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Sample records for retrovir zidovudine azt

  1. Combination strategies to enhance transdermal permeation of zidovudine (AZT).

    PubMed

    Thomas, N S; Panchagnula, R

    2003-12-01

    The objective of this study was to evaluate the effect of simultaneous application of two penetration enhancers of different chemical classes or a chemical penetration enhancer and current application on permeation of zidovudine (AZT) across rat skin. Ex vivo permeation of AZT using combinations of cineole or menthol in vehicle with either oleic acid/linolenic acid or 0.5 mA/cm2 anodal current application for 6 h was studied. Penetration enhancers were significantly different in enhancing the permeability of AZT across rat skin and are in the decreasing order of activity: linolenic acid > menthol > oleic acid > cineole > vehicle. The combination of cineole and oleic acid synergistically enhanced transdermal flux of AZT in addition to reducing lag time. However, this was not observed for combinations of menthol with oleic or linolenic acid. On the other hand, the simultaneous application of current with menthol and cineole significantly increased cumulative amounts of AZT permeating during the course of current application and reduced the lag time but failed to further increase steady state flux of AZT. These results suggest that a combination of two penetration enhancers of different classes or the simultaneous use of iontophoresis and a penetration enhancer may be advantageous to achieve permeation enhancement with low risk of skin damage. PMID:14703969

  2. Reversal of brain metabolic abnormalities following treatment of AIDS dementia complex with 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine): a PET-FDG study

    SciTech Connect

    Brunetti, A.; Berg, G.; Di Chiro, G.; Cohen, R.M.; Yarchoan, R.; Pizzo, P.A.; Broder, S.; Eddy, J.; Fulham, M.J.; Finn, R.D.

    1989-05-01

    Brain glucose metabolism was evaluated in four patients with acquired immunodeficiency syndrome (AIDS) dementia complex using (/sup 18/F)fluorodeoxyglucose (FDG) and positron emission tomography (PET) scans at the beginning of therapy with 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine), and later in the course of therapy. In two patients, baseline, large focal cortical abnormalities of glucose utilization were reversed during the course of therapy. In the other two patients, the initial PET study did not reveal pronounced focal alterations, while the post-treatment scans showed markedly increased cortical glucose metabolism. The improved cortical glucose utilization was accompanied in all patients by immunologic and neurologic improvement. PET-FDG studies can detect cortical metabolic abnormalities associated with AIDS dementia complex, and may be used to monitor the metabolic improvement in response to AZT treatment.

  3. Spectroscopic identification of AZT derivative obtained from biotransformation of AZT by Stenotrophomonas maltophilia

    NASA Astrophysics Data System (ADS)

    Kruszewska, Hanna; Chmielowiec, Urszula; Bednarek, Elżbieta; Witowska-Jarosz, Janina; Dobrowolski, Jan Cz.; Misicka, Aleksandra

    2003-06-01

    The 3'-azido-2',3'-dideoxy-β-ribosylthymine (AZT, Zidovudine) is a cytostatic antivirial drug worldwide used in AIDS treatment or, in combination with other antiproliferative drugs, in treatment of cancer. About 30-40% of AZT is metabolised by conjunction with glucuronic acid in liver and about 70% is eliminated untouched by urinary system. In this work a possible fate of the AZT in the environment is studied. To this end, a product of AZT biotransformation by an environmental strain, Stenotrophomonas maltophilia, (aerobic, Gram(-) rod, common in soil and water) is found and isolated by HPLC and TLC techniques and identified by NMR and mass spectroscopy. All the molecular spectroscopy methods confirm presence of the product, which is AZT molecule hydroxylated in the position 2' of the deoxyribose ring.

  4. Zidovudine and isoniazid induced liver toxicity and oxidative stress: Evaluation of mitigating properties of silibinin.

    PubMed

    Raghu, Ramanathan; Karthikeyan, Sivanesan

    2016-09-01

    HIV/AIDS patients are more prone for opportunistic TB infections and they are administered the combined regimen of anti-retroviral drug zidovudine (AZT) and isoniazid (INH) for therapy. However, AZT+INH treatment has been documented to induce injury and remedial measures to prevent this adversity are not clearly defined. Silibinin (SBN) is a natural hepatoprotective principle isolated from medicinal plant Silybum marianum and is currently used for therapy of various liver diseases. This study investigate the hepatotoxic potentials of AZT alone, INH alone and AZT+INH treatments and the mitigating potentials of SBN against these drugs induced toxic insults of liver in rats. Separate groups of rats (n=6 in each group) were administered AZT alone (50mg/kg b.w.), INH alone (25mg/kg, b.w.), AZT+INH (50mg/kg, b.w. and 25mg/kg, b.w.), SBN alone (100mg/kg, b.w.) and SBN+AZT+INH daily for sub-chronic period of 45days orally. The control rats received saline/propylene glycol. INH alone and AZT+INH-induced parenchymal cell injury and cholestasis of liver was evidenced by highly significant increase in the activities of marker enzymes (aspartate and alanine transaminase, alkaline phosphatase, argino succinic acid lyase), bilirubin, protein, oxidative stress parameters (lipid peroxidation, superoxide dismutase, catalase, reduced glutathione, vitamins C and E) and membrane bound ATPases were evaluated in serum/liver tissue homogenates. Histopathological studies show ballooning degradation, inflammatory lesions, lipid deposition and hydropic changes in the liver tissue. All the above biochemical and pathological changes induced by AZT+INH treatments were mitigated in rats receiving SBN simultaneously with these hepatotoxins, indicating its hepatoprotective and antioxidant potentials against AZT+INH-induced hepatotoxicity. The moderate hepatoprotective and oxidant potentials of SBN could be due to its low bioavailability and this deficiency could be prevented by supplementation of

  5. Synergistic inhibition of replication of human immunodeficiency virus type 1, including that of a zidovudine-resistant isolate, by zidovudine and 2',3'-dideoxycytidine in vitro.

    PubMed Central

    Eron, J J; Johnson, V A; Merrill, D P; Chou, T C; Hirsch, M S

    1992-01-01

    The combination of zidovudine (AZT) and 2',3'-dideoxycytidine synergistically inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro with AZT-sensitive and AZT-resistant clinical isolates and HIV-1IIIB. Synergy was determined by the median-effect principle and isobologram techniques. Cytotoxicity of the agents was not observed. Clinical trials are ongoing to define the combination's role in HIV-1 therapy. PMID:1324648

  6. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  7. Zidovudine Injection

    MedlinePlus

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  8. AZT treatment induces molecular and ultrastructural oxidative damage to muscle mitochondria. Prevention by antioxidant vitamins.

    PubMed

    de la Asunción, J G; del Olmo, M L; Sastre, J; Millán, A; Pellín, A; Pallardó, F V; Viña, J

    1998-07-01

    AIDS patients who receive zidovudine (AZT) frequently suffer from myopathy. This has been attributed to mitochondrial (mt) damage, and specifically to the loss of mtDNA. This study examines whether AZT causes oxidative damage to DNA in patients and to skeletal muscle mitochondria in mice, and whether this damage may be prevented by supranutritional doses of antioxidant vitamins. Asymptomatic HIV-infected patients treated with AZT have a higher urinary excretion (355+/-100 pmol/kg/d) of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxo-dG) (a marker of oxidative damage to DNA) than untreated controls (asymptomatic HIV-infected patients) (182+/-29 pmol/kg/d). This was prevented (110+/-79 pmol/kg/d) by simultaneous oral treatment with AZT plus antioxidant vitamins (C and E). Mice treated with AZT also had a significantly higher urinary excretion of 8-oxo-dG than controls. Skeletal muscle mtDNA of mice treated with AZT had more 8-oxo-dG than controls. mt lipoperoxidation was also increased and skeletal muscle glutathione was oxidized. These effects may be due to an increased peroxide production by muscle mitochondria of AZT-treated animals. Dietary supplements with vitamins C and E at supranutritional doses protect against oxidative damage to skeletal muscle mitochondria caused by AZT. PMID:9649550

  9. Human inter-individual variability in metabolism and genotoxic response to zidovudine

    SciTech Connect

    Olivero, Ofelia A. Ming, Jessica M.; Das, Shreyasi; Vazquez, Irma L.; Richardson, Diana L.; Weston, Ainsley; Poirier, Miriam C.

    2008-04-15

    A mainstay of the antiretroviral drugs used for therapy of HIV-1, zidovudine (AZT) is genotoxic and becomes incorporated into DNA. Here we explored host inter-individual variability in AZT-DNA incorporation, by AZT radioimmunoassay (RIA), using 19 different strains of normal human mammary epithelial cells (NHMECs) exposed for 24 h to 200 {mu}M AZT. Twelve of the 19 NHMEC strains showed detectable AZT-DNA incorporation levels (16 to 259 molecules of AZT/10{sup 6} nucleotides), while 7 NHMEC strains did not show detectable AZT-DNA incorporation. In order to explore the basis for this variability, we compared the 2 NHMEC strains that showed the highest levels of AZT-DNA incorporation (H1 and H2) with 2 strains showing no detectable AZT-DNA incorporation (L1 and L2). All 4 strains had similar ({>=} 80%) cell survival, low levels of accumulation of cells in S-phase, and no relevant differences in response to the direct-acting mutagen bleomycin (BLM). Finally, when levels of thymidine kinase 1 (TK1), the first enzyme in the pathway for incorporation of AZT into DNA, were determined by Western blot analysis in all 19 NHMEC strains at 24 h of AZT exposure, higher TK1 protein levels were found in the 12 strains showing AZT-DNA incorporation, compared to the 7 showing no incorporation (p = 0.0005, Mann-Whitney test). Furthermore, strains L1 and L2, which did not show AZT-DNA incorporation at 24 h, did have measurable incorporation by 48 and 72 h. These data suggest that variability in AZT-DNA incorporation may be modulated by inter-individual differences in the rate of induction of TK1 in response to AZT exposure.

  10. Nasal chitosan microparticles target a zidovudine prodrug to brain HIV sanctuaries.

    PubMed

    Dalpiaz, Alessandro; Fogagnolo, Marco; Ferraro, Luca; Capuzzo, Antonio; Pavan, Barbara; Rassu, Giovanna; Salis, Andrea; Giunchedi, Paolo; Gavini, Elisabetta

    2015-11-01

    Zidovudine (AZT) is an antiretroviral drug that is a substrate of active efflux transporters (AETs) that extrude the drug from the central nervous system (CNS) and macrophages, which are considered to be sanctuaries of HIV. The conjugation of AZT to ursodeoxycholic acid is known to produce a prodrug (UDCA-AZT) that is able to elude the AET systems, indicating the potential ability of this prodrug to act as a carrier of AZT in the CNS and in macrophages. Here, we demonstrate that UDCA-AZT is able to permeate and remain in murine macrophages with an efficiency twenty times higher than that of AZT. Moreover, we propose the nasal administration of this prodrug in order to induce its uptake into the CNS. Chitosan chloride-based microparticles (CP) were prepared by spray-drying and were characterized with respect to size, morphology, density, water uptake and the dissolution profile of UDCA-AZT. The CP sample was then nasally administered to rats. All in vitro and in vivo measurements were also performed for a CP parent physical mixture. The CP sample was able to increase the dissolution rate of UDCA-AZT and to reduce water uptake with respect to its parent physical mixture, inducing better uptake of UDCA-AZT into the cerebrospinal fluid of rats, where the prodrug can act as an AZT carrier in macrophages. PMID:26427553

  11. Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells

    PubMed Central

    Borojerdi, Jennifer P.; Ming, Jessica; Cooch, Catherine; Ward, Yvona; Semino-Mora, Cristina; Yu, Mia; Braun, Hannan M.; Taylor, Barbara J.; Poirier, Miriam C.; Olivero, Ofelia A.

    2009-01-01

    The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese Hamster Ovary (CHO) and Normal Human Mammary Epithelial (NHMEC) cells exposed to the antiretroviral drug zidovudine (3’-azido-3’-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC), strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (High incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (Low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound. PMID:19427513

  12. Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine.

    PubMed

    Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Staud, Frantisek

    2016-01-01

    Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26390406

  13. AZT, rodent somatic and germ cell mutagenicity and reproductive toxicity tests

    SciTech Connect

    Shelby, M.D.; Russell, L.B.; Generoso, W.

    1995-11-01

    AZT (3`-axido-3`-deoxythymidine, Zidovudine) is the most widely used therapeutic agent in the treatment of Acquired Immune Deficiency Syndrome (AIDS). Use of AZT has not been limited to HIV-seropositive individuals or to those with symptoms of AIDS. It has also been used as a chemoprophylactic agent in people accidentally exposed to HIV-contaminated body fluids, and to HIV-seropositive pregnant women to prevent infection of the fetus. Because of these latter uses, it is particularly important to determine whether long-term health effects might be associated with AZT exposure. Tests have been conducted to determine the in vivo genetic toxicity of AZT in mice. Dominant-lethal and morphological-specific-locus tests were conducted in males using 2 daily initraperitoneal injections of 750 mg/kg. The dominant-lethal test was negative for all germ cell stages from differentiating spermatogonia to mature sperm. Likewise, no evidence of the induction of specific locus mutations was observed in either spermatogonial stem cells or poststem-cell stages. Further, tests for effects on male and female reproduction and in utero development indicate a lack of effects. These results, along with preliminary clinical reports that birth outcomes are normal in newborns exposed to AZT in utero, are encouraging with regard to the risks to offspring of parents exposed to AZT, either prior to or during pregnancy. However, positive results in mouse bone marrow micronucleus tests and one report on the induction of chromosomal aberrations in the lymphocytes of AIDS patients on AZT therapy indicate that further studies are needed on the potential of AZT to adversely affect the long-term health of exposed individuals.

  14. Long-Term Exposure to AZT, but not d4T, Increases Endothelial Cell Oxidative Stress and Mitochondrial Dysfunction

    PubMed Central

    Kline, Erik R.; Bassit, Leda; Hernandez-Santiago, Brenda I.; Detorio, Mervi A.; Liang, Bill; Kleinhenz, Dean J.; Walp, Erik R.; Dikalov, Sergey; Jones, Dean P.; Schinazi, Raymond F.

    2009-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 μM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects. PMID:19067249

  15. Preparation and Characterization of Chitosan Nanoparticles for Zidovudine Nasal Delivery.

    PubMed

    Barbi, Mariana Da Silva; Carvalho, Flávia Chiva; Kiill, Charlene Priscila; Barud, Hernane Da Silva; Santagneli, Sílvia Helena; Ribeiro, Sidney José Lima; Gremião, Maria Palmira Daflon

    2015-01-01

    Zidovudine (AZT) is the antiretroviral drug most frequently used for the treatment of Acquired Immunodeficiency Syndrome. Its low oral bioavailability demands the development of innovative strategies to overcome the first pass metabolism. The nasal route is an option for enhanced therapeutic efficacy and to reduce the extent of the first-pass effect. In this article, AZT loaded chitosan nanoparticles were prepared by a modified ionotropic gelation method with sodium tripolyphosphate. The increase proportion of CS (NP1 10:01 (w/w)) promoted the formation of smaller nanoparticles (260 nm), while raising the proportion of TPP (NP2 5:1 w/w) increased the nanoparticles size (330 nm). The incorporation of AZT increased the nanoparticles size for both AZT-loaded nanoparticles AZT-loaded NP1 (406 nm) and AZT-loaded NP2 (425 nm). The incorporation of AZT into NP1 did not change the electrophoretic mobility, however, in AZT-loaded NP2 there was a significant increase. The positive surface of the nanoparticles is very important for the mucoadhesive properties due interaction with the sialic groups of the mucin. Nuclear resonance magnetic data showed that the higher concentration of chitosan in the nanoparticles favored the interaction of few phosphate units (pyrophosphate) by ionic interaction Scanning electron microscopy, revealed that the nanoparticles are nearly spherical shape with porous surface. The entrapment efficiency of AZT, was 17.58% ± 1.48 and 11.02% ± 2.05 for NP1 and NP2, respectively. The measurement of the mucoadhesion force using mucin discs and nasal tissue obtained values of NP1 = 2.12 and NP2 = 4.62. In vitro permeation study showed that the nanoparticles promoted an increase in the flux of the drug through the nasal mucosa. In view of these results, chitosan nanoparticles were found to be a promising approach for the incorporation of hydrophilic drugs and these results suggest that the CS-containing nanoparticles have great potential for nasal AZT

  16. Zidovudine-poly(L-lactic acid) solid dispersions with improved intestinal permeability prepared by supercritical antisolvent process.

    PubMed

    Yoshida, Valquíria M H; Balcão, Victor M; Vila, Marta M D C; Oliveira Júnior, José M; Aranha, Norberto; Chaud, Marco V; Gremião, Maria P D

    2015-05-01

    A supercritical antisolvent (SAS) process for obtaining zidovudine-poly(L-lactic acid) (PLLA) solid dispersions (SDs) was used to attain a better intestinal permeation of this drug. A 3(2) factorial design was used, having as independent variables the ratio 3'-azido-2'3'-dideoxythymidine (AZT)-PLLA and temperature/pressure conditions, as dependent variables the process yield and particle macroscopic morphology. AZT-PLLA production batches were carried out by the SAS process, and the resulting products evaluated via scanning electron microscope, X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared analyses. From the nine possible combinations of tests performed experimentally, only one combination did not produced a solid. The L3 batch of SD, produced with 1:2 (AZT-PLLA) ratio, resulted in a 91.54% yield, with 40% AZT content. Intestinal permeability studies using the AZT-PLLA from L3 batch led to an AZT permeability of approximately 9.87%, which was higher than that of pure AZT (∼3.84%). AZT remained in crystalline form, whereas PLLA remained in semicrystalline form. AZT release is controlled by a diffusion mechanism. It has been demonstrated that it is possible to use PLLA carrier and SAS process to obtain SD, in a single step. PMID:25676038

  17. Improved Safety, Bioavailability and Pharmacokinetics of Zidovudine through Lactoferrin Nanoparticles during Oral Administration in Rats

    PubMed Central

    C., Bhaskar; Golla, Kishore; Kondapi, Anand K.

