of 12 weeks of administration of lamivudine alone, followed by 12 weeks of simultaneous dosing with both drugs, followed by 12 weeks of therapy with rHu alpha B/D IFN alone. Both treatment regimens with combinations of lamivudine and rHu alpha B/D IFN were more effective at reducing WHV replication in chronically infected wood-chucks than the corresponding monotherapies. Both combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. The staggered treatment regimen reduced viraemia and intrahepatic WHV replication significantly more than that expected for additive interactions, indicating synergistic antiviral effects. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent.

Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2-fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L-FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment.

Are there protective enzymatic pathways to regulate high local nitric oxide (NO) concentrations in cells under stress conditions?

This paper examines, from a chemical perspective, the hypothesis of the existence of protective enzymes whose role would be to regulate the high local nitric oxide (NO) concentrations that are released in NO-generating cells in situations of response to oxidative stress. These enzymes should play the role, with respect to NO, either of a reductase or of a dismutase. The energetics of the intervening transformations is herein presented, along with a review of pertinent literature. An attempt is made in order to describe the physiognomy of such enzymes, in relation with the literature data. Experimental investigation is needed to further evaluate the validity of such a hypothesis.

Monocytes influence the fate of T cells challenged with oxidised low density lipoproteins towards apoptosis or MHC-restricted proliferation.

Atherosclerosis has been implicated in myocardial infarction, stroke and a host of cardiovascular diseases. The presence of activated T lymphocytes and macrophages, and the increased expression of HLA-DR antigen are consistent with the notion of immune activity in the atherosclerotic plaque. The nature of the causative antigen has not been established although oxidised low density lipoproteins (oxLDL) that accumulate in atherosclerotic plaques could fulfil this role. Here, we report that monocytes play a key role in influencing the fate of purified peripheral human T lymphocytes from healthy donors when the cells are exposed to LDL oxidised under the controlled conditions of water radiolysis. Our data showed that oxLDL generated under these conditions were chemoattractants for T cells. However, they induced a state of apoptosis in T lymphocytes cultured in the absence of monocytes. The extent of apoptosis was related to the degree of oxidation of LDL and the time of T cell exposure to oxLDL. OxLDL-dependent apoptosis did not involve a scavenger-like receptor. CD4(+) cells were more sensitive to the apoptotic effect of oxLDL than CD8(+) cells. OxLDL-primed (12 h) autologous monocytes triggered a robust proliferation of T lymphocytes cultured in the absence of oxLDL. The strength of T cell stimulation was related to the degree of oxidation of the LDL used in priming. Heterologous monocytes exposed to oxLDL under similar conditions induced a response that was not different than monocytes exposed to untreated LDL (natLDL) which did not induce T cell proliferation. Fucoidan did not modify the oxLDL-, monocyte-dependent T cell response to proliferation, suggesting that a scavenger-like receptor was not involved. The expression of the HLA-DR marker and the B7.2 protein were up-regulated in monocytes exposed to oxLDL but not to natLDL. The levels of B7.1 were unchanged. Our data are consistent with the notion that monocytes are critical for T cell survival in the presence of oxLDL and MHC-restricted T cell proliferative response to oxLDL.

Dehydroepiandrosterone protects low density lipoproteins against peroxidation by free radicals produced by gamma-radiolysis of ethanol-water mixtures.

Oxidized low density lipoproteins (LDL) are believed to play a central role in the events that initiate atherosclerosis. Antioxidants have been shown to decrease the oxidation of LDL, leading to the diminution of atherosclerosis. Since it is well-known that decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, we studied the modulation of the oxidation of LDL by DHEA. LDL were obtained from 10 healthy subjects and oxidized by free radicals produced by gamma-radiolysis of ethanol-water mixtures. The formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), the vitamin E content, as well as the incorporation of 4-[14C]DHEA in LDL and the chemotactic effect of oxidized LDL in the presence of DHEA towards monocytes, were investigated. It was found that DHEA was able to inhibit the oxidation of LDL by reducing over 90% of the conjugated dienes and TBARS formation, as well as by reducing the vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes. Our results suggest that DHEA exerts its antioxidative effect by protecting the endogenous vitamin E of LDL.

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

Altered glycosylation of alpha-fetoprotein in hepadnavirus-induced hepatocellular carcinoma of the woodchuck.

Altered glycosylation of alpha-fetoprotein (AFP) has been proposed as a marker of hepatocellular carcinoma (HCC) in humans. The lectin-binding properties of woodchuck AFP were investigated to determine if woodchuck hepatitis virus (WHV)-induced HCCs are also accompanied by changes in AFP glycosylation. Ninety-eight to 100% of the AFP from normal, WHV-free woodchucks with physiologic AFP elevations and from WHV-carrier woodchucks with HCC bound to concanavalin A, indicating that virtually all of the AFP was glycosylated. Three percent or less of the serum AFP of normal woodchucks bound to Lens culinaris agglutinin (LCA). In contrast, the AFP from woodchucks with HCC had an increased LCA-binding fraction (range, 8-77%). The increased LCA-binding AFP in WHV-induced HCC is analogous to that which frequently accompanies hepatitis B virus (HBV)-induced HCC in humans. This study corroborates the relationship of altered glycoconjugate synthesis to virus-induced malignant transformation, confirms the importance of AFP glycoforms as markers of HCC, and demonstrates that the WHV-infected woodchuck should be useful in investigating changes in AFP glycosylation during hepadnavirus hepatocarcinogenesis and HCC growth.

Antibodies to polymerized albumin in woodchuck hepatitis virus infection.

Polymerized human serum albumin may play a role in the entry of hepatitis B virus into hepatocytes, and antibodies to polyalbumin that frequently appear during acute hepatitis may aid the process of viral clearance. We developed an enzyme-linked immunosorbent assay for antibodies to polymerized woodchuck albumin to enable us to evaluate further the role of these antibodies in an animal model system. Sera from 17 uninfected adult woodchucks and 8 newborns showed no binding to control plates coated with woodchuck transferrin, woodchuck albumin, or polymerized human serum albumin. One of 8 newborn animals demonstrated a significant antibody titer to polymerized woodchuck albumin, and 16 of 17 adults without evidence of prior woodchuck hepatitis virus infection had measurable serum antibody titers. Antibodies to polymerized woodchuck albumin could be adsorbed by prior incubation with the antigen. In 2 animals subjected to experimental infection, significant rises in polyalbumin antibody were seen. When 4 adult woodchucks were immunized with woodchuck polyalbumin, significant increases in antibody titer were observed in 2 of the 4 animals. Of the 4 immunized and 4 controls subsequently challenged with woodchuck hepatitis virus, 7 became viremic and all 8 developed antibody to woodchuck hepatitis virus core antigen. We conclude that naturally occurring antibodies to polymerized woodchuck albumin are observed in most adult woodchucks in the absence of woodchuck hepatitis virus infection and do not seem to confer immunity against infection with this virus.