Administration Of Nucleoside Analogues Research Articles

Specific Disruption of Memory Reconsolidation of Conditioned Food Aversion in Snails by DNA Synthesis Inhibitors.

The involvement of DNA synthesis in the mechanisms of long-term memory reconsolidation in edible snails trained for conditioned food aversion was investigated. Administration of nucleoside analogs, such as 5-bromo-2'-deoxyuridine or 3'-azido-3'-deoxythymidine, which inhibit DNA synthesis, 1 h before or 1-3 h, but not 5 h after reminder with the conditioned stimulus led to memory impairment. One day after the inhibitor application and memory reactivation, a weakly expressed memory impairment (amnesia) was observed, which progressed over the next few days to the complete disappearance of behavioral memory expression. In snails with formed aversive memory for two food conditioned stimulus, a specific memory impairment was observed only for the stimulus that was paired with the presentation of an inhibitor during the reminder, while the memory for the other non-reactivated conditioned stimulus remained intact. It is suggested that DNA synthesis in the brain plays a specific role in the genetic mechanisms of reconsolidation and maintenance of long-term conditioned food aversion memory in snails.

Immune responses of human T lymphocytes to novel hepatitis B virus-derived peptides.

Background & aimsMany individuals are infected with hepatitis B virus (HBV) worldwide, and this virus is commonly controlled by treatments with interferon (IFN)-alpha and nucleoside analogues (NA). However, the complete elimination of HBV by these treatments is difficult and, thus, the development of new treatments is needed. Host immune responses are closely involved in the elimination of HBV, suggesting the usefulness of immunotherapy. In the present study, we attempted to identify novel cytotoxic T-lymphocyte (CTL) epitopes that are useful for immunotherapy against HBV.MethodsCTL epitopes were predicted using computer software. Immune responses to each peptide were evaluated by IFN-γ ELISPOT and cytotoxic assays. The relationships between the immune responses to these newly identified CTL epitopes and the clinical backgrounds of patients and administration of NA were analyzed. Peptides were administered to mice as vaccines and peptide-specific T-cell induction was measured in vivo.ResultsPositive reactions to 10 synthesized peptides were detected in 3 or more patients using the IFN-γ ELISPOT assay, and concentration-dependent cytotoxicity against 2 of these peptides was observed in the cytotoxic assay. Some peptides that correlated with serum ALT, HBsAg, and HBV core-related antigen (HBcrAg) levels were identified. Immune reactions against some peptides were enhanced by the administration of NA. Regarding their effects as a vaccine, peptide-specific T-cells were induced by four peptides in vivo.ConclusionsNovel HBV epitopes that correlated with HBsAg and HBcrAg levels were identified. These newly identified epitopes may be useful in the analysis of immune responses to HBV and development of immunotherapy against HBV.

Impatto sul budget di un trattamento personalizzato dell’epatite B cronica HBeAg-negativa in Italia mediante peg-interferone alfa-2a associato alla stopping-rule alla 12a settimana

Background and objective Treatment options for chronic hepatitis B (CHB) are the direct inhibition of viral replication by continuous administration of nucleoside analogues (NUCs) or a finite 48-week course of peg-interferon (PEG). PEG can induce the off-therapy immune control of CHB leading to HBsAg loss/anti-HBs seroconversion, but with a low success rate. On the other hand life-long treatment with NUCs is expensive. Currently in Italy, around 67% of naive patients receive treatment with NUCs. However, exploiting the early identification of PEG-non-responders by combined HBV-DNA and HBsAg quantification at week 12 (stopping-rule) is a new sequential therapeutic strategy that may benefit both patients and third payers. We measured the impact on the Italian National Health Service (NHS) budget of the strategy PEG-week-12-stopping-rule in the treatment of HBeAg-negative CHB.

Prevention of hepatitis B virus reactivation in immunosuppressive therapy or chemotherapy

In recent years, hepatitis B virus (HBV) has been found to reproliferate either during or following immunosuppressive therapy or chemotherapy, with hepatitis caused by HBV reactivation now considered a serious issue. HBV reactivation is categorized into occurrence in HBsAg- and anti-HBe-positive asymptomatic carriers, and in HBsAg-negative, anti-HBc low-titer-positive, and/or anti-HBs-positive resolved HBV infection cases. Despite the fact that "clinical cure" is claimed for such resolved HBV cases, low levels of ongoing HBV production are now recognized as being sustained within the liver or in peripheral blood mononuclear cells, with the infection thus now considered to be virologically persistent. The risk of HBV reactivation rises as the level of immunosuppression intensifies, but in recent years HBV reactivation risk has been clearly shown to increase in cases of rituximab plus steroid-containing regimen for treatment of malignant lymphoma. In particular, the incidence of fulminant hepatitis caused by HBV reactivation in cases with resolved HBV infection is reported to be higher than that brought about by acute hepatitis B. Therefore, for all cases in which immunosuppressive therapy or chemotherapy treatment regimens are used, screening for HBV infection and appropriate management in accordance with the status of HBV-related markers are crucial, aimed at preventing occurrence of HBV reactivation. The foundation of the aforementioned management, regardless of HBsAg status, is administration of nucleoside analogues, with their powerful anti-viral properties, when HBV DNA levels reach detectable levels.