    2015-01-01

    Zidovudine (AZT) is one of the most referred antiretroviral drug. In spite of its higher bioavailability (50–75%) the most important reason of its cessation are bone marrow suppression, anemia, neutropenia and various organs related toxicities. This study aims at the improvement of oral delivery of AZT through its encapsulation in lactoferrin nanoparticles (AZT-lactonano). The nanoparticles (NPs) are of 50–60 nm in size and exhibit 67% encapsulation of the AZT. They are stable in simulated gastric and intestinal fluids. Anti-HIV-1 activity of AZT remains unaltered in nanoformulation in acute infection. The bioavailability and tissue distribution of AZT is higher in blood followed by liver and kidney. AZT-lactonano causes the improvement of pharmacokinetic profile as compared to soluble AZT; a more than 4 fold increase in AUC and AUMC in male and female rats. The serum Cmax for AZT-lactonano was increased by 30%. Similarly there was nearly 2-fold increase in Tmax and t1/2. Our in vitro study confirms that, the endosomal pH is ideal for drug release from NPs and shows constant release from up to 96h. Bone marrow micronucleus assay show that nanoformulation exhibits approximately 2fold lower toxicity than soluble form. Histopathological and biochemical analysis further confirms that less or no significant organ toxicities when nanoparticles were used. AZT-lactonano has shown its higher efficacy, low organs related toxicities, improved pharmacokinetics parameter while keeping the antiviral activity intact. Thus, the nanoformulation are safe for the target specific drug delivery. PMID:26461917

  18. Transdermal delivery system for zidovudine: in vitro, ex vivo and in vivo evaluation.

    PubMed

    Narishetty, Sunil Thomas Kumar; Panchagnula, Ramesh

    2004-01-01

    The objective of this study was to prepare a transdermal delivery system (TDS) for zidovudine (AZT) with a combination of menthol and oleic acid as penetration enhancers incorporated in hydroxypropyl methylcellulose, and to evaluate ex vivo as well as in vivo permeation across rat skin. It was found that AZT in gel formulation was stable in both refrigerated as well as accelerated stability conditions for 3 months and further, the gel did not significantly retard the permeability of AZT across the skin in comparison with solution formulation. Ex vivo steady state flux of AZT across rat skin from gel was 2.26 mg cm(-2) h(-1), which is sufficient to achieve therapeutic plasma concentrations. Intravenous pharmacokinetic parameters of AZT in rats were determined and used together with ex vivo flux data to generate theoretical plasma profiles of AZT and compared with plasma concentrations achieved after application of TDS. Further, steady state plasma concentrations of drug following multiple applications of TDS were determined and good correlations between ex vivo and in vivo data were observed. In addition, the combination of penetration enhancers used at 2.5% w/w in this study proved efficient in achieving sufficient enhancement in the transdermal permeability of AZT across rat skin with reduced skin irritation potential when compared with individual penetration enhancers at higher concentrations. PMID:14716748

  19. Radiosensitization effect of zidovudine on human malignant glioma cells

    SciTech Connect

    Zhou Fuxiang; Liao Zhengkai; Dai Jing; Xiong Jie; Xie CongHua; Luo Zhiguo; Liu Shiquan; Zhou Yunfeng . E-mail: yfzhouwhu@163.com

    2007-03-09

    Telomeres are shortened with each cell division and play an important role in maintaining chromosomal integrity and function. Telomerase, responsible for telomere synthesis, is activated in 90% of human tumor cells but seldom in normal somatic cells. Zidovudine (AZT) is a reverse transcriptase inhibitor. In this study, we have investigated the effects of {gamma}-radiation in combination with AZT on telomerase activity (TA), telomere length, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and the changes in radiosensitivity of human malignant glioma cell line U251. The results showed that the TA was suppressed by AZT but enhanced by irradiation, resulting in a deceleration of restored rate of shortened telomere, decreased repair rate of DNA strand breaks, and increased radiosensitivity of U251 cells. Our results suggested that telomerase activity and telomere length may serve as markers for estimating the efficacy of cancer radiotherapy and reverse transcriptase inhibitors, such as AZT, may be used clinically as a new radiosensitizer in cancer radiotherapy.

  20. pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo.

    PubMed Central

    Eron, J J; Chow, Y K; Caliendo, A M; Videler, J; Devore, K M; Cooley, T P; Liebman, H A; Kaplan, J C; Hirsch, M S; D'Aquila, R T

    1993-01-01

    Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations. PMID:7689822

  1. Acrolein enhances epigenetic modifications, FasL expression and hepatocyte toxicity induced by anti-HIV drug Zidovudine.

    PubMed

    Ghare, Smita S; Donde, Hridgandh; Chen, Wei-Yang; Barker, David F; Gobejishvilli, Leila; McClain, Craig J; Barve, Shirish S; Joshi-Barve, Swati

    2016-09-01

    Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity. PMID:27238871

  2. Transdermal delivery of zidovudine: effect of terpenes and their mechanism of action.

    PubMed

    Narishetty, Sunil Thomas Kumar; Panchagnula, Ramesh

    2004-03-24

    The effect of various oxygen-containing monoterpenes such as cineole, menthol, alpha-terpineol, menthone, pulegone and carvone was investigated on ex vivo permeation of zidovudine (AZT) across rat skin. Furthermore, saturation solubility of AZT, its stratum corneum (SC)/vehicle partition coefficient and activation energy for diffusion across skin with or without terpene(s) in vehicle (66.6% ethanol in water) were determined to understand their mechanism of action. All the terpenes studied significantly increased transdermal flux of AZT in comparison to vehicle (p<0.05) and their enhancement activities are in the following decreasing order: cineole>menthol>menthone approximately pulegone approximately alpha-terpineol>carvone>vehiclewater. On the other hand, saturation solubility and SC/vehicle partition coefficient of AZT were not significantly altered (p>0.05) by terpenes. Activation energies of AZT permeation across rat skin from water, vehicle and cineole in vehicle were measured to be 20.4, 18.6 and 10.6 kcal/mol, respectively. Interactions between terpenes and SC lipids were studied with molecular modeling and found that terpenes form hydrogen bonds (bond lengths<2 A) with lipid head groups. The mechanism of permeation enhancement of AZT by terpenes was explained with thermodynamic activity, SC/vehicle partition coefficient, activation energy and molecular modeling studies. PMID:15023449

  3. Molecular geometry, vibrational spectra, atomic charges, frontier molecular orbital and Fukui function analysis of antiviral drug zidovudine

    NASA Astrophysics Data System (ADS)

    Ramkumaar, G. R.; Srinivasan, S.; Bhoopathy, T. J.; Gunasekaran, S.

    2012-12-01

    The solid phase FT-IR and FT-Raman spectra of zidovudine (AZT) were recorded in the regions 4000-400 and 3500-100 cm-1, respectively. The optimized geometry, frequency and intensity of the vibrational bands of zidovudine were obtained by the Restricted Hartree-Fock (RHF) density functional theory (DFT) with complete relaxation in the potential energy surface using 6-31G(d,p) basis set. The harmonic vibrational frequencies for zidovudine were calculated and the scaled values have been compared with experimental values of FTIR and FT-Raman spectra. The observed and the calculated frequencies are found to be in good agreement. The harmonic vibrational wave numbers and intensities of vibrational bands of zidovudine with its cation and anion were calculated and compared with the neutral AZT. The DFT calculated HOMO and LUMO energies shows that charge transfer occurs within the molecule. The electron density-based local reactivity descriptors such as Fukui functions were calculated to explain the chemical selectivity or reactivity site in AZT.

  4. Antiretroviral Drugs Used in the Treatment of HIV Infection

    MedlinePlus

    ... 6 months Retrovir zidovudine, azidothymidine, AZT, ZDV 19-Mar-87 3.5 months Trizivir abacavir, zidovudine, and ... months Viramune XR (Extended Release) nevirapine, NVP 25-Mar-11 9.9 months Protease Inhibitors (PIs) Brand ...

  5. Parameterization of AZT-A widely used nucleoside inhibitor of HIV-1 reverse transcriptase

    NASA Astrophysics Data System (ADS)

    Carvalho, Alexandra T. P.; Fernandes, Pedro A.; Ramos, Maria J.

    Seven nucleoside reverse transcriptase (RT) inhibitors are currently used in the clinical treatment of acquired immunodeficiency syndrome (AIDS). These substrate analogues block DNA synthesis by the viral enzyme RT. However, the emergence of resistant variants of RT allied to their long-term toxicity requires the design of new and better RT inhibitors, with long-term in vivo efficacy. In this work we used density functional theory (DFT) calculations to develop a set of molecular mechanics (MM) parameters committed to the AMBER force field for one of the most used in the clinic nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine (AZT). These parameters were tested by comparing the optimized geometries of AZT at both the DFT and MM levels of theory. The ability of the new parameters to reproduce the torsional energy of the azide group was also verified by scanning the surface in MM with the new parameters and comparing the results with the same potential energy surface (PES) at the DFT level. Finally, the parameters were validated through classical MD simulations of AZT in aqueous environment.

  6. XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells

    SciTech Connect

    Wu Qiangen; Beland, Frederick A.; Chang, Ching-Wei; Fang Jialong

    2011-03-01

    Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 {mu}M AZT treatment significantly increased [{sup 3}H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

  7. Fourier transform infrared spectra and normal mode analysis of drug molecules: Zidovudine

    NASA Astrophysics Data System (ADS)

    Jain, Nivedita; Prabhakar, Santosh; Singh, R. A.

    2013-03-01

    The FTIR spectra of zidovudine molecule have been recorded in the range 4000-400 cm-1. The title compound is used as a drug against AIDS or HIV. The molecular structure, fundamental vibrational frequencies and intensities of vibrational bands are evaluated using density functional theory (DFT) using BLYP, B3LYP, B3PW91 and MPW1PW91 methods with 6-31+G(d,p) standard basis set. Comparison of simulated spectra with the experimental spectrum provides important informations and the ability of the computational method to describe the vibrational modes. These calculations have allowed finding most stable conformational structure of AZT. Calculated results of the title compound indicate that the drug molecule has syn orientation. The glycosidic bond in AZT and a minimum-energy structure in which the glycosy torsion angle χ and torsion angle γ values are consistent with those in the conformation of AZT in the AZT5-triphosphate bound to HIV RT is determined.

  8. Mucoadhesive Nanostructured Polyelectrolyte Complexes as Potential Carrier to Improve Zidovudine Permeability.

    PubMed

    Pedreiro, Liliane Neves; Stringhetti, Beatriz; Cury, Ferreira; Gremião, Maria Palmira Daflon

    2016-02-01

    Mucoadhesive drug delivery systems have been widely investigated as a strategic to allow the raising of intestinal residence time of drugs and the intimate contact with the intestinal mucosa, both factors that increase the local concentration gradient. Zidovudine (AZT) mucoadhesive nanostructured polyelectrolyte complexes were obtained by chitosan (CS)-hypromellose phthalate (HP) interactions in order to favor the permeability through biological membranes and the AZT absorption. Particle size and morphology analyses showed the obtaining of nanoparticulate delivery systems, with AZT loaded about of 65%. The characterization by DSC, X-ray diffraction and FTIR showed a new crystalline structure formed in which the drug remained molecularly dispersed, without changing this structure. The reduced release rates in the simulated gastric medium and the control of release rates in simulated intestinal medium of AZT were demonstrated by in vitro release studies. The nanoparticles liquid uptake ability associated to the mucoadhesiveness by electronic interaction between the particles and mucus revealed that the drug delivery system developed in this work is a promising approach to improve the permeation of this drug throughout the intestinal mucosa. PMID:27433574

  9. Quantitative determination and sampling of lamivudine and zidovudine residues for cleaning validation in a production area.

    PubMed

    Santoro, Maria Inês Rocha Miritello; Fazio, Tatiana Tatit; Singh, Anil Kumar; Kedor-Hackmann, Erica Rosa Maria

    2007-01-01

    Lamivudine (3TC) and zidovudine (AZT) are systemic antiviral substances extensively used in human immunodeficiency virus (HIV) infected patients. Nowadays, 3TC, AZT, and several other pharmacologically potent pharmaceuticals are manufactured in the same production area. To assure quality of drug products and patient safety, properly validated cleaning methodology is necessary. A carefully designed cleaning validation and its evaluation can ensure that residues of 3TC and AZT will not carry over and cross contaminate the subsequent product. The aim of this study was to validate a simple analytical method for verification of residual 3TC and AZT in equipment used in the production area and to confirm the efficiency of the cleaning procedure. The liquid chromatography method was validated using a Nova-Pak C18 column (3.9 x 150 mm, 4 microm particle size) and methanol-water (20 + 80, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Ultraviolet detection was made at 266 nm. The calibration curve was linear over a concentration range of 2.0-22.0 microg/mL with a correlation coefficient of 0.9998. The detection and quantitation limits were 0.36 and 1.21 microg/mL, respectively. The intra-day and interday precision expressed as relative standard deviation were below 2.0%. The mean recovery of the method was 99.19%. The mean extraction recovery from manufacturing equipment was 83.5%. PMID:17580624

  10. Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model

    PubMed Central

    Snowdin, Jacob W.; Hsiung, Chia-Heng; Kesterson, Daniel G.; Kamath, Vasudeva G.

    2015-01-01

    The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3′-azido-3′-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis. PMID:26248377

  11. Evaluation of Microemulsion and Lamellar Liquid Crystalline Systems for Transdermal Zidovudine Delivery.

    PubMed

    Carvalho, André Luis Menezes; Silva, José Alexsandro da; Lira, Ana Amélia Moreira; Conceição, Tamara Matos Freire; Nunes, Rogéria de Souza; de Albuquerque Junior, Ricardo Luiz Cavalcanti; Sarmento, Victor Hugo Vitorino; Leal, Leila Bastos; de Santana, Davi Pereira

    2016-07-01

    This study proposed to investigate and to compare colloidal carrier systems containing Zidovudine (3'-azido-3'-deoxythymidine) (AZT) for transdermal administration and optimization of antiretroviral therapy. Microemulsion (ME) and lamellar phase (LP) liquid crystal were obtained and selected from pseudoternary diagrams previously developed. Small-angle X-ray scattering and rheology analysis confirmed the presence of typical ME and liquid crystalline structures with lamellar arrangement, respectively. Both colloidal carrier systems, ME, and LP remained stable, homogeneous, and isotropic after AZT addition. In vitro permeation study (using pig ear skin) showed that the amount of permeated drug was higher for ME compared to the control and LP, obtaining a permeation enhancing effect on the order of approximately 2-fold (p < 0.05). Microscopic examination after in vivo skin irritation studies using mice suggested few histological changes in the skin of animals treated with the ME compared to the control group (hydrogel). Thus, ME proved to be adequate and have promising effects, being able to promote the drug permeation without causing apparent skin irritation. On the order hand, LP functioned as a drug reservoir reducing AZT partitioning into the skin. PMID:27220471

  12. Neurotoxic effects of AZT on developing and adult neurogenesis

    PubMed Central

    Demir, Meryem; Laywell, Eric D.

    2015-01-01

    Azidothymidine (AZT) is a synthetic, chain-terminating nucleoside analog used to treat HIV-1 infection. While AZT is not actively transported across the blood brain barrier, it does accumulate at high levels in cerebrospinal fluid, and subsequently diffuses into the overlying parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system, we hypothesize that diffusion from CSF exposes neural stem/progenitor cells and their progeny to biologically relevant levels of AZT sufficient to perturb normal cell functions. We employed in vitro and in vivo models of mouse neurogenesis in order to assess the effects of AZT on developing and adult neurogenesis. Using in vitro assays we show that AZT reduces the population expansion potential of neural stem/progenitor cells by inducing senescence. Additionally, in a model of in vitro neurogenesis AZT severely attenuates neuroblast production. These effects are mirrored in vivo by clinically-relevant animal models. We show that in utero AZT exposure perturbs both population expansion and neurogenesis among neural stem/progenitor cells. Additionally, a short-term AZT regimen in adult mice suppresses subependymal zone neurogenesis. These data reveal novel negative effects of AZT on neural stem cell biology. Given that the sequelae of HIV infection often include neurologic deficits—subsumed under AIDS Dementia Complex (Brew, 1999)—it is important to determine to what extent AZT negatively affects neurological function in ways that contribute to, or exacerbate, ADC in order to avoid attributing iatrogenic drug effects to the underlying disease process, and thereby skewing the risk/benefit analysis of AZT therapy. PMID:25852464

  13. Comparative studies of lamivudine-zidovudine nanoparticles for the selective uptake by macrophages.

    PubMed

    Sankar, V; Nareshkumar, Parmar Nilaykumar; Ajitkumar, Gohel Nishit; Penmetsa, Shalini Devi; Hariharan, Sivaram

    2012-09-01

    The present study investigates the specific drug targeting of anti retroviral drugs, such as lamivudine and zidovudine, after intraperitoneal (i.p) injection by incorporation into polymeric nanoparticles (PNs) and solid lipid nanoparticles (SLNs). Our results showed that Glyceryl Monosterate-Poloxamer 188 SLNs (average diameter of 522.466 nm) showed slow drug release rates (63.18% of lamivudine and 62.37% of zidovudine were released in 12 hrs) among all the SLN formulations. For Poly lactic-co-glycolic acid (PLGA)-Poloxamer 188 PNs (average diameter of 70.348 nm), there were faster release rates of both lamivudine and zidovudine (97% and 94.06%, respectively, in 12 hrs). Tissue distribution studies were carried out in mice and concentrations of drugs in different organs were determined using high performance liquid chromatography (HPLC) after i.p. administration. Glyceryl Monosterate-Poloxamer 188 SLNs and PLGA-Poloxamer 188 PNs showed increase in the distribution of lamivudine and zidovudine to liver and spleen when compared to the drugs in solution. Also, Glyceryl Monosterate-P 188 SLNs showed higher concentration of drugs in RES organs than PLGA-P 188 PNs. PMID:22452408

  14. Metabolism and pharmacokinetics of the combination Zidovudine plus Lamivudine in the adult Erythrocebus patas monkey determined by liquid chromatography-tandem mass spectrometric analysis

    SciTech Connect

    Divi, Rao L.; Doerge, Daniel R.; Twaddle, Nathan C.; Shockley, Marie E.; St Claire, Marisa C.; Harbaugh, Jeffrey W.; Harbaugh, Steven W.; Poirier, Miriam C.

    2008-01-15

    Because of their similarity to humans, non-human primates constitute useful preclinical models in which to examine potential human drug toxicities. Antiretroviral nucleoside reverse transcriptase inhibitor (NRTI) toxicity is currently under investigation in Erythrocebus patas monkeys, and whereas NRTI pharmacokinetics have been studied in other monkey species, pharmacokinetics for Zidovudine plus Lamivudine (AZT/3TC) dosing have not been reported in the patas. Here we present 24 h serum pharmacokinetic parameters after a single oral exposure to the combination of AZT (40 mg) and 3TC (24 mg), doses equivalent to a human daily dose of Combivir (registered) . The patas (n = 3) AZT/3TC pharmacokinetic profiles were similar to those seen in other primate species. Average maximum serum concentrations (C{sub max}) for AZT and 3TC were 2.35 and 2.65 {mu}g/ml, respectively, and were observed at 0.83 h (T{sub max}). C{sub max} was 13.34 {mu}g/ml for the AZT-glucuronide (AZT-G) and was 0.023 {mu}g/ml for the potentially toxic minor metabolite 3'-amino-3'-deoxythymidine (AMT), both occurring at about 1 h after dosing. Similar elimination half-times, 0.70 and 0.68 h{sup -1}, were found for AZT and AZT-G, respectively, while 3TC was eliminated about half as fast (0.33 h{sup -1}) resulting in AUC{sub (0-{infinity})} values of 6.97 {mu}g/ml h for 3TC, 2.99 {mu}g/ml h for AZT, 20.5 {mu}g/ml h for AZT-G and 0.002 for AMT 6.97 {mu}g/ml h. This study shows similar metabolism and pharmacokinetics for oral administration of AZT/3TC in the adult patas monkey, other primate species and humans. The data validate the use of the patas monkey for studies of NRTI toxicity.

  15. The impact of zidovudine on dementia-free survival in a population of HIV-positive men and women on antiretroviral therapy.

    PubMed

    Cornelisse, P G; Montessori, V; Yip, B; Craib, K J; O'Shaughnessy, M V; Montaner, J S; Hogg, R S

    2000-01-01

    Our objective was to characterize the effect of zidovudine therapy on AIDS dementia complex (dementia) free survival among HIV-infected men and women in a population-based cohort with free access to antiretroviral therapy in the province of British Columbia. Time to diagnosis of dementia among individuals was examined on the basis of zidovudine duration, CD4+ cell count at first treatment, gender, and transmission group [men having sex with men (MSM), intravenous drug users (IDU), heterosexuals]. We restricted the analysis to subjects with CD4+ cells counts within 12 months prior to treatment start date. Among 641 participants eligible for analysis, median duration of follow-up was 3.6 years, under which 86 (9.3%) events of dementia occurred. Participants were less likely to develop dementia with: increased zidovudine exposure (OR=0.26, 95% CI: 0.14-0.49), at least 260 CD4+ cells/mm3 (median) (OR=0.52, 95% CI: 0.34-0.78), and MSM risk group (OR=0.57, 95% CI: 0.35-0.94). Those infected through heterosexual contact had an increased risk (RR=2.04, 95% CI: 1.02-4.07). Using Cox's proportional hazards model, controlling for CD4+ cell count at treatment start date, independent predictors of dementia-free survival were: duration of zidovudine (OR=0.28, 95% CI: 0.15-0.52) and MSM transmission group (OR=0.61, 95% CI: 0.37-1.00). In this observational treatment cohort, factors associated with dementia-free survival include duration of zidovudine (AZT) therapy and MSM transmission group. It is not clear from these data whether the AZT protective effect is exclusive to this agent or whether other therapies might offer a similar protective effect. PMID:10667902

  16. Effects of zidovudine on B lymphocyte activation.

    PubMed

    Widney, D; Yawetz, S; van der Meyden, M; Miles, S A; Kishimoto, T; Martínez-Maza, O

    1994-10-01

    B cell dysfunction associated with HIV infection includes polyclonal B cell activation and hypergammaglobulinemia. There is also an elevated frequency of B cell malignancies, especially non-Hodgkin's lymphoma, in HIV infection. It is believed that chronic polyclonal activation of B cells might increase the chances for the occurrence of a genetic accident, resulting in tumorigenesis. Long-term zidovudine use in people with HIV infection has been reported to be associated with a particularly high incidence of B cell lymphoma. This may be due to an increase in life span associated with antiretroviral treatment, placing treated individuals at risk for developing lymphoma for a greater period of time. However, zidovudine could be directly contributing to lymphoma-genesis in HIV-infected individuals, perhaps by enhancing B cell activation, since B cell hyperactivation and elevated levels of IL-6, a B cell stimulatory cytokine, are seen in HIV infection. Also, people treated with zidovudine may inherently be at higher risk for developing lymphoma because of the relatively greater degree of immune impairment seen in those that receive treatment with this drug. To examine if exposure to zidovudine resulted in enhanced B cell activation, we determined whether or not the presence of zidovudine enhanced B cell activation or IL-6 production in vitro or in vivo. Exposure to zidovudine in vitro did not enhance spontaneous immunoglobulin or IL-6 secretion by cells from HIV-infected (or uninfected) subjects and did not enhance B cell activation induced by EBV or affect the ability of T cells to regulate EBV-activated B cells. Neither serum immunoglobulin or IL-6 levels, nor the expression of cell surface activation markers on circulating B cells, were seen to increase following zidovudine treatment. These results indicate that zidovudine does not induce B cell activation in vivo or in vitro, suggesting that zidovudine treatment does not contribute to lymphomagenesis by enhancing B

  17. Human Serum Albumin Complexed with Myristate and AZT

    SciTech Connect

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong

    2008-06-16

    3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

  18. Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

    PubMed Central

    Boyer, Paul L.; Das, Kalyan; Arnold, Eddy

    2015-01-01

    Although anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3′ OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3′ end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration. PMID:26324274

  19. Zidovudine and ursodeoxycholic acid conjugation: design of a new prodrug potentially able to bypass the active efflux transport systems of the central nervous system.

    PubMed

    Dalpiaz, Alessandro; Paganetto, Guglielmo; Pavan, Barbara; Fogagnolo, Marco; Medici, Alessandro; Beggiato, Sarah; Perrone, Daniela

    2012-04-01

    We have synthesized a new prodrug obtained by the 5'-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA-AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half-life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA-AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA-AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 μM AZT across this monolayer was regulated by apparent permeability coefficients (P(E)) higher from the apical to basolateral compartments (P(E) = 209 ± 4 × 10⁻⁵ cm/min) than in the opposite way (P(E) = 133 ± 8 × 10⁻⁵ cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P(E) = 39.1 ± 1.2 × 10⁻⁵ cm/min) and efflux (P(E) = 31.3 ± 3.6 × 10⁻⁵ cm/min) permeability coefficients of 30 μM UDCA-AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P(E) values of UDCA-AZT were associated with a partial hydrolysis during its permeation across the cell monolayer. PMID:22356133

  20. An unusual phenotypic and genotypic expression in F2 generation following one stage zidovudine exposure during pregnancy and lactation- an experiment in mice.

    PubMed

    Rajlakshmi, Chongtham; Roy, Jagat Kumar; Rai, Amit Kumar; Bhattacharyya, Asima; Pandey, Bajarang Lal

    2012-02-01

    Zidovudine (3'-Azido-2', 3'-dideoxythymidine, AZT, ZDV) is routinely used as one of the component of antiretroviral therapy to prevent transmission of the HIV infection from mother to child. The drug, when given during pregnancy can give rise to myriad toxicities as reported in previous studies on human, animal and in-vitro experiments. The present study was an attempt to explore the Zidovudine teratogenesis in F1 and F2 generation of mice following initial maternal exposure to Zidovudine during pregnancy, through delivery and lactation. The F1 generation actually would have got the exposure during embryonic development and infant stages. Pregnant Swiss mice were treated orally with ZDV 50 mg/kg/day or distilled water (control), from day eighth of gestation, through delivery and continued for first ten days of lactation. The F1 generation litters were raised and mated to produce F2 generation mice. An interesting phenotype of "healthy" and "sick" was noted in F2 generation but not in the F1 generation. In F2 generation 35% died on different postnatal day during 120 days of follow up period. Chromosomal study from bone marrow of F1 and F2 showed various chromosomal aberrations. Lipodystrophy and hepatotoxicity was observed in "sick" mice. The study generated a hypothesis of recessive mutation and concludes that Zidovudine is a transplacental genotoxic agent. The result of present study therefore suggests the need to study the effect of zidovudine in human subjects for a longer period of time to rule out similar genotoxic effect. PMID:22293411

  1. AZT side effect on mitochondria does not depend on either inhibition of electron flow or mitochondrial uncoupling.

    PubMed

    Atlante, A; Passarella, S

    1998-03-01

    The mitochondrial myopathy associated with long-term AZT therapy limits the clinical efficacy of this drug in AIDS therapy. Thus, in order to determine how AZT can affect mitochondria bioenergetics, the capability of AZT to both uncouple oxidative phosphorylation and inhibit electron flow in isolated rat liver mitochondria was investigated. The failure of AZT to oxidize intramitochondrial pyridine nucleotides, to stimulate mitochondrial swelling in K+-acetate plus valinomycin or to cause ATP hydrolysis shows that AZT is not an uncoupler. PMID:9852271

  2. Effect of L-menthol and 1,8-cineole on phase behavior and molecular organization of SC lipids and skin permeation of zidovudine.

    PubMed

    Narishetty, Sunil T K; Panchagnula, Ramesh

    2005-01-20

    The aim of this investigation was to study the effect of 1,8-cineole and L-menthol on phase behavior and molecular organization of Stratum corneum (SC) lipids and permeation of zidovudine (AZT) across human cadaver skin (HCS). Permeation studies were conducted across HCS using Franz diffusion cells at 37 degrees C. Differential scanning calorimetry (DSC) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were employed to understand the effect of terpenes on phase behavior and molecular organization of a model SC lipid system consisting of an equimolar mixture of ceramide, palmitic acid and cholesterol. Both 1,8-cineole and L-menthol applied at 5% w/v in 66.6% ethanol as a vehicle significantly enhanced the pseudosteady state flux of AZT across HCS. The vehicle reduced the number of endothermic transitions observed in the DSC thermogram of a hydrated model SC lipid system from three to two with a lowered midtransition temperature (Tm), while the inclusion of terpenes resulted in a single but very broad endothermic transition for the model SC lipid system. Correspondingly, ATR-FTIR studies revealed that both 1,8-cineole and L-menthol increased CH2 stretching frequencies on either side of lipid transition in addition to lowering the Tm of model SC lipid system by approximately 2-8 degrees C. The alterations observed in the amide-I frequencies of model SC lipid system after the inclusion of terpenes suggest that they disrupt the interlamellar hydrogen-bonding network at the polar head group region. Further, terpenes also increased the hydration levels of the lipid system probably by forming new aqueous channels. These results indicate that terpenes enhance transdermal permeation of AZT and other drugs by transforming SC lipids from a highly ordered orthorhombic perpendicular subcellular packing to a less ordered hexagonal subcell packing. PMID:15653134

  3. Zidovudine Adherence in Persons with AIDS

    PubMed Central

    Smith, Meredith Y; Rapkin, Bruce D; Morrison, Anne; Kammerman, Sandra

    1997-01-01

    OBJECTIVE To examine the relation of patient beliefs about medication usage and adherence to zidovudine (ZDV) therapy in persons with AIDS. DESIGN Face-to-face interviews were used to determine attitudes of persons with AIDS toward ZDV and other prescribed medications, history of ZDV usage, and sociodemographics. SETTING A public hospital infectious disease clinic, an AIDS day care program, and an inpatient unit in a voluntary hospital where care was provided cooperatively by staff and an informal–care partner. PATIENTS/PARTICIPANTS One hundred forty-one people with the acquired immunodeficiency syndrome agreed to be reinterviewed as part of a longitudinal, New York City–based study examining outcomes related to quality of life. Initial recruitment procedures were to approach all active AIDS patients at each of the three sites between January and July of 1992; reinterviews, which were conducted an average of 6 months later, occurred from mid-1992 through May of 1993. MEASUREMENTS AND MAIN RESULTS The Zidovudine Drug Attitude Inventory was used to assess subjective feelings and attitudes concerning ZDV and prescribed medications in general. Respondents were grouped into five categories on the basis of their ZDV usage history: (1) “short-term” users (i.e., those who had been taking ZDV for 25 months or less); (2) “long-term” users (i.e., those who had been taking ZDV for more than 25 months); (3) self-terminated users; (4) doctor-terminated users; and (5) never users. Long-term users were likely to view ZDV as an illness prophylactic. In contrast, self-terminated users and never users were most likely to believe that ZDV caused adverse side effects and that medicine need not be taken as prescribed. CONCLUSIONS Patients’ beliefs about ZDV were significantly associated with adherence-related behavior. In particular, those who had self-terminated ZDV treatment believed that taking the drug was harmful, were skeptical of its ability to prevent illness, and

  4. Antiproliferative potential of zidovudine in human keratinocyte cultures.

    PubMed

    Bonnekoh, B; Wevers, A; Geisel, J; Rasokat, H; Mahrle, G

    1991-09-01

    Because the beneficial effects of zidovudine in human immunodeficiency virus infection-associated psoriasis have recently been observed, this study focused on the drug's action on the rapidly proliferating human HaCaT keratinocyte line as an in vitro model for epidermal hyperproliferation. Cultures in log growth phase were exposed to zidovudine for 2 days. Zidovudine slowed proliferation in a dose-dependent fashion as evidenced by 50% inhibition concentrations of 33 mumol/L (cell number), 30 mumol/L (protein content), 0.9 mumol/L (protein synthesis), and 0.7 mumol/L (DNA synthesis). Significant (p less than 0.01) reduction of cell viability to 94.6% and 87.2%, as well as morphologic manifestations of cytotoxicity, were first evident after 2 days' exposure to maximal drug concentrations of 10 and 100 mumol/L, respectively. Control viability, assayed by trypan blue exclusion, was 98.0%. Direct cytotoxic plasma membrane injury could be ruled out by the absence of any increase in cytoplasmic lactate dehydrogenase release into supernatants at least during the 1 day of maximal dosage exposure. The drug-induced inhibition of proliferation was reversible within 7 days after a 2-day exposure to 100 mumol/L zidovudine. Two days of treatment with a 10 mumol/L dose did not alter the pattern and synthesis of keratins in vitro. Thus the known antipsoriatic efficacy of zidovudine might be explained, at least partly, by the drug's cytostatic potency. PMID:1918488

  5. Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells

    SciTech Connect

    Szebeni, J.; Patel, S.S.; Hung, K.; Wahl, L.M.; Weinstein, J.N. )

    1991-01-01

    The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed ({sup 3}H)AZT phosphorylation in the same concentration range (20 to 100 microM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.

  6. A model for the immune system response to HIV: AZT treatment studies

    SciTech Connect

    Kirschner, D.E. . Dept. of Mathematics); Perelson, A.S. )

    1993-01-01

    We use mathematical models to describe the interaction of the immune system with the human immunodeficiency virus (HIV). Our model includes T-lymphocytes and macrophages, cells which can be infected with the virus. Using our model we compare the efficacy of AZT treatments given at different stages of disease progression in order to predict when treatment should be initiated.

  7. A model for the immune system response to HIV: AZT treatment studies

    SciTech Connect

    Kirschner, D.E.; Perelson, A.S.

    1993-05-01

    We use mathematical models to describe the interaction of the immune system with the human immunodeficiency virus (HIV). Our model includes T-lymphocytes and macrophages, cells which can be infected with the virus. Using our model we compare the efficacy of AZT treatments given at different stages of disease progression in order to predict when treatment should be initiated.