Reactivation of resolved hepatitis B virus infection after immunosuppression: Is it time to adopt pre-emptive therapy?

New therapeutic options like monoclonal antibodies (anti-CD20/rituximab) and hematopoietic stem cell transplantation (HSCT) have increased both the effectiveness of therapies and the risk for reactivation of Hepatitis B virus (HBV). We describe two cases with serological evidence of resolved HBV infection (hepatitis B surface antigen (HBsAg) negative/antibody to hepatitis B core antigen (anti-HBc) and antibody to hepatitis B surface antigen (anti-HBs) positive), who developed reverse seroconversion (clearance of HBsAb/appearance of HBsAg) with active HBV infection after treatment with combination of conventional chemotherapy, rituximab and autologous HSCT for hematological malignancies. Review of the literature highlights the increasing incidence of HBV reactivation in patients with resolved infection and raises concerns as to whether current guidelines for pre-chemotherapy screening with sensitive HBV-DNA assays and serial monitoring for anti-HBs titres should be implemented also for patients with resolved infection. Future studies should aim at clarifying the cost-benefit from administration of nucleoside analogues in these patients.

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

The RNase H activity of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) cleaves the viral genome concomitant with minus strand synthesis. We previously analyzed RT-mediated pausing and RNase H cleavage on a hairpin-containing RNA template system and reported that RT generated 3' end-directed primary and secondary cuts while paused at the base of the hairpin during synthesis. Here, we report that all of the prominent cleavage products observed during primer extension on this template correlated with pause induced cuts. Products that persisted throughout the reaction corresponded to secondary cuts, about eight nucleotides in from the DNA primer terminus. This distance allows little overlap of intact template with the primer terminus. We considered whether secondary cuts could inactivate further synthesis by promoting dissociation of the primer from the template. As anticipated, 3' end-directed secondary cuts decreased primer extendibility. This provides a plausible mechanism to explain the persistence of secondary cut products in our hairpin template system. Improving the efficiency of synthesis by increasing the concentration of dNTPs or addition of nucleocapsid protein (NC) reduced pausing and the generation of pause related secondary cuts on this template. Further studies reveal that 3' end-directed primary and secondary cleavages were also generated when synthesis was stalled by the presence of 3'-azido-3'-deoxythymidine at the primer terminus, possibly contributing to 3'-azido-3'-deoxythymidine inhibition. Considered together, the data reveal a role for NC and other factors that enhance DNA synthesis in the prevention of RNase H cleavages that could be detrimental to viral replication.

Fidelity of the Human Mitochondrial DNA Polymerase

We have quantified the fidelity of polymerization of DNA by human mitochondrial DNA polymerase using synthetic DNA oligonucleotides and recombinant holoenzyme and examining each of the possible 16-base pair combinations. Although the kinetics of incorporation for all correct nucleotides are similar, with an average Kd of 0.8 microM and an average k(pol) of 37 s(-1), the kinetics of misincorporation vary widely. The ground state binding Kd of incorrect bases ranges from a low of 25 microM for a dATP:A mispair to a high of 360 microM for a dCTP:T mispair. Similarly, the rates of incorporation of incorrect bases vary from 0.0031 s(-1) for a dCTP:C mispair to 1.16 s(-1) for a dGTP:T mispair. Due to the variability in the kinetic parameters for misincorporation, the estimates of fidelity range from 1 error in 3563 nucleotides for dGTP:T to 1 error in 2.3 x 10(6) nucleotides for dCTP:C. Interestingly, the discrimination against a dGTP:T mismatch is 16.5 times lower than that of a dTTP:G mismatch due to a tighter Kd for ground state binding and a faster rate of incorporation of the dGTP:T mismatch relative to the dTTP:G mismatch. We calculate an average fidelity of 1 error in 440,000 nucleotides.

High hepatitis B virus (HBV) DNA viral load as the most important risk factor for HBV reactivation in patients positive for HBV surface antigen undergoing autologous hematopoietic cell transplantation