  8. Gene targeting with retroviral vectors

    SciTech Connect

    Ellis, J.; Bernstein, A. )

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  9. RETROVIRAL INTEGRASE: THEN AND NOW

    PubMed Central

    Andrake, Mark D.; Skalka, Anna Marie

    2016-01-01

    The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell’s DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus (HIV). We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions. PMID:26958915

  10. Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.

    PubMed

    Kharytonchyk, Siarhei; King, Steven R; Ndongmo, Clement B; Stilger, Krista L; An, Wenfeng; Telesnitsky, Alice

    2016-06-01

    A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC-rC pairs either by excising the mismatched base or switching templates prematurely, while AZT(R) RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT's high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZT(R) RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis. PMID:27075671

  11. Continuum Theory of Retroviral Capsids

    NASA Astrophysics Data System (ADS)

    Nguyen, T. T.; Bruinsma, R. F.; Gelbart, W. M.

    2006-02-01

    We present a self-assembly phase diagram for the shape of retroviral capsids, based on continuum elasticity theory. The spontaneous curvature of the capsid proteins drives a weakly first-order transition from spherical to spherocylindrical shapes. The conical capsid shape which characterizes the HIV-1 retrovirus is never stable under unconstrained energy minimization. Only under conditions of fixed volume and/or fixed spanning length can the conical shape be a minimum energy structure. Our results indicate that, unlike the capsids of small viruses, retrovirus capsids are not uniquely determined by the molecular structure of the constituent proteins but depend in an essential way on physical constraints present during assembly.

  12. Pharmacokinetic interaction of megestrol acetate with zidovudine in human immunodeficiency virus-infected patients.

    PubMed Central

    Van Harken, D R; Pei, J C; Wagner, J; Pike, I M

    1997-01-01

    This nonrandomized, two-period crossover study was performed to assess whether concomitant administration of megestrol acetate influences the steady-state pharmacokinetics of zidovudine and its inactive 5'-O-glucuronide metabolite. Twelve HIV-positive, asymptomatic male volunteers received a 100-mg oral capsule dose of zidovudine at least 30 min before meals five times a day at 0700, 1100, 1500, 1900, and 2300 h on study days 1 to 3 and a single 100-mg dose at 0700 h on day 4. On days 5 to 17, 800 mg of megestrol acetate, as a 40-mg/ml aqueous suspension, was administered orally immediately before the 0700 h dose of zidovudine. On days 5 to 16, zidovudine was also administered at 1100, 1500, 1900, and 2300 h. Serial blood samples were collected for 12 h after the single 100-mg dose of zidovudine on days 4 and 17; trough samples were also obtained just before the 0700 h dose on days 2 to 4 and 15 to 17. Levels of zidovudine and its glucuronide in plasma were assayed by a validated radioimmunoassay. Statistical analysis of trough plasma level data indicated that steady-state levels of zidovudine and its glucuronide in plasma had been attained when pharmacokinetic assessments were made on days 4 and 17. When megestrol acetate and zidovudine were coadministered for 13 days, differences of -14, -6.5, and -4.6% in mean zidovudine peak concentration and areas under the curve at 0 to 4 and 0 to 12 h, respectively, +22.5% in mean trough concentration, +2.6% in mean plasma half-life, and no change in median time to peak were observed compared to conditions when zidovudine was administered alone; for zidovudine 5'-O-glucuronide the respective differences were -9, -7.3, -4.4, +2.3, and +10% and no change. None of the differences were statistically significant (P > 0.05). Concomitant therapy with megestrol acetate, at the dose employed to treat anorexia, cachexia, or an unexplained, significant weight loss in AIDS patients, did not alter the steady-state pharmacokinetics of

  13. The effect of AZT and chloroquine on the activities of ricin and a saporin-transferrin chimeric toxin.

    PubMed

    Lizzi, A R; D'Alessandro, A M; Zeolla, N; Brisdelli, F; D'Andrea, G; Pitari, G; Oratore, A; Bozzi, A; Ippoliti, R

    2005-08-15

    This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3'-azido-3'-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells ( approximately 50% inhibition of protein synthesis for TS alone, and TS with AZT and approximately 70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients. PMID:15982641

  14. Clinical pharmacokinetics of 3'-azido-3'-deoxythymidine (zidovudine) and catabolites with formation of a toxic catabolite, 3'-amino-3'-deoxythymidine.

    PubMed

    Stagg, M P; Cretton, E M; Kidd, L; Diasio, R B; Sommadossi, J P

    1992-06-01

    This study investigated pharmacokinetics and metabolism of 3'-azido-3'-deoxythymidine (zidovudine) in patients after a 1-hour intravenous infusion of 2.5 mg/kg zidovudine with a radiolabeled tracer amount of [5-3H]-zidovudine. In addition to unchanged drug and its 5'-O-glucuronide (zidovudine glucuronide), two novel catabolites of zidovudine were detected as 3'-amino-3'-deoxythymidine (AMT), and its 5'-O-glucuronide (GAMT). The AMT apparent plasma elimination half-life (2.70 +/- 0.7 hours) was longer than that of zidovudine (1.20 +/- 0.30 hours) and zidovudine glucuronide (1.60 +/- 0.5 hours). The zidovudine/AMT plasma peak concentration and area under the concentration-time curve ratios were approximately 8 and 5, respectively. Urinary recovery of radioactivity was essentially complete within 24 hours. AMT glucuronide was not detected in urine or plasma, and only low levels of this catabolite were detected in bile. In contrast, AMT was not detected in bile. The substantial levels of AMT in the plasma of patients after zidovudine administration suggests that this catabolite may affect the pharmacodynamic properties of zidovudine in relation to its activity against human immunodeficiency virus replication and cytotoxicity to host cells. PMID:1611806

  15. In vivo efficacy of zidovudine (3'-azido-3'-deoxythymidine) in experimental gram-negative-bacterial infections.

    PubMed Central

    Keith, B R; White, G; Wilson, H R

    1989-01-01

    The therapeutic efficacy of orally administered zidovuldine (3'-azido-3'-deoxythymidine) was determined in animals infected with Escherichia coli and Salmonella dublin. The 50% effective dose (ED50) of zidovudine (9.6 to 11.8 mg/kg of body weight) compared favorably with that of trimethoprim (19.4 to 22.2 mg/kg) in mice with systemic E. coli infection. At 50 mg/kg, both zidovudine and ampicillin reduced the number of bacteria in the kidneys of mice and prevented lethal infection in mice with ascending pyelonephritis caused by E. coli. Zidovudine prevented a lethal S. dublin infection in calves over a wide dose range (8.0 to 31.0 mg/kg per day). Zidovudine levels in plasma of uninfected mice were 28.2 +/- 4.5 and 7.9 +/- 2.2 micrograms/ml at 30 and 60 min, respectively, exceeding the MICs for the bacteria used in the infections. Few zidovudine-resistant strains were observed. The in vivo data raise the possibility that zidovudine may have an antibacterial effect in patients receiving this therapy. PMID:2658792

  16. The Combined Use of Known Antiviral Reverse Transcriptase Inhibitors AZT and DDI Induce Anticancer Effects at Low Concentrations

    PubMed Central

    Aschacher, Thomas; Sampl, Sandra; Käser, Lisa; Bernhard, David; Spittler, Andreas; Holzmann, Klaus; Bergmann, Michael

    2012-01-01

    A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTIs) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, nontoxic concentrations. Biologic effects of AZT and ddI were analyzed at concentrations that correspond to minimal plasma levels achieved during human immunodeficiency virus therapy. Long-term coapplication of low-dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso, and Jurkat. Treatment of cells with both RTI, but not with single RTI, led to a significant accumulation of γH2AX, to p53 phosphorylation, and to cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This antiproliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer. PMID:22355273

  17. The combined use of known antiviral reverse transcriptase inhibitors AZT and DDI induce anticancer effects at low concentrations.

    PubMed

    Aschacher, Thomas; Sampl, Sandra; Käser, Lisa; Bernhard, David; Spittler, Andreas; Holzmann, Klaus; Bergmann, Michael

    2012-01-01

    A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTIs) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, nontoxic concentrations. Biologic effects of AZT and ddI were analyzed at concentrations that correspond to minimal plasma levels achieved during human immunodeficiency virus therapy. Long-term coapplication of low-dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso, and Jurkat. Treatment of cells with both RTI, but not with single RTI, led to a significant accumulation of γH2AX, to p53 phosphorylation, and to cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This antiproliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer. PMID:22355273

  18. Exposure of Allium cepa Root Cells to Zidovudine or Nevirapine Induces Cytogenotoxic Changes

    PubMed Central

    Onwuamah, Chika K.; Ekama, Sabdat O.; Audu, Rosemary A.; Ezechi, Oliver C.; Poirier, Miriam C.; Odeigah, Peter G C.

    2014-01-01

    Antiretroviral drugs have proved useful in the clinical management of HIV-infected persons, though there are concerns about the effects of exposure to these DNA-reactive drugs. We investigated the potential of the plant model Allium cepa root tip assay to demonstrate the cytogenotoxicity of zidovudine and nevirapine and as a replace-reduce-refine programme amenable to resource–poor research settings. Cells mitotic index were determined in squashed root cells from Allium cepa bulbs exposed to zidovudine or nevirapine for 48 hr. The concentration of zidovudine and nevirapine inhibiting 50% root growth after 96 hr exposure was 65.0 µM and 92.5 µM respectively. Root length of all antiretroviral-exposed roots after 96 hr exposure was significantly shorter than the unexposed roots while additional root growth during a subsequent 48 hr recovery period in the absence of drug was not significantly different. By ANOVA, there was a significant association between percentage of cells in mitosis and zidovudine dose (p = 0.004), but not nevirapine dose (p = 0.68). Chromosomal aberrations such as sticky chromosomes, chromatin bridges, multipolar mitoses and binucleated cells were observed in root cells exposed to zidovudine and nevirapine for 48 hr. The most notable chromosomal aberration was drug-related increases in sticky chromosomes. Overall, the study showed inhibition in root length growth, changes in the mitotic index, and the induction of chromosomal aberrations in Allium bulbs treated for 96 hr or 48 hr with zidovudine and nevirapine. The study reveals generalized cytogenotoxic damage induced by exposure to zidovudine and nevirapine, and further show that the two compounds differ in their effects on mitosis and the types of chromosomal aberrations induced. PMID:24599327

  19. Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility.

    PubMed Central

    Harrigan, P R; Kinghorn, I; Bloor, S; Kemp, S D; Nájera, I; Kohli, A; Larder, B A

    1996-01-01

    Amino acid variation at reverse transcriptase (RT) codon 210 (generally Leu-210 to Trp [L210W], TTG-->TGG) is occasionally detected after the initiation of azidothymidine (AZT) therapy. The impact of this variation on AZT resistance and viral replication was addressed by four different approaches. The frequency and genetic background of the L210W mutation in vivo were assessed by analyzing sera of AZT-naive and AZT-experienced patients by RT-PCR and DNA sequencing. The degree of AZT resistance (50% infective concentration [IC50]) of recombinant viruses constructed by using the RT of 21 clinical isolates was stratified by the presence or absence of the 210 mutation. The AZT IC50S of a panel of mutant viruses (with or without W-210) constructed by site-directed mutagenesis in an HXB2 background were assayed by using a HeLa CD4 plaque reduction assay. Finally, the effect of the 210 mutation on viral replication was assessed by replication competition of an AZT-resistant virus, RTMN (L-41/Y-215), and RTMN with the W-210 mutation in the presence and in the absence of AZT. In AZT-naive patients, tryptophan at RT residue 210 was rare. After AZT exposure, W-210 appeared in a minority of those patients, most commonly in association with L-41 and Y-215. The presence of W-210 increased the AZTIC50 by two- to fourfold, as determined by both the recombinant virus assay and site-directed mutagenesis. A significant replication advantage in favor of the wild-type L-210 over W-210 was observed, although the selection against the 210 mutant was two- to threefold lower when the viruses were grown in the presence of 5 microM AZT. In summary, the L210W mutation appears to be of marginal significance, conferring approximately two- to fourfold-reduced sensitivity to AZT compared with similar AZT-resistant genomes with L-210. The selection pressure against W-210 may account for the modest proportion of patients in which W-210 appears in vivo. PMID:8709214

  20. Transplacental exposure to AZT induces adverse neurochemical and behavioral effects in a mouse model: protection by L-acetylcarnitine.

    PubMed

    Zuena, Anna Rita; Giuli, Chiara; Venerosi Pesciolini, Aldina; Tramutola, Antonella; Ajmone-Cat, Maria Antonietta; Cinque, Carlo; Alemà, Giovanni Sebastiano; Giovine, Angela; Peluso, Gianfranco; Minghetti, Luisa; Nicolai, Raffaella; Calamandrei, Gemma; Casolini, Paola

    2013-01-01

    Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for

  1. Studies of retroviral infection in humanized mice

    PubMed Central

    Marsden, Matthew D.; Zack, Jerome A.

    2015-01-01

    Many important aspects of human retroviral infections cannot be fully evaluated using only in vitro systems or unmodified animal models. An alternative approach involves the use of humanized mice, which consist of immunodeficient mice that have been transplanted with human cells and/or tissues. Certain humanized mouse models can support robust infection with human retroviruses including different strains of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). These models have provided wide-ranging insights into retroviral biology, including detailed information on primary infection, in vivo replication and pathogenesis, latent/persistent reservoir formation, and novel therapeutic interventions. Here we describe the humanized mouse models that are most commonly utilized to study retroviral infections, and outline some of the important discoveries that these models have produced during several decades of intensive research. PMID:25680625

  2. Bats and Rodents Shape Mammalian Retroviral Phylogeny.

    PubMed

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  3. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  4. Immobilization of Zidovudine Derivatives on the SBA-15 Mesoporous Silica and Evaluation of Their Cytotoxic Activity

    PubMed Central

    Lewandowski, Dawid

    2015-01-01

    Novel zidovudine derivatives, able to be covalently conjugated to silica surface, have been obtained and grafted to SBA-15 mesoporous silica. Cytotoxic activity of the hybrid organic-inorganic (zidovudine derivatives-silica) systems against HeLa and KB cell lines has been analyzed. Addition of folic acid had a positive influence on the cytotoxicity. Up to 69% of HeLa and 65% of KB tumor cells growth inhibition has been achieved at low silica concentration used (10 μg/mL). PMID:25942021

  5. Immobilization of Zidovudine Derivatives on the SBA-15 Mesoporous Silica and Evaluation of Their Cytotoxic Activity.

    PubMed

    Lewandowski, Dawid; Lewandowska, Marta; Ruszkowski, Piotr; Pińska, Anita; Schroeder, Grzegorz

    2015-01-01

    Novel zidovudine derivatives, able to be covalently conjugated to silica surface, have been obtained and grafted to SBA-15 mesoporous silica. Cytotoxic activity of the hybrid organic-inorganic (zidovudine derivatives-silica) systems against HeLa and KB cell lines has been analyzed. Addition of folic acid had a positive influence on the cytotoxicity. Up to 69% of HeLa and 65% of KB tumor cells growth inhibition has been achieved at low silica concentration used (10 μg/mL). PMID:25942021

  6. Ultrasound-Targeted Retroviral Gene Delivery

    NASA Astrophysics Data System (ADS)

    Taylor, Sarah L.; Rahim, Ahad A.; Bush, Nigel L.; Bamber, Jeffrey C.; Porter, Colin D.

    2007-05-01

    This study demonstrates the ability of focused ultrasound to target retroviral gene delivery. Key to our experiments was the use of non-infectious virus particles lacking the envelope protein required for receptor-mediated entry. The novelty of our approach is that spatial control at a distance is exerted upon viral delivery by subsequent exposure to ultrasound, leading to stable gene delivery. The technology is ideally suited to controlling gene delivery in vivo following systemic vector administration. Our data provide a solution to the critical issue of obtaining tissue specificity with retroviral vectors and impart stability of expression to ultrasound-mediated gene delivery.

  7. Structure and conformational analysis of the anti-HIV AZT 5‧-aminocarbonylphosphonate prodrug using DFT methods

    NASA Astrophysics Data System (ADS)

    Tamara Molina, A.; Alcolea Palafox, M.

    2011-08-01

    A comprehensive theoretical conformational analysis of the anti-HIV AZT 5'-aminocarbonylphosphonate prodrug was carried out, due to this prodrug has noticeable advantage over approved drugs AZT and Nikavir. The whole conformational parameters ( χ, γ, β, α, δ, ɛ, τ, P, νmax) were analysed as well as the NBO Natural atomic charges. The calculations were carried out by means of B3LYP/6-31G ∗∗ and B3LYP/6-311++G(3df,pd) DFT levels of theory with full relaxation of all geometrical parameters. The search located at least 86 stable structures, 6 of which are within a 1 kcal/mol electronic energy range of the global minimum and 11 conformers are within a 1 kcal/mol Gibbs energy range. The global minimum with the 6-311++G(3df,pd) basis set corresponds to the calculated values of the exocyclic torsional angles χ = -121.6°, β = 153.0°, γ = -152.0° and α = -74.1°. The results obtained are in accordance to those found in related anti-HIV nucleoside Analogs. Comparisons of the conformers with those determined in the common anti-HIV drug AZT were carried out. Several correlations and general conclusions were emphasized.

  8. Impact of HIV Infection and Zidovudine Therapy on RBC Parameters and Urine Methylmalonic Acid Levels

    PubMed Central

    Adediran, Adewumi; Osunkalu, Vincent; Wakama, Tamunomieibi; John-Olabode, Sarah; Akinbami, Akinsegun; Uche, Ebele; Akanmu, Sulaimon

    2016-01-01

    Background. Anaemia is a common complication of human immunodeficiency virus (HIV) infection. The aim of this study was to investigate the impact of HIV infection and zidovudine on red blood cells (RBC) parameters and urine methylmalonic acid (UMMA) levels in patients with HIV infection. Material and Methods. A cross-sectional study involving 114 subjects, 94 of which are HIV-infected nonanaemic and 20 HIV negative subjects (Cg) as control. Full blood count parameters and urine methylmalonic acid (UMMA) level of each subject were determined. Associations were determined by Chi-square test and logistic regression statistics where appropriate. Results. Subjects on zidovudine-based ART had mean MCV (93 fL) higher than that of control group (82.9 fL) and ART-naïve (85.9 fL) subjects and the highest mean RDW. Mean UMMA level, which reflects vitamin B12 level status, was high in all HIV-infected groups but was significantly higher in ART-naïve subjects than in ART-experienced subjects. Conclusion. Although non-zidovudine therapy may be associated with macrocytosis (MCV > 95 fL), zidovudine therapy and ART naivety may not. Suboptimal level of vitamin B12 as measured by high UMMA though highest in ART-naïve subjects was common in all HIV-infected subjects. PMID:26989408

  9. Impact of HIV Infection and Zidovudine Therapy on RBC Parameters and Urine Methylmalonic Acid Levels.

    PubMed

    Adediran, Adewumi; Osunkalu, Vincent; Wakama, Tamunomieibi; John-Olabode, Sarah; Akinbami, Akinsegun; Uche, Ebele; Akanmu, Sulaimon

    2016-01-01

    Background. Anaemia is a common complication of human immunodeficiency virus (HIV) infection. The aim of this study was to investigate the impact of HIV infection and zidovudine on red blood cells (RBC) parameters and urine methylmalonic acid (UMMA) levels in patients with HIV infection. Material and Methods. A cross-sectional study involving 114 subjects, 94 of which are HIV-infected nonanaemic and 20 HIV negative subjects (Cg) as control. Full blood count parameters and urine methylmalonic acid (UMMA) level of each subject were determined. Associations were determined by Chi-square test and logistic regression statistics where appropriate. Results. Subjects on zidovudine-based ART had mean MCV (93 fL) higher than that of control group (82.9 fL) and ART-naïve (85.9 fL) subjects and the highest mean RDW. Mean UMMA level, which reflects vitamin B12 level status, was high in all HIV-infected groups but was significantly higher in ART-naïve subjects than in ART-experienced subjects. Conclusion. Although non-zidovudine therapy may be associated with macrocytosis (MCV > 95 fL), zidovudine therapy and ART naivety may not. Suboptimal level of vitamin B12 as measured by high UMMA though highest in ART-naïve subjects was common in all HIV-infected subjects. PMID:26989408

  10. Effect of Zidovudine on the Primary Cytolytic T-Lymphocyte Response and T-Cell Effector Function

    PubMed Central

    Francke, Sabine; Orosz, Charles G.; Hayes, Kathleen A.; Mathes, Lawrence E.

    2000-01-01

    Azidothymidine (AZT) and other nucleoside analogues, used to treat AIDS, can cause severe clinical side effects and are suspected of suppressing immune cell proliferation and effector immune cell function. The purpose of the present study was to quantitatively measure the effects of AZT on cytotoxic T-lymphocyte (CTL) priming and to determine if the major histocompatibility complex-restricted CTL killing was affected by AZT exposure. For this purpose, we employed a murine alloantigen model and limiting-dilution analysis (LDA) to estimate cytotoxic effector cell frequencies of alloreactive splenocytes treated with drug during antigen sensitization. This noninfectious model was chosen to avoid analysis of a virus-compromised immune system. Exposure of splenocytes to therapeutic concentrations of AZT (2 to 10 μM) caused a two- to threefold dose-dependent reduction in CLT precursor frequency. This reduction was caused by decreased proliferation of alloantigen-specific CTLs rather than loss of function, because full cytolytic function could be restored by adjusting the AZT-treated effector/target cell ratios to that of untreated cells. In addition, when AZT was added to the assay system at various times during antigen sensitization there was a time-related loss of the suppressive effect on the generation of cytolytic effector function, suggesting that functional CTLs are not affected by even high doses of AZT. Taken together, the data indicate that the reduction of CTL function associated with AZT treatment is due to a quantitative decrease of effector cell precursor frequency rather than to direct drug cytotoxicity or interference with mediation of cytolysis. Furthermore, antigen-naive immune cells were most sensitive to this effect during the first few days following antigen encounter. PMID:10858351

  11. Molecular Architecture of the Retroviral Capsid.

    PubMed

    Perilla, Juan R; Gronenborn, Angela M

    2016-05-01

    Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores. PMID:27039020

  12. Retroviral Transduction of Murine Primary T Lymphocytes

    PubMed Central

    Lee, James; Sadelain, Michel; Brentjens, Renier

    2016-01-01

    Summary In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti-CD3 and CD28 antibodies. Activated T cells are subsequently centrifuged (spinoculated) on RetroNectin-coated tissue culture plates in the context of retroviral supernatant. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spinoculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. PMID:19110621

  13. Pharmacokinetics of zidovudine dosed twice daily according to World Health Organization weight bands in Ugandan HIV-infected children.

    PubMed

    Fillekes, Quirine; Kendall, Lindsay; Kitaka, Sabrina; Mugyenyi, Peter; Musoke, Philippa; Ndigendawani, Milly; Bwakura-Dangarembizi, Mutsa; Gibb, Diana M; Burger, David; Walker, Ann Sarah

    2014-05-01

    Data on zidovudine pharmacokinetics in children dosed using World Health Organization weight bands are limited. About 45 HIV-infected, Ugandan children, 3.4 (2.6-6.2) years, had intensive pharmacokinetic sampling. Geometric mean zidovudine AUC0-12h was 3.0 h.mg/L, which is higher than previously observed in adults, and was independently higher in those receiving higher doses, younger and underweight children. Higher exposure was also marginally associated with lower hemoglobin. PMID:24736440

  14. Retroviral DNA Transposition: Themes and Variations

    PubMed Central

    Skalka, Anna Marie

    2015-01-01

    SUMMARY Retroviruses and LTR retrotransposons are transposable elements that encapsidate the RNAs that are intermediates in the transposition of DNA copies of their genomes (proviruses), from one cell (or one locus) to another. Mechanistic similarities in DNA transposase enzymes and retroviral/retrotransposon integrases underscore the close evolutionary relationship among these elements. The retroviruses are very ancient infectious agents, presumed to have evolved from Ty3/Gypsy LTR retrotransposons (1), and DNA copies of their sequences can be found embedded in the genomes of most, if not all, members of the tree of life. All retroviruses share a specific gene arrangement and similar replication strategies. However, given their ancestries and occupation of diverse evolutionary niches, it should not be surprising that unique sequences have been acquired in some retroviral genomes and that the details of the mechanism by which their transposition is accomplished can vary. While every step in the retrovirus lifecycle is, in some sense, relevant to transposition, this Chapter focuses mainly on the early phase of retroviral replication, during which viral DNA is synthesized and integrated into its host genome. Some of the initial studies that set the stage for current understanding are highlighted, as well as more recent findings obtained through use of an ever-expanding technological toolbox including genomics, proteomics, and siRNA screening. Persistence in the area of structural biology has provided new insight into conserved mechanisms as well as variations in detail among retroviruses, which can also be instructive. PMID:25844274

  15. The design of artificial retroviral restriction factors

    SciTech Connect

    Yap, Melvyn W.; Mortuza, Gulnahar B.; Taylor, Ian A.; Stoye, Jonathan P.

    2007-09-01

    In addition to the ability to bind the retroviral capsid protein, the retroviral restriction factors Fv1, Trim5{alpha} and Trim5-CypA share the common property of containing sequences that promote self-association. Otherwise Fv1 and Trim5{alpha} appear unrelated. Mutational analyses showed that restriction was invariably lost when changes designed to disrupt the sequences responsible for multimerization were introduced. A novel restriction protein could be obtained by substituting sequences from the self-associating domain of Fv1 for the Trim5 sequences in Trim5-CypA. Similarly, a fusion protein containing cyclophilin A joined to arfaptin2, a protein known to form extended dimers, was also shown to restrict HIV-1. Hence, multimerization of a capsid-binding domain could be the common minimum design feature for capsid-dependent retroviral restriction factors. However, not all domains that promote multimerization can substitute for the N-terminal domains of Fv1 and Trim5{alpha}. Moreover, only CypA can provide a capsid-binding site with different N-terminal domains. It is suggested that the spatial relationship between the multiple target binding sites may be important for restriction.

  16. In Utero Exposure of Female CD-1 Mice to AZT and/or 3TC: I. Persistence of Microscopic Lesions in Cardiac Tissue

    PubMed Central

    Torres, Salina M.; March, Thomas H.; Carter, Meghan M.; McCash, Consuelo L.; Seilkop, Steven K.; Poirier, Miriam C.; Walker, Dale M.

    2010-01-01

    The current study was designed to delineate temporal changes in cardiomyocytes and mitochondria at the light and electron microscopic levels in hearts of mice exposed transplacentally to commonly used nucleoside analogs (NRTIs). Pregnant CD-1 mice were given 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during the last 7 days of gestation, and hearts from female mouse pups were examined at 13 and 26 weeks postpartum for histopathological or ultrastructural changes in cross-sections of both the ventricles and the interventricular septum. Using light microscopy and special staining techniques, transplacental exposure to AZT, 3TC, or AZT/3TC was shown to induce significant histopathological changes in myofibrils; these changes were more widespread at 13 weeks than at 26 weeks postpartum. While most light microscopic lesions resolved, some became more severe between 13 and 26 weeks postpartum. Transplacental NRTI exposure also resulted in progressive drug-specific changes in the number and ultrastructural integrity of cardiac mitochondria. These light and electron microscopic findings show that a subset of changes in cardiac mitochondria and myofibrils persisted and progressed months after transplacental exposure of an animal model to NRTIs, with combined AZT/3TC exposure yielding additive effects compared with either drug alone. PMID:20101476

  17. Antiviral drugs other than zidovudine and immunomodulating therapies in human immunodeficiency virus infection. An overview.

    PubMed

    Clumeck, N; Hermans, P

    1988-08-29

    Although the management of patients with human immunodeficiency virus infections has focused on the treatment of opportunistic infections, or acquired immune deficiency syndrome (AIDS)-related cancers in end stages of the disease, therapies now aim at preventing the natural progression of the underlying disease. In addition to zidovudine many investigational drugs are proposed to treat AIDS-related complex patients. Most of these therapies can be divided into two major groups: (1) The first group includes agents with antiretroviral properties: nucleoside analogues, such as 2'-3'-dideoxycytidine and ribavirin, suramin, antimoniotungstate (heteropolyanion-23), foscarnet (phosphonoformate), interferons, peptide T, castanospermine, dextran sulfate, AL721, or ampligen. (2) The second group aims to restore the defective immune system; it includes thymosin (thymopentin), interleukin-2, cyclosporine, plasmapheresis, bone marrow transplantation, inosine, sodium diethyldithiocarbamate, methionine-enkephalin and carrisyn. At present, no drug other than zidovudine has proved as monotherapy to lengthen survival of human immunodeficiency virus-infected patients. PMID:2457313

  18. Relative Replicative Fitness of Zidovudine-Resistant Human Immunodeficiency Virus Type 1 Isolates In Vitro

    PubMed Central

    Harrigan, P. Richard; Bloor, Stuart; Larder, Brendan A.

    1998-01-01

    Replication of mixtures of two or more human immunodeficiency virus type 1 (HIV-1) variants would be expected to result in the eventual selection of the fittest virus due to Darwinian competition among the variants. The relative proportions of known HIV-1 variants (which may differ only by a single nucleotide from a standard “wild-type” virus, HIV-1HXB2) in mixed viral cultures were quantified by analysis of automated sequence signals of reverse transcriptase PCR products. With this method, the relative levels of replicative fitness of several zidovudine (3′-azidothymidine)-resistant HIV-1HXB2 variants were estimated under controlled in vitro conditions by measuring the rate of change in the proportions of viral variants as they replicated in cell cultures both in the presence and in the absence of drug selection pressure. These variants were engineered to contain commonly observed zidovudine resistance mutations in the HIV-1 reverse transcriptase (M41L, K70R, T215Y, and M41L+T215Y). In the absence of zidovudine, all variants tested displayed reduced replicative fitness compared to wild-type HIV-1HXB2. The order of relative fitness was wild type > K70R ≫ T215Y = M41L+T215Y > M41L. Mixed cultures in the presence of zidovudine showed a dose-dependent selection pressure against the wild-type virus which varied according to the resistance profile of each virus. The information gathered from this approach provides insight into competition among multiple HIV-1 variants, which likely occurs in vivo with drug selection pressure, and may be applicable in more complex mathematical models for predicting the emergence of HIV-1 variants after the initiation of antiretroviral therapy. PMID:9557659

  19. Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

    PubMed

    Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

    2016-07-01

    Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer. PMID:27220342

  20. Effect of penetration enhancers on gel formulation of Zidovudine: in vivo and ex vivo studies.

    PubMed

    Pokharkar, Varsha; Dhar, Sheetal; Singh, Nripendra

    2010-01-01

    To overcome many challenges associated with antiretroviral drug therapy, novel drug delivery systems present an opportunity for formulation scientists to improve the management of patients with HIV/AIDS. The purpose of this study was to prepare a transdermal delivery system for zidovudine using different penetration enhancers incorporated in carbopol 971P gel and to evaluate the same for rheology, percent drug content, drug deposition, in vitro, ex vivo, and in vivo permeation across rat skin. The rheology studies indicated that 1% w/w carbopol gel had a higher linear viscoelastic region, good creep recovery, and desirable viscosity. Among all gel formulations, gel containing cineole and menthol as penetration enhancers attained a steady-state flux of 5.9 mg/cm(2)/h and 5.4 mg/cm(2)/h of zidovudine, respectively, leading to plasma concentration in the therapeutic range. The drug deposition was also found to be highest in the case of gel containing cineole and menthol as penetration enhancers. The results indicated a linear relationship between in vitro flux and in vivo bioavailability of zidovudine transdermal gel. PMID:21502034

  1. Photolysis of three antiviral drugs acyclovir, zidovudine and lamivudine in surface freshwater and seawater.

    PubMed

    Zhou, Chengzhi; Chen, Jingwen; Xie, Qing; Wei, Xiaoxuan; Zhang, Ya-nan; Fu, Zhiqiang

    2015-11-01

    Photodegradation is an important elimination process for many pharmaceuticals in surface waters. In this study, photodegradation of three antiviral drugs, acyclovir, zidovudine, and lamivudine, was investigated in pure water, freshwater, and seawater under the irradiation of simulated sunlight. Results showed that zidovudine was easily transformed via direct photolysis, while acyclovir and lamivudine were mainly transformed via indirect photolysis. We found that in freshwater, nitrate enhanced the photodegradation of the three antiviral drugs, bicarbonate promoted the photodegradation of acyclovir, and dissolved organic matter (DOM) accelerated the photolysis of acyclovir and lamivudine. In seawater, the photolysis of acyclovir was not susceptible to Cl(-), Br(-) and ionic strength; however, the photolysis of zidovudine was inhibited by Cl(-) and Br(-), and the photolysis of lamivudine was enhanced by Cl(-), Br(-) and ionic strength. Second-order reaction rate constants for the three antiviral drugs with (1)O2 (k1O2) and OH (kOH) were also measured. These results are important for fate and ecological risk assessment of the antiviral drugs in natural waters. PMID:26295538

  2. Formation of aminyl radicals on electron attachment to AZT: Abstraction from the sugar phosphate backbone vs. one-electron oxidation of Guanine

    PubMed Central

    Adhikary, Amitava; Khanduri, Deepti; Pottiboyina, Venkata; Rice, Cory T.; Sevilla, Michael D.

    2010-01-01

    Employing electron spin resonance (ESR) spectroscopy, we have characterized the radicals formed in 3′-Azido-3′-deoxythymidine (3′-AZT) and in its 5′-analog 5′-azido-5′-deoxythymidine (5′-AZT) after electron attachment in γ-irradiated aqueous (H2O or D2O) glassy (7.5 M LiCl) systems. ESR spectral studies and theoretical calculations show that the predominant site of electron capture in 3′-AZT and in 5′-AZT is at the azide group and not at the thymine moiety. The azide group in AZT is therefore more electron affinic than the most electron affinic DNA base, thymine. Electron attachment to 3′-AZT and 5′-AZT results in an unstable azide anion radical intermediate (RN3•−) that is too short lived to be observed in our work even at 77 K. At 77 K we observe the neutral aminyl radical (RNH•) after loss of N2 from RN3•− followed by protonation of nitrene anion radical (RN•−) to give RNH•. The expected RN•− intermediate is not observed as protonation from water is complete at 77 K even in under highly basic conditions. Formation of RND• in D2O solutions confirms water as the source of the NH proton in the RNH•. Our assignments to these radicals are aided by DFT calculations for hyperfine coupling constants which closely match the experimental values. On annealing to higher temperatures (ca. 160–170 K), RNH• undergoes bimolecular hydrogen abstraction reactions from the thymine methyl group and the sugar moiety resulting in the formation of the thymine allyl radical (UCH2•) and two sugar radicals - C3′•, C5′•. RNH• also results in one-electron oxidation of the guanine base in 3′-AZG. This work provides a potential mechanism for the reported radiosensitization effects of AZT. PMID:20575557

  3. Effect of gamma radiation on retroviral recombination.

    PubMed

    Hu, W S; Temin, H M

    1992-07-01

    To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism. PMID:1602553

  4. Retroviral Integrations in Gene Therapy Trials

    PubMed Central

    Biasco, Luca; Baricordi, Cristina; Aiuti, Alessandro

    2012-01-01

    γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis. PMID:22252453

  5. Haemoglobin recovery among HIV-1 infected patients on zidovudine-based antiretroviral therapy and other regimens in north-central Nigeria.

    PubMed

    Parrish, Deidra D; Blevins, Meridith; Megazzini, Karen M; Shepherd, Bryan E; Mohammed, Mukhtar Y; Wester, C William; Vermund, Sten H; Aliyu, Muktar H

    2014-04-01

    We conducted a study to assess trends in haemoglobin recovery among HIV-infected patients initiated on zidovudine-based combination antiretroviral therapy (cART) stratified by baseline haemoglobin level. Haemoglobin data from non-pregnant adult patients initiating cART in rural north-central Nigeria between June 2009 and May 2011 were analysed using a linear mixed effects model to assess the interaction between time, zidovudine-containing regimen and baseline haemoglobin level on the outcome of subsequent haemoglobin level. Best-fit curves were created for baseline haemoglobin in the 10th, 25th, 75th and 90th percentiles. We included 313 patients with 736 measures of haemoglobin in the analysis (239 on zidovudine and 74 on non-zidovudine-containing regimens). Median haemoglobin increased over time in both groups, with differences in haemoglobin response over time related to baseline haemoglobin levels and zidovudine use (p = 0.003). The groups of patients on zidovudine at the 10th and 90th percentiles had downward sloping curves while all other groups had upward trending haemoglobin levels. Although haemoglobin levels increased overall for patients on zidovudine-containing regimens, for those in the 10th and 90th percentiles haemoglobin levels trended downward over time. These results have implications for decisions regarding when to initiate, switch from or avoid the use of zidovudine. PMID:24104694

  6. Pharmacokinetics of Zidovudine Dosed Twice Daily According to World Health Organization Weight Bands in Ugandan HIV-infected Children

    PubMed Central

    2014-01-01

    Data on zidovudine pharmacokinetics in children dosed using World Health Organization weight bands are limited. About 45 HIV-infected, Ugandan children, 3.4 (2.6–6.2) years, had intensive pharmacokinetic sampling. Geometric mean zidovudine AUC0–12h was 3.0 h.mg/L, which is higher than previously observed in adults, and was independently higher in those receiving higher doses, younger and underweight children. Higher exposure was also marginally associated with lower hemoglobin. PMID:24736440

  7. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  8. Retroviral infections transmitted by blood transfusion.

    PubMed Central

    Sandler, S. G.; Fang, C.; Williams, A.

    1990-01-01

    Modifications in donor screening and the introduction of laboratory testing of donated blood for anti-HIV-1 and anti-HTLV-I have resulted in a significant reduction in the risks of retroviral infections from blood transfusion. Presently, the American Red Cross detects an average of eight carriers of human immunodeficiency virus, type 1 (HIV-1) per 100,000 otherwise acceptable blood donors (0.008 percent), compared with an average of 35 per 100,000 (0.035 percent) when testing for HIV-1 antibodies began in 1985. Surveillance studies in the United States indicate a small likelihood that HIV-2 carriers will pass current screening procedures and be accepted as blood donors. Even if an HIV-2-infected person were to be accepted as a blood donor, there is a 42-92 percent likelihood that this person's blood would be detected as infective for HIV-2 and excluded because of serological cross-reactions that occur in the EIA for HIV-1 antibodies. During 1989, which was the first year that donated blood was routinely tested for antibodies to human T-lymphotropic virus, type I (HTLV-I) in the United States, approximately nine in 100,000 donors (0.009 percent) were confirmed positive for antibodies to HTLV-I, and their donated blood was excluded. Subsequent testing has revealed that a significant number of these persons whose sera was reactive by the HTLV-I EIA were, in fact, infected by HTLV-II. Epidemiological studies of human retroviral infections (HIV-1, HIV-2, HTLV-I, and HTLV-II) continue to provide important data and direction for improving criteria for qualifying blood donors. PMID:1981409

  9. Effect of zidovudine on survival of patients with AIDS in Australia.

    PubMed

    Solomon, P J; Wilson, S R; Swanson, C E; Cooper, D A

    1990-09-01

    Since the first case of AIDS in Australia was diagnosed in December 1982, there have been substantial improvements in the treatment of AIDS-related conditions. In particular, zidovudine was widely introduced into clinical practice in Australia in June 1987. In order to evaluate its effect, we compared the survival of patients diagnosed before and after July 31, 1987 using data available in early 1989. Survival distributions were compared by means of Kaplan-Meier curves and by fitting exponential survival models incorporating a special feature of the data. Before August 1, 1987 the overall distribution of survival times for patients with AIDS in Australia is well described by an exponential distribution with a mean of 1.04 years. The corresponding median survival time for this period was 8.8 months. For patients diagnosed with AIDS after July 31, 1987 the median survival time had not been attained by December 31, 1988. However, the estimated mean survival time increased to 2.7 years. Survival times were found to be remarkably stable over the different regions of Australia. We have shown that substantial improvements in survival of patients diagnosed with AIDS in Australia are associated with the widespread availability of zidovudine from mid 1987. To the best of our knowledge this study is the first of its kind to show a major shift in the distribution of survival associated with the introduction of antiviral therapy. PMID:2392071

  10. Simple and rapid RP-HPLC method for simultaneous determination of acyclovir and zidovudine in human plasma.

    PubMed

    Sharma, Megha; Nautiyal, Pragya; Jain, Surendra; Jain, Deepti

    2010-01-01

    Combination therapy with acyclovir and zidovudine is used for the treatment of herpes-infected immunocompromised patients. In the view of the optimal drug concentrations (minimum effective concentrations) for viral suppression and avoidance of drug toxicity, monitoring of drug levels has been considered essential to determine drug concentrations in plasma after administration of a dose of acyclovir and zidovudine. A simple, precise, and rapid RP-HPLC method has been developed for this purpose. Chromatographic separation was performed using methanol-water (50 + 50, v/v), pH 2.5 adjusted with orthophosphoric acid, as an isocratic mobile phase at a flow rate of 0.8 mL/min with an Inertsil ODS (C18) column (5 microm particle size, 250 x 4.60 mm id). Detection was carried out using a UV photo diode array detector at 258 nm. The plasma samples were prepared by a protein precipitation method. The retention time for acyclovir and zidovudine was 3.5 +/- 0.2 and 6.2 +/- 0.3 min, respectively. The method was linear in the range of 200-1800 and 400-3600 ng/mL with LOQ of 200 ng (SD = +/-1.4) and 400 ng (SD = +/-0.9) for zidovudine and acyclovir, respectively, in plasma. The mean accuracy was 98.0 and 96.4%, with average extraction recovery of 64.8 +/- 2.1 and 77.5 +/- 1.7% for lower nominal concentrations of acyclovir and zidovudine, respectively. PMID:21140658

  11. The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer

    PubMed Central

    Lavie, Arnon; Yanagisawa, Teruyuki; Medin, Jeffrey A.

    2013-01-01

    We previously described a novel suicide (or ‘cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs. PMID:24194950

  12. The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

    PubMed

    Sato, Takeya; Neschadim, Anton; Lavie, Arnon; Yanagisawa, Teruyuki; Medin, Jeffrey A

    2013-01-01

    We previously described a novel suicide (or 'cell fate control') gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs. PMID:24194950

  13. 'Click' chemistry synthesis and capillary electrophoresis study of 1,4-linked 1,2,3-triazole AZT-systemin conjugate.

    PubMed

    Dobkowski, Michał; Szychowska, Aleksandra; Pieszko, Małgorzata; Miszka, Anna; Wojciechowska, Monika; Alenowicz, Magdalena; Ruczyński, Jarosław; Rekowski, Piotr; Celewicz, Lech; Barciszewski, Jan; Mucha, Piotr

    2014-09-01

    The Cu(I) catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was applied for a nucleoside-peptide bioconjugation. Systemin (Sys), an 18-aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N-propynoic acid functionalized analog (Prp-Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp-Sys with 3'-azido-2',3'-dideoxythymidine (AZT), a model cargo molecule. 1,4-Linked 1,2,3-triazole AZT-Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by-products. Under typical separation conditions of phosphate 'buffer' at low pH and uncoated fused bare-silica capillary, an increasing peak intensity assigned to triazole-linked AZT-Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE-ESI-MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. PMID:24889517

  14. Membrane-mediated interaction between retroviral capsids

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan

    2012-02-01

    A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

  15. Structural basis for retroviral integration into nucleosomes

    PubMed Central

    Maskell, Daniel P.; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2015-01-01

    Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. PMID:26061770

  16. Zidampidine, an aryl phosphate derivative of AZT: in vivo pharmacokinetics, metabolism, toxicity, and anti-viral efficacy against hemorrhagic fever caused by Lassa virus.

    PubMed

    Uckun, F M; Venkatachalam, T K; Erbeck, D; Chen, C L; Petkevich, A S; Vassilev, A

    2005-05-01

    The pharmacokinetics, metabolism, and toxicity of Zidampidine, an aryl phosphate derivative of AZT, 3'-azidothymidine-5'-[p-bromophenyl methoxyalaninyl phosphate] were investigated in CD-1 mice. Following iv injection, Zidampidine was rapidly converted to its metabolites Ala-AZT-MP and AZT. Zidampidine was not toxic to mice at doses up to 250mg/kg. We next examined the therapeutic effect of Zidampidine in CBA mice challenged with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or non-toxic doses of Zidampidine administered intraperitoneally 24h prior, 1h prior, and 24, 48, 72, and 96h after virus inoculation. The probability of survival following the Lassa challenge was significantly improved for Zidampidine-treated mice (Kaplan Meier, Log-Rank p value<0.0001). This pilot study provides the basis for future preclinical evaluation of Zidampidine and its potential as a new agent for the treatment of viral hemorrhagic fevers caused by Lassa virus. PMID:15809163

  17. Zidovudine Oral

    MedlinePlus

    ... HIV, it may decrease your chance of developing acquired immunodeficiency syndrome (AIDS) and HIV-related illnesses such as serious infections or cancer. Taking these medications along with practicing ...

  18. Zidovudine Injection

    MedlinePlus

    ... effectiveness and side effects of your treatment using laboratory tests and physical examinations. It is important to keep all appointments with your doctor and the laboratory. The length of treatment depends on how your ...

  19. Zidovudine Oral

    MedlinePlus

    ... to the liver and a blood condition called lactic acidosis.Call your doctor immediately if you experience ... interferon, lorazepam (Ativan), oxazepam (Serax), probenecid (Benemid), valproic acid (Depakene, Depakote), and vitamins.tell your doctor if ...

  20. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    SciTech Connect

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-08-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

  1. The Host RNAs in Retroviral Particles.

    PubMed

    Telesnitsky, Alice; Wolin, Sandra L

    2016-01-01

    As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs' packaging determinants differ from the viral genome's, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs-if any-have remained elusive. PMID:27548206

  2. The Host RNAs in Retroviral Particles

    PubMed Central

    Telesnitsky, Alice; Wolin, Sandra L.

    2016-01-01

    As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive. PMID:27548206

  3. Sporadic ALS/MND: a global neurodegeneration with retroviral involvement?

    PubMed

    Westarp, M E; Ferrante, P; Perron, H; Bartmann, P; Kornhuber, H H

    1995-05-01

    Sporadic amyotrophic lateral sclerosis may be an aetiologically heterogenous disease. We confirmed elevated circulating IgG immune complexes, and altered IgG seroreactivities against human retroviral antigens (HIV-2 and HTLV immunoblots) in overlapping subgroups of patients. Together with preliminary findings of a positive polymerase chain reactivity for human T-lymphotropic virus (HTLV.tax/rex) in blood leukocytes of 5 out of 14 sALS patients, we interpret this as evidence for a retroviral involvement in this relentlessly progressive, often asymmetrically spreading neurodegeneration. The possibility of a secondary phenomenon seems unlikely, yet cannot be completely ruled out. PMID:7595609

  4. Caring for the retrovirus infected cat.

    PubMed

    McCaw, D

    1995-11-01

    No commercial vaccine [correction of vacine] exists for feline immunodeficiency virus (FIV), and although feline leukemia virus (FeLV) vaccines are available, they are neither 100% effective nor used in all cats. These realities clearly indicate the veterinarian will be required to treat either FeLV- or FIV-positive cats for some time to come. The management of FIV- or FeLV-positive cats may require supportive therapies as well as virus-specific therapies such as zidovudine (AZT; Retrovir, Burroughs Wellcome, Research Triangle Park, NC). PMID:8820595

  5. Concurrent zidovudine levels in semen and serum determined by radioimmunoassay in patients with AIDS or AIDS-related complex

    SciTech Connect

    Henry, K.; Chinnock, B.J.; Quinn, R.P.; Fletcher, C.V.; de Miranda, P.; Balfour, H.H.

    1988-05-27

    Zidovudine was present in the semen and serum of six patients with acquired immunodeficiency syndrome or the related complex who were receiving 200 mg of the drug orally every four to six hours. Mean semen zidovudine levels (as measured by a new radioimmunoassay) in samples collected 0.75 to 1.25 hours after oral dosing were 3.63 to 7.19 ..mu..mol/L. Levels in semen samples collected 3.0 to 4.5 hours after oral dosing were 1.68 to 6.43 ..mu..mol/L. These values are above the in vitro minimum inhibitory concentration for the human immunodeficiency virus type 1 (HIV-1). Mean serum concentrations at the early and late times after oral dosing were 0.22 to 3.07 ..mu..mol/L and 0.10 to 1.42 ..mu..mol/L, respectively. Ratios of semen/serum zidovudine levels ranged from 1.3 to 20.4. It is possible that a pH-dependent trapping mechanism, which has been described in the prostate for other antibiotics, was responsible for the relatively high semen levels observed.

  6. In vitro Evaluation of the Effect of Combination of Hydrophilic and Hydrophobic Polymers on Controlled Release Zidovudine Matrix Tablets.

    PubMed

    Ganesh, S; Radhakrishnan, M; Ravi, M; Prasannakumar, B; Kalyani, J

    2008-01-01

    The aim of the present study was to prepare and characterize controlled-release matrix tablets of zidovudine using hydrophilic HPMC K4 M or Carbopol 934 alone or in combination with hydrophobic ethyl cellulose. Release kinetics was evaluated by using USP XXIV dissolution apparatus No.2 (paddle) type. Scanning electron microscopy was used to visualize the effect of dissolution medium on matrix tablet surface. The in vitro results of controlled - release zidovudine tablets were compared with conventional marketed tablet Zidovir. The in vitro drug release study revealed that HPMC K4 M or Carbopol 934 preparation was able to sustain the drug release near to 6 hours. Combining HPMC K4 M or Carbopol 934 with ethyl cellulose sustained the drug release for nearly 12 h. The in vitro evaluation showed that the drug release may be by diffusion along with erosion. Results suggest that the developed controlled-release tablets of zidovudine could perform therapeutically better than marketed dosage forms, leading to improve efficacy, controlling the release and better patient compliance. PMID:20046771

  7. TRIM5 acts as more than a retroviral restriction factor.

    PubMed

    de Silva, Suresh; Wu, Li

    2011-07-01

    The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction. PMID:21866272

  8. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

    PubMed

    Palesch, David; Boldt, Felix; Müller, Janis A; Eisele, Klaus; Stürzel, Christina M; Wu, Yuzhou; Münch, Jan; Weil, Tanja

    2016-08-17

    Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer. PMID:27239020

  9. Early-initiated zidovudine therapy prevents disease but not low levels of persistent retrovirus in mice.

    PubMed

    Morrey, J D; Okleberry, K M; Sidwell, R W

    1991-01-01

    An F1 hybrid mouse strain containing the Rfv-3r/s genotype was inoculated with Friend virus complex (FV) and treated with zidovudine (ZDV) intraperitoneally three times daily for 20 days beginning as early as 10 min after initial viral exposure. This strain of mice develops FV-specific neutralizing antibodies that aid in reducing viremia and splenic virus titers but do not prevent splenomegaly and eventual FV-associated death. The virally exposed mice treated with ZDV did not develop splenomegaly or have detectable viremia after the last drug treatment. On day 21, a single animal had demonstrable virus in the spleen as determined by a focal immunoenzyme assay; 57% had detectable virus at 5 weeks, but non displayed splenic virus after 35 weeks. None of the animals died after the 35-week holding period, compared to 38% dying in placebo-treated mice. To detect low levels of the virus, or potentially latent virus, splenocytes were cocultivated with a cell line known to readily propagate FV, and the cells were subsequently passaged four times to amplify replication of the virus. After amplification, a significant increase was seen in the number of mice testing positive for virus. Thus, ZDV treatment initiated early after virus exposure was effective in preventing FV-induced splenomegaly and death, but did not prevent low levels of persistent retrovirus in the mice. PMID:2016687

  10. Composite alginate hydrogel microparticulate delivery system of zidovudine hydrochloride based on counter ion induced aggregation

    PubMed Central

    Roy, Harekrishna; Rao, P. Venkateswar; Panda, Sanjay Kumar; Biswal, Asim Kumar; Parida, Kirti Ranjan; Dash, Jharana

    2014-01-01

    Aim: The present study deals with preparation of zidovudine loaded microparticle by counter ion induced aggregation method. During this study effect of polyacrylates and hypromellose polymers on release study were investigated. Materials and Methods: The ion induced aggregated alginate based microparticles were characterized for surface morphology, particle size analysis, drug entrapment study, in-vitro study, Fourier-transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC) study. Results and Discussion: The result showed Eudragit RL-100 (ERL) based formulations had smoother surface as well as their mean particle sizes were found greater compared with Eudragit RS-100 (ERS) microparticles. Furthermore, drug entrapments were found to be more in ERL formulae as compared with ERS. RL3 released 101.05% drug over a period of 8th h and followed Higuchi profile and Fickian diffusion. Moreover, data obtained illustrated that, higher amount of quaternary ammonium group, alkali value, and glass transition temperature may be possible reason for improving permeability of ERL based formulations. It was also noticed, hyroxypropyl methylcellulose (HPMC) K4M premium grade polymer sustained drug release more than HPMC K15M. In addition, drug-excipient interaction study was carried out by FTIR and DSC study. PMID:25298940

  11. Evaluation of Bystander Cell Killing Effects in Suicide Gene Therapy of Cancer: Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis.

    PubMed

    Sato, Takeya; Neschadim, Anton; Nakagawa, Ryo; Yanagisawa, Teruyuki; Medin, Jeffrey A

    2015-01-01

    Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant cells, where they can also mediate destruction. This phenomenon, termed "bystander killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander cell killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target cells, and various approaches for the evaluation of bystander cell killing effects in SGCT in both in vitro and in vivo models. PMID:26072401

  12. Convergent capture of retroviral superantigens by mammalian herpesviruses.

    PubMed

    Aswad, Amr; Katzourakis, Aris

    2015-01-01

    Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV. PMID:26400439

  13. Convergent capture of retroviral superantigens by mammalian herpesviruses

    PubMed Central

    Aswad, Amr; Katzourakis, Aris

    2015-01-01

    Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV. PMID:26400439

  14. Retroviral Transduction of T Cells and T Cell Precursors.

    PubMed

    Simmons, Amie; Alberola-Ila, José

    2016-01-01

    Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors. PMID:26294401

  15. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.

    PubMed

    Grow, Edward J; Flynn, Ryan A; Chavez, Shawn L; Bayless, Nicholas L; Wossidlo, Mark; Wesche, Daniel J; Martin, Lance; Ware, Carol B; Blish, Catherine A; Chang, Howard Y; Pera, Renee A Reijo; Wysocka, Joanna

    2015-06-11

    Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. PMID:25896322

  16. Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

    PubMed

    Unsinger, J; Kröger, A; Hauser, H; Wirth, D

    2001-11-01

    Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells. PMID:11708885

  17. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  18. Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3β-Snail pathway.

    PubMed

    Namba, T; Kodama, R; Moritomo, S; Hoshino, T; Mizushima, T

    2015-01-01

    Pancreatic cancer is one of the most difficult malignancies to treat owing to the rapid acquisition of resistance to chemotherapy. Gemcitabine, a first-line treatment for pancreatic cancer, prolongs patient survival by several months, and combination treatment with gemcitabine and other anti-cancer drugs in the clinic do not show any significant effects on overall survival. Thus, identification of a drug that resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine and a better understanding of the molecular mechanisms of gemcitabine resistance are critical to develop new therapeutic options for pancreatic cancer. Here, we report that zidovudine resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine as shown by screening a compound library, including clinical medicine, using gemcitabine-resistant cells. In analyzing the molecular mechanisms of zidovudine effects, we found that the epithelial-to-mesenchymal transition (EMT)-like phenotype and downregulation of human equilibrative nucleoside transporter 1 (hENT1) are essential for the acquisition of gemcitabine resistance, and zidovudine restored these changes. The chemical biology investigations also revealed that activation of the Akt-GSK3β-Snail1 pathway in resistant cells is a key signaling event for gemcitabine resistance, and zidovudine resensitized resistant cells to gemcitabine by inhibiting this activated pathway. Moreover, our in vivo study demonstrated that co-administration of zidovudine and gemcitabine strongly suppressed the formation of tumors by gemcitabine-resistant pancreatic cancer and prevented gemcitabine-sensitive pancreatic tumors from acquiring gemcitabine-resistant properties, inducing an EMT-like phenotype and downregulating hENT1 expression. These results suggested that co-treatment with zidovudine and gemcitabine may become a novel therapeutic strategy for pancreatic cancer by inhibiting chemoresistance-specific signaling. PMID:26111057

  19. Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3β-Snail pathway

    PubMed Central

    Namba, T; Kodama, R; Moritomo, S; Hoshino, T; Mizushima, T

    2015-01-01

    Pancreatic cancer is one of the most difficult malignancies to treat owing to the rapid acquisition of resistance to chemotherapy. Gemcitabine, a first-line treatment for pancreatic cancer, prolongs patient survival by several months, and combination treatment with gemcitabine and other anti-cancer drugs in the clinic do not show any significant effects on overall survival. Thus, identification of a drug that resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine and a better understanding of the molecular mechanisms of gemcitabine resistance are critical to develop new therapeutic options for pancreatic cancer. Here, we report that zidovudine resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine as shown by screening a compound library, including clinical medicine, using gemcitabine-resistant cells. In analyzing the molecular mechanisms of zidovudine effects, we found that the epithelial-to-mesenchymal transition (EMT)-like phenotype and downregulation of human equilibrative nucleoside transporter 1 (hENT1) are essential for the acquisition of gemcitabine resistance, and zidovudine restored these changes. The chemical biology investigations also revealed that activation of the Akt-GSK3β-Snail1 pathway in resistant cells is a key signaling event for gemcitabine resistance, and zidovudine resensitized resistant cells to gemcitabine by inhibiting this activated pathway. Moreover, our in vivo study demonstrated that co-administration of zidovudine and gemcitabine strongly suppressed the formation of tumors by gemcitabine-resistant pancreatic cancer and prevented gemcitabine-sensitive pancreatic tumors from acquiring gemcitabine-resistant properties, inducing an EMT-like phenotype and downregulating hENT1 expression. These results suggested that co-treatment with zidovudine and gemcitabine may become a novel therapeutic strategy for pancreatic cancer by inhibiting chemoresistance-specific signaling. PMID:26111057

  20. Multiple-Dose Pharmacokinetics and Pharmacodynamics of Abacavir Alone and in Combination with Zidovudine in Human Immunodeficiency Virus-Infected Adults

    PubMed Central

    McDowell, James A.; Lou, Yu; Symonds, William S.; Stein, Daniel S.

    2000-01-01

    Abacavir (1592U89) is a nucleoside reverse transcriptase inhibitor with potent activity against human immunodeficiency virus type 1 (HIV-1) when used alone or in combination with other antiretroviral agents. The present study was conducted to determine the multiple-dose pharmacokinetics and pharmacodynamics of abacavir in HIV-1-infected subjects following oral administration of daily doses that ranged from 600 to 1,800 mg, with and without zidovudine. Seventy-nine subjects received abacavir monotherapy for 4 weeks (200, 400, or 600 mg every 8 hours [TID] and 300 mg every 12 h [BID]) and thereafter received either zidovudine (200 mg TID or 300 mg BID) or matching placebo with abacavir for 8 additional weeks. Pharmacokinetic parameters were calculated for abacavir after administration of the first dose and at week 4 and for abacavir, zidovudine, and its glucuronide metabolite at week 12. The concentrations of abacavir in cerebrospinal fluid were determined in a subset of subjects. Steady-state plasma abacavir concentrations were achieved by week 4 of monotherapy and persisted to week 12. At steady state, abacavir pharmacokinetic parameters (area under the plasma concentration-time curve for a dosing interval [AUCtau] and peak concentration [Cmax]) were generally proportional to dose over the range of a 600- to 1,200-mg total daily dose. Coadministration of zidovudine with abacavir produced a small and inconsistent effect on abacavir pharmacokinetic parameters across the different doses. At the clinical abacavir dose (300 mg BID) zidovudine coadministration had no effect on the abacavir AUCtau, which is most closely associated with efficacy. Zidovudine pharmacokinetics appeared to be unaffected by abacavir. Statistically significant but weak relationships were found for the change in the log10 HIV-1 RNA load from the baseline to week 4 versus total daily AUCtau and Ctau (P < 0.05). The incidence of nausea was significantly associated with total daily AUCtau and Cmax

  1. Comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1.

    PubMed

    Sperber, K; Chiang, G; Chen, H; Ross, W; Chusid, E; Gonchar, M; Chow, R; Liriano, O

    1997-01-01

    Hydroxychloroquine (HCQ), an antimalarial agent used to treat patients with autoimmune diseases, has been shown to suppress human immunodeficiency virus type 1 (HIV-1) replication in T cells and monocytes in vitro by inhibiting posttranscriptional modification of the virus. An initial randomized, placebo-controlled clinical trial conducted in 38 asymptomatic HIV-1-infected patients who had CD4+ counts between 200 and 500 cells/mm3 demonstrated that the amount of recoverable virus declined significantly in the HCQ group compared with the placebo group over the 8-week study period. These preliminary observations were expanded into a second 16-week clinical trial comparing the efficacy of HCQ with that of zidovudine (ZDV) in 72 asymptomatic HIV-1-infected patients with CD4+ counts between 200 and 500 cells/mm3. Patients were randomly assigned to receive either HCQ 800 mg/d (n = 35) or ZDV 500 mg/d (n = 37) for 16 weeks. No adverse reactions to the study medications were observed in either the HCQ or ZDV group. Patients in both groups had reduced levels of recoverable HIV-1 RNA in the plasma, reduced levels of cultured virus, and reduced levels of serum p24 antigen after the 16-week study period. However, no difference was noted in absolute CD4+ counts between the two groups. Interleukin-6 and serum immunoglobulin G levels were significantly reduced in the HCQ group but not in the ZDV group. These findings support the results of the previous clinical trial. Thus HCQ may be potentially useful in the treatment of patients with HIV-1 infection. PMID:9385480

  2. Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

    PubMed

    Chung, Jie-Yu; Mackay, Fabienne; Alderuccio, Frank

    2015-01-01

    Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals. PMID:25445328

  3. Population Pharmacokinetics Study of Recommended Zidovudine Doses in HIV-1-Infected Children

    PubMed Central

    Treluyer, Jean-Marc; Frange, Pierre; Urien, Saik; Foissac, Frantz; Bouazza, Naim; Benaboud, Sihem; Blanche, Stephane; Hirt, Déborah

    2013-01-01

    The aims of this study were to describe the pharmacokinetics of zidovudine (ZDV) and its biotransformation to its metabolite, 3*-azido-3*-deoxy-5*-glucuronylthymidine (G-ZDV), in HIV-infected children, to identify factors that influence the pharmacokinetics of ZDV, and to compare and evaluate the doses recommended by the World Health Organization (WHO) and the Food and Drug Administration (FDA). ZDV concentrations in 782 samples and G-ZDV concentrations in 554 samples from 247 children ranging in age from 0.5 to 18 years were retrospectively measured. A population pharmacokinetic model was developed with NONMEM software (version 6.2), and the pharmacokinetics of ZDV were best described by a one-compartment model with first-order absorption and elimination. The effect of body weight on the apparent elimination clearance and volume of distribution was significant. The mean population parameter estimates were as follows: absorption rate, 2.86 h−1; apparent elimination clearance, 89.7 liters · h−1 (between-subject variability, 0.701 liters · h−1); apparent volume of distribution, 229 liters (between-subject variability, 0.807 liters); metabolic formation rate constant, 12.6 h−1 (between-subject variability, 0.352 h−1); and elimination rate constant of G-ZDV, 2.27 h−1. On the basis of simulations with FDA and WHO dosing recommendations, the probabilities of observing efficient exposures (doses resulting in exposures of between 3 and 5 mg/liter · h) with less adverse events (doses resulting in exposures below 8.4 mg/liter · h) were higher when the FDA recommendations than when the WHO recommendations were followed. In order to improve the FDA recommendations, ZDV doses should be reconsidered for the weight band (WB) of 20 to 40 kg. The most appropriate doses should be decreased from 9 to 8 mg/kg of body weight twice a day (BID) for the WB from 20 to 29.9 kg and from 300 to 250 mg BID for the WB from 30 to 39.9 kg. The highest dose, 300 mg BID, should be

  4. Formulation and in vitro, in vivo evaluation of extended- release matrix tablet of zidovudine: influence of combination of hydrophilic and hydrophobic matrix formers.

    PubMed

    Kuksal, Atul; Tiwary, Ashok K; Jain, Narendra K; Jain, Subheet

    2006-01-01

    The aim of the present study was to prepare and characterize extended-release matrix tablets of zidovudine using hydrophilic Eudragit RLPO and RSPO alone or their combination with hydrophobic ethyl cellulose. Release kinetics was evaluated by using United States Pharmacopeia (USP)-22 type I dissolution apparatus. Scanning electron microscopy was used to visualize the effect of dissolution medium on matrix tablet surface. Furthermore, the in vitro and in vivo newly formulated sustained-release zidovudine tablets were compared with conventional marketed tablet (Zidovir, Cipla Ltd, Mumbai, India). The in-vitro drug release study revealed that either Eudragit preparation was able to sustain the drug release only for 6 hours (94.3% +/- 4.5% release). Combining Eudragit with ethyl cellulose sustained the drug release for 12 hours (88.1% +/- 4.1% release). Fitting the in vitro drug release data to Korsmeyer equation indicated that diffusion along with erosion could be the mechanism of drug release. In vivo investigation in rabbits showed sustained-release pharmacokinetic profile of zidovudine from the matrix tablets formulated using combination of Eudragits and ethylcellulose. In conclusion, the results suggest that the developed sustained-release tablets of zidovudine could perform therapeutically better than conventional dosage forms, leading to improve efficacy and better patient compliance. PMID:16584139

  5. Split-Intron Retroviral Vectors: Enhanced Expression with Improved Safety

    PubMed Central

    Ismail, Said I.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2000-01-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37–48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650–3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5′-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5′ end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications. PMID:10666267

  6. Split-intron retroviral vectors: enhanced expression with improved safety.

    PubMed

    Ismail, S I; Kingsman, S M; Kingsman, A J; Uden, M

    2000-03-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37-48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650-3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5'-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5' end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications. PMID:10666267

  7. Abacavir/lamivudine/zidovudine as a combined formulation tablet: bioequivalence compared with each component administered concurrently and the effect of food on absorption.