The risk factors for hepatitis due to hepatitis B virus (HBV) reactivation in patients positive for hepatitis B surface antigen (HBsAg) treated with autologous hematopoietic cell transplantation (HCT) are unknown. We evaluated 137 consecutive patients (23 positive for HBsAg, 37 positive for hepatitis B surface antibody, and 77 negative for HBV) who underwent HCT. Serial serum ALT were measured before transplant and after transplant at 1 to 4 weekly intervals for the first year and then at 2 to 12 weekly intervals thereafter. Before HCT, basic core promoter (T(1762)/A(1764)) and precore (A(1896)) HBV variants were determined in HBsAg-positive and HBV DNA-positive (by polymerase chain reaction assay) patients by direct sequencing and serum HBV DNA quantitation using the Digene Hybrid Capture II assay. Cox proportional hazards analysis was used to assess the association between pretransplantation HBV virologic and host factors and occurrence of hepatitis due to HBV reactivation. After HCT, hepatitis due to HBV reactivation was more common in HBsAg-positive patients than in HBsAg-negative patients (hazard ratio, 33.3; 95% confidence interval [CI], 7.35-142.86; P <.0001). HBsAg-positive patients with detectable serum HBV DNA before HCT (on Digene assay) had a significantly higher risk of hepatitis due to HBV reactivation than HBsAg-positive patients with no detectable serum HBV DNA (adjusted hazard ratio, 9.35; 95% CI, 1.65-52.6; P =.012). Thus, we found that hepatitis due to HBV reactivation is common in HBsAg-positive patients undergoing autologous HCT. A high HBV DNA level (>10(5) copies/mL) was the most important risk factor for HBV reactivation, and its lowering by administration of nucleoside analogues before transplantation should be considered.

A new acyclic heterodinucleotide active against Human Immunodeficiency Virus and Herpes Simplex Virus

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1) infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice against HSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity in a phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of both HSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should be welcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug bound by a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase their phagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protection against both HSV-1 and HIV-1 replication in human macrophages.

Macrophage protection against human immunodeficiency virus or herpes simplex virus by red blood cell-mediated delivery of a heterodinucleotide of azidothymidine and acyclovir.

Human herpesvirus (HSVs) are distributed worldwide and are among the most frequent causes of viral infection in HIV-1-immunocompromised patients. Hence, therapeutic strategies able to inhibit HSV-1 and HIV-1 replication are sorely needed. Until now, the most common therapies against HSV-1 and HIV-1 infectivity have been based on the administration of nucleoside analogs; however, to be active, these antiviral drugs must be converted to their triphosphorylated derivatives by viral and/or cellular kinases. At the cellular level, the main problems involved in the use of such drugs are their limited phosphorylation in some cells (e.g., antiretroviral drugs in macrophages) and the cytotoxic side effects of nucleoside analog triphosphates. To overcome these limitations, a new heterodinucleotide (AZTp2ACV) consisting of both an antiretroviral and an antiherpetic drug, bound by a pyrophosphate bridge, was designed and synthesized. The impermeant AZTp2ACV was encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages. Once inside macrophages, metabolic activation of the drug occurred. The addition of AZTp2ACV-loaded erythrocytes to human macrophages provided effective and almost complete in vitro protection from HIV-1 and HSV-1 replications, respectively. Therefore, AZTp2ACV acts as an efficient antiviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

FIV infection of macrophages: in vitro and in vivo inhibition by dideoxycytidine 5′-triphosphate

We have evaluated in vitro and in vivo whether it is possible to protect cat macrophages from feline immunodeficiency virus (FIV) infection by the administration of dideoxycytidine 5′-triphosphate (DDCTP). Since cell membranes are impermeable to phosphorylated drugs we have encapsulated DDCTP into autologous erythrocytes and modified erythrocyte membranes to target these drug-loaded cells to macrophages. DDCTP-loaded erythrocytes reduced FIV production by macrophages infected in vitro or obtained from naturally or experimentally infected cats. The same treatment protected the majority of peritoneal macrophages during a 7 month experimental FIV infection and reduced the percentage of circulating lymphocytes stained with an anti-p24 antibody. These results suggest that the administration of nucleoside analogues in phosphorylated form is feasible and their targeting to macrophages reduces FIV infection in vitro and in vivo.

Feline Immunodeficiency Virus Infection of Macrophages:In Vitroandin VivoInhibition by Dideoxycytidine-5′-triphosphate-Loaded Erythrocytes

Although HIV-1 and other mammalian lentiviruses infect macrophages, they are not cytopathic. Consequently, these infected long-lived cells serve as major virus reservoirs with a key role in the propagation of the virus throughout the body as well as in the pathogenesis of AIDS. Furthermore, well-differentiated macrophages possess low abilities to phosphorylate the most common reverse transcriptase inhibitors of the nucleoside analog family. In an attempt to overcome these problems we have evaluated in vitro and in vivo in a feline immunodeficiency animal model whether it is possible to protect macrophages from FIV infection by direct administration of dideoxycytidine-5'-triphosphate (ddCTP). Because the cell membranes are impermeable to phosphorylated drugs we have encapsulated ddCTP into autologous erythrocytes. The drug-loaded erythrocyte membranes were then modified to target these carrier cells to macrophages. ddCTP-loaded erythrocytes were able to reduce FIV production by macrophages infected in vitro or obtained from naturally or experimentally infected cats. Furthermore, the administration of ddCTP-loaded erythrocytes protected the majority of peritoneal macrophages during a 7-month experimental FIV infection and reduced the percentage of circulating lymphocytes stained by an anti-p24 antibody. These results suggest that the administration of nucleoside analogs in phosphorylate form is feasible and their targeting to macrophages reduces FIV infection both in vitro and in vivo.