    PubMed

    Yuen, G J; Lou, Y; Thompson, N F; Otto, V R; Allsup, T L; Mahony, W B; Hutman, H W

    2001-03-01

    A single-center, open-label, three-way crossover study was conducted in 24 healthy subjects to assess (1) the bioequivalence of a combined abacavir 300 mg/lamivudine 150 mg/zidovudine 300 mg (A/L/Z) combination tablet relative to the separate brand-name components administered simultaneously and (2) the effect of food on the bioavailability of the drugs from the combination tablet. The subjects were randomly assigned to receive each of the following three treatments, separated by a 2-day washout period: one A/L/Z combination tablet after an overnight fast, one abacavir 300 mg tablet + one lamivudine 150 mg tablet + one zidovudine 300 mg tablet sequentially after an overnight fast, or one A/L/Z combination tablet 5 minutes after completing a standardized high-fat breakfast (67 g fat, 58 g carbohydrate, and 33 g protein). Serial blood samples were collected up to 24 hours postdose for determination of abacavir, lamivudine, and zidovudine serum concentrations. Standard pharmacokinetic parameters were estimated. Treatments were considered bioequivalent if 90% confidence intervals (CI) for geometric least squares (GLS) mean ratios for abacavir, lamivudine, and zidovudine area under the serum concentration-time curve (AUC(infinity)) and maximum observed serum concentration (Cmax) fell entirely within 0.80 to 1.25 for log-transformed parameters. The combined A/L/Z tablet was bioequivalent in the extent (AUC) and rate of absorption (Cmax and time of Cmax [tmax]) to the individual brand-name drug components administered concurrently under fasted conditions. GLS ratios and 90% CI for AUC(infinity) and Cmax were 0.99 (0.96, 1.03) and 1.00 (0.90, 1.11), respectively, for abacavir; 0.95 (0.91, 0.99) and 0.90 (0.84, 0.99), respectively, for lamivudine; and 0.95 (0.89, 1.02) and 0.96 (0.80, 1.15), respectively, for zidovudine. The extent of absorption of abacavir, lamivudine, and zidovudine from the combination tablet was not altered by administration with meals, indicating that

  8. Abacavir, zidovudine, or stavudine as paediatric tablets for African HIV-infected children (CHAPAS-3): an open-label, parallel-group, randomised controlled trial

    PubMed Central

    Mulenga, Veronica; Musiime, Victor; Kekitiinwa, Adeodata; Cook, Adrian D; Abongomera, George; Kenny, Julia; Chabala, Chisala; Mirembe, Grace; Asiimwe, Alice; Owen-Powell, Ellen; Burger, David; McIlleron, Helen; Klein, Nigel; Chintu, Chifumbe; Thomason, Margaret J; Kityo, Cissy; Walker, A Sarah; Gibb, Diana M

    2016-01-01

    Summary Background WHO 2013 guidelines recommend universal treatment for HIV-infected children younger than 5 years. No paediatric trials have compared nucleoside reverse-transcriptase inhibitors (NRTIs) in first-line antiretroviral therapy (ART) in Africa, where most HIV-infected children live. We aimed to compare stavudine, zidovudine, or abacavir as dual or triple fixed-dose-combination paediatric tablets with lamivudine and nevirapine or efavirenz. Methods In this open-label, parallel-group, randomised trial (CHAPAS-3), we enrolled children from one centre in Zambia and three in Uganda who were previously untreated (ART naive) or on stavudine for more than 2 years with viral load less than 50 copies per mL (ART experienced). Computer-generated randomisation tables were incorporated securely within the database. The primary endpoint was grade 2–4 clinical or grade 3/4 laboratory adverse events. Analysis was intention to treat. This trial is registered with the ISRCTN Registry number, 69078957. Findings Between Nov 8, 2010, and Dec 28, 2011, 480 children were randomised: 156 to stavudine, 159 to zidovudine, and 165 to abacavir. After two were excluded due to randomisation error, 156 children were analysed in the stavudine group, 158 in the zidovudine group, and 164 in the abacavir group, and followed for median 2·3 years (5% lost to follow-up). 365 (76%) were ART naive (median age 2·6 years vs 6·2 years in ART experienced). 917 grade 2–4 clinical or grade 3/4 laboratory adverse events (835 clinical [634 grade 2]; 40 laboratory) occurred in 104 (67%) children on stavudine, 103 (65%) on zidovudine, and 105 (64%), on abacavir (p=0·63; zidovudine vs stavudine: hazard ratio [HR] 0·99 [95% CI 0·75–1·29]; abacavir vs stavudine: HR 0·88 [0·67–1·15]). At 48 weeks, 98 (85%), 81 (80%) and 95 (81%) ART-naive children in the stavudine, zidovudine, and abacavir groups, respectively, had viral load less than 400 copies per mL (p=0·58); most ART

  9. Retroviral Transcriptional Regulation and Embryonic Stem Cells: War and Peace

    PubMed Central

    Schlesinger, Sharon

    2014-01-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. PMID:25547290

  10. Comparative Application of PLS and PCR Methods to Simultaneous Quantitative Estimation and Simultaneous Dissolution Test of Zidovudine - Lamivudine Tablets.

    PubMed

    Üstündağ, Özgür; Dinç, Erdal; Özdemir, Nurten; Tilkan, M Günseli

    2015-01-01

    In the development strategies of new drug products and generic drug products, the simultaneous in-vitro dissolution behavior of oral dosage formulations is the most important indication for the quantitative estimation of efficiency and biopharmaceutical characteristics of drug substances. This is to force the related field's scientists to improve very powerful analytical methods to get more reliable, precise and accurate results in the quantitative analysis and dissolution testing of drug formulations. In this context, two new chemometric tools, partial least squares (PLS) and principal component regression (PCR) were improved for the simultaneous quantitative estimation and dissolution testing of zidovudine (ZID) and lamivudine (LAM) in a tablet dosage form. The results obtained in this study strongly encourage us to use them for the quality control, the routine analysis and the dissolution test of the marketing tablets containing ZID and LAM drugs. PMID:26085428

  11. [Prevalence of transmission of zidovudine-resistant viruses in Switzerland. l'Etude suisse de cohorte VIH].

    PubMed

    Yerly, S; Rakik, A; Kinloch-de-Loes, S; Erb, P; Vernazza, P; Hirschel, B; Perrin, L

    1996-10-26

    Zidovudine (ZDV) was the most widely used anti-HIV drug between 1987 and 1995, and, as already reported, transmission of ZDV-resistant viruses occurs. Several mutations of the reverse transcriptase gene have been identified; one of them affects the 215 codon and is associated with a high degree of resistance. We have determined, using selective PCR, the prevalence of transmission of 215 mutant isolates in 134 patients with primary HIV infection (PHI) and have identified 8 patients with 215 mutant virus between 1989 and 1995 in Switzerland. Mutant resistant viruses have been isolated from patients treated with most antiviral drugs. A systematic search for mutant viruses may provide useful information for the adaptation of treatment strategies. PMID:9005525

  12. Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells

    PubMed Central

    Yang, Kuo H.; Kendrick, Racheal; Reddy, Y. Sunila; Kashuba, Angela D. M.; Troiani, Luigi; Bridges, Arlene S.; Fiscus, Susan A.; Forrest, Alan; Cohen, Myron S.

    2015-01-01

    The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure. PMID:26239974

  13. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    PubMed

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. PMID:24462869

  14. Generation of high-titer pseudotyped retroviral vectors with very broad host range.

    PubMed

    Yee, J K; Friedmann, T; Burns, J C

    1994-01-01

    Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future. PMID:7823872

  15. Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.

    PubMed

    Gama-Norton, L; Herrmann, S; Schucht, R; Coroadinha, A S; Löw, R; Alves, P M; Bartholomae, C C; Schmidt, M; Baum, C; Schambach, A; Hauser, H; Wirth, D

    2010-08-01

    The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line. PMID:20222806

  16. Detection of a human intracisternal A-type retroviral particle antigenically related to HIV

    NASA Technical Reports Server (NTRS)

    Garry, R. F.; Fermin, C. D.; Hart, D. J.; Alexander, S. S.; Donehower, L. A.; Luo-Zhang, H.

    1990-01-01

    Sjogren's syndrome is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. A human intracisternal A-type retroviral particle that is antigenically related to HIV was detected in lymphoblastoid cells exposed to homogenates of salivary tissue from patients with Sjogren's syndrome. Comparison of this retroviral particle to HIV indicates that they are distinguishable by several ultrastructural, physical, and enzymatic criteria.

  17. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    PubMed Central

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801

  18. Adherence to Anti Retroviral Therapy (ART) During Muslim Ramadan Fasting

    PubMed Central

    Habib, A. G.; Shepherd, J. C.; Eng, M. K. L.; Babashani, M.; Jumare, J.; Yakubu, U.; Gebi, U. I.; Saad, M.; Ibrahim, H.; Blattner, W. A.

    2010-01-01

    Annual fasting during the month of Ramadan is observed in Muslim countries, some of which have widespread HIV infection. We studied treatment adherence and customary practices among 142 fasting `FT' and 101 non-fasting `NFT' patients on anti-retroviral therapy (ART) in Nigeria. Adherence on ART among FT and NFT patients was similar during Ramadan, 96% and 98%, and ever since commencement of ART, 80% and 88%, respectively. FT patients altered their typical daily behaviors by advancing morning and delaying evening doses thereby prolonging dosing intervals, eating heavier meals pre-dawn and on breakfast at sunset (78%), and changing or reducing their sleeping and waking times (40%). This preliminary study suggests that adherence and drug taking frequency appear uncompromised in FT HIV infected patients on ARVs. PMID:18521736

  19. Fully-spliced HIV-1 RNAs are reverse transcribed with similar efficiencies as the genomic RNA in virions and cells, but more efficiently in AZT-treated cells

    PubMed Central

    Houzet, Laurent; Morichaud, Zakia; Mougel, Marylène

    2007-01-01

    We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery. PMID:17474982

  20. Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

    PubMed Central

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M.

    2016-01-01

    We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs. PMID:26850643

  1. Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors.

    PubMed

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M

    2016-03-18

    We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs. PMID:26850643

  2. Prevalence and Risk Factors of Low Bone Mineral Density in Korean HIV-Infected Patients: Impact of Abacavir and Zidovudine

    PubMed Central

    Kim, Hee-Sung; Chin, Bum Sik

    2013-01-01

    Low bone mineral density (BMD) is common in HIV-infected patients. We aimed to describe the prevalence of low BMD and risk factors in Korean HIV-infected patients and to assess the effects of antiretroviral therapy (ART) on BMD. We retrospectively evaluated 224 HIV infected-patients. The prevalence of osteopenia and osteoporosis were 41.5% and 12.9%. These were much higher in 53 patients aged 50 yr and older (52.8% and 34.0%). Older age, lower body mass index, and ART > 3 months were independent risk factors for low BMD. Osteoporosis was more prevalent in patients on the abacavir-based regimen for < 1 yr than ≥ 1 yr; however, it was more prevalent in patients on the zidovudine-based regimen for ≥ 1 yr than < 1 yr (P = 0.017). Osteoporosis in patients on the abacavir-based regimen was more common in the spine than in the femur (P = 0.01). Given such a high prevalence of low BMD, close monitoring of BMD for HIV-infected patients on ART is required. The different prevalence of osteoporosis over time and affected areas between two regimens suggest they may play roles in different mechanisms in bone loss. PMID:23772145

  3. Effects of stoichiometry of retroviral components on virus production.

    PubMed

    Yap, M W; Kingsman, S M; Kingsman, A J

    2000-09-01

    A study was conducted to investigate the effects of increasing the amount of each retroviral component on vector production. It was found that, while the components of both amphotropic and ecotropic vectors were expressed independently of each other in a transient transfection system, increasing the amount of the gag/gag-pol component resulted in a decrease in virus titres for the amphotropic particles but not ecotropic particles. Analyses of the virus stocks produced indicated that the negative effect on titres was closely linked to the availability of envelope proteins for virion incorporation. The negative effect was not observed for ecotropic particle production in 293T cells, where the ecotropic receptor was absent, but was manifested when production was conducted in 293/12 cells expressing the ecotropic receptor. This suggested that the premature interaction between envelope and receptor in producer cells could limit the amount of envelope available for virion incorporation. In designing optimal vector production systems it is essential, therefore, to balance the concentration of the vector components and to ensure that there is never an excess of Gag/Gag-Pol. PMID:10950977

  4. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  5. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    PubMed Central

    Rawson, Jonathan M.O.; Mansky, Louis M.

    2014-01-01

    Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved. PMID:25254386

  6. Epigenetics, drugs of abuse, and the retroviral promoter

    PubMed Central

    Shirazi, Jasmine; Shah, Sonia; Sagar, Divya; Nonnemacher, Michael R.; Wigdahl, Brian; Khan, Zafar K.; Jain, Pooja

    2013-01-01

    Drug abuse alone has been shown to cause epigenetic changes in brain tissue that have been shown to play roles in addictive behaviors. In conjunction with HIV-1 infection, it can cause epigenetic changes at the viral promoter that can result in altered gene expression, and exacerbate disease progression overall. This review entails an in-depth look at research conducted on the epigenetic effects of three of the most widely abused drugs (cannabinoids, opioids, and cocaine), with a particular focus on the mechanisms through which these drugs interact with HIV-1 infection at the viral promoter. Here we discuss the impact of this interplay on disease progression from the point of view of the nature of gene regulation at the level of chromatin accessibility, chromatin remodeling, and nucleosome repositioning. Given the importance of chromatin remodeling and DNA methylation in controlling the retroviral promoter, and the high susceptibility of the drug abusing population of individuals to HIV infection, it would be beneficial to understand the way in which the host genome is modified and regulated by drugs of abuse. PMID:24218017

  7. The Effect of Life History on Retroviral Genome Invasions

    PubMed Central

    Kanda, Ravinder K.; Coulson, Tim

    2015-01-01

    Endogenous retroviruses (ERV), or the remnants of past retroviral infections that are no longer active, are found in the genomes of most vertebrates, typically constituting approximately 10% of the genome. In some vertebrates, particularly in shorter-lived species like rodents, it is not unusual to find active endogenous retroviruses. In longer-lived species, including humans where substantial effort has been invested in searching for active ERVs, it is unusual to find them; to date none have been found in humans. Presumably the chance of detecting an active ERV infection is a function of the length of an ERV epidemic. Intuitively, given that ERVs or signatures of past ERV infections are passed from parents to offspring, we might expect to detect more active ERVs in species with longer generation times, as it should take more years for an infection to run its course in longer than in shorter lived species. This means the observation of more active ERV infections in shorter compared to longer-lived species is paradoxical. We explore this paradox using a modeling approach to investigate factors that influence ERV epidemic length. Our simple epidemiological model may explain why we find evidence of active ERV infections in shorter rather than longer-lived species. PMID:25692467

  8. A revised nomenclature for transcribed human endogenous retroviral loci

    PubMed Central

    2011-01-01

    Background Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved. Results Here we present a revised nomenclature of transcribed human endogenous retroviral loci that sorts loci into groups based on Repbase classifications. Each symbol is of the format ERV + group symbol + unique number. Group symbols are based on a mixture of Repbase designations and well-supported symbols used in the literature. The presented guidelines will allow newly identified loci to be easily incorporated into the scheme. Conclusions The naming system will be employed by the HUGO Gene Nomenclature Committee for naming transcribed human ERV loci. We hope that the system will contribute to clarifying a certain aspect of a sometimes confusing nomenclature for human endogenous retroviruses. The presented system may also be employed for naming transcribed loci of human non-ERV repeat loci. PMID:21542922

  9. Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement.

    PubMed

    Verhoeyen, E; Hauser, H; Wirth, D

    2001-05-20

    Successful retroviral vector construction is still empirical. Test systems for vector efficiency are based on statistical comparison of numerous infectants with single proviral integrates, since their expression depends on the chromosomal surroundings. More reliable data would be obtained if different vector constructs were studied in an identical chromosomal context. Here, we demonstrate the use of a new method, in which chromosomal sites are provirally tagged in such a way that they can be targeted with other expression cassettes. The original tagging integrate is replaced in one step by the targeting element. This permits a reliable comparison of different retroviral vector configurations, eliminating the influence of neighboring chromosomal elements. We compared different retroviral vector types for coexpression of two genes: a vector containing an internal promoter and a vector with an internal ribosome entry site (IRES) element. In contrast to bicistronic retroviral vectors, dual-promoter proviruses exhibited rapid inactivation of the long terminal repeat (LTR)-driven gene expression. Targeted exchange of the dual-promoter provirus with a bicistronic retroviral cassette resulted in gain of expression stability. The reverse experiment confirmed this promoter interaction phenomenon since initial expression stability from a single-promoter bicistronic provirus was lost by targeted exchange with a dual-promoter cassette. In addition, targeting exchange of the dual-promoter provirus, replacing the LTR with an artificial (Tet) promoter restored expression stability. These observations, valid for various integration sites, prove the strong interaction between the LTR and the internal promoter. Our results have implications for retroviral vector design and suggest that retroviral coexpression of two genes is more predictable in the bicistronic configuration. PMID:11387058

  10. Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

    NASA Astrophysics Data System (ADS)

    Yolamanova, Maral; Meier, Christoph; Shaytan, Alexey K.; Vas, Virag; Bertoncini, Carlos W.; Arnold, Franziska; Zirafi, Onofrio; Usmani, Shariq M.; Müller, Janis A.; Sauter, Daniel; Goffinet, Christine; Palesch, David; Walther, Paul; Roan, Nadia R.; Geiger, Hartmut; Lunov, Oleg; Simmet, Thomas; Bohne, Jens; Schrezenmeier, Hubert; Schwarz, Klaus; Ständker, Ludger; Forssmann, Wolf-Georg; Salvatella, Xavier; Khalatur, Pavel G.; Khokhlov, Alexei R.; Knowles, Tuomas P. J.; Weil, Tanja; Kirchhoff, Frank; Münch, Jan

    2013-02-01

    Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

  11. Larger Mammalian Body Size Leads to Lower Retroviral Activity

    PubMed Central

    Katzourakis, Aris; Magiorkinis, Gkikas; Lim, Aaron G.; Gupta, Sunetra; Belshaw, Robert; Gifford, Robert

    2014-01-01

    Retroviruses have been infecting mammals for at least 100 million years, leaving descendants in host genomes known as endogenous retroviruses (ERVs). The abundance of ERVs is partly determined by their mode of replication, but it has also been suggested that host life history traits could enhance or suppress their activity. We show that larger bodied species have lower levels of ERV activity by reconstructing the rate of ERV integration across 38 mammalian species. Body size explains 37% of the variance in ERV integration rate over the last 10 million years, controlling for the effect of confounding due to other life history traits. Furthermore, 68% of the variance in the mean age of ERVs per genome can also be explained by body size. These results indicate that body size limits the number of recently replicating ERVs due to their detrimental effects on their host. To comprehend the possible mechanistic links between body size and ERV integration we built a mathematical model, which shows that ERV abundance is favored by lower body size and higher horizontal transmission rates. We argue that because retroviral integration is tumorigenic, the negative correlation between body size and ERV numbers results from the necessity to reduce the risk of cancer, under the assumption that this risk scales positively with body size. Our model also fits the empirical observation that the lifetime risk of cancer is relatively invariant among mammals regardless of their body size, known as Peto's paradox, and indicates that larger bodied mammals may have evolved mechanisms to limit ERV activity. PMID:25033295

  12. Mean Corpuscular Volume as a Marker for Adherence to Zidovudine-Containing Therapy in HIV-Infected Adults

    PubMed Central

    Mugisha, Joseph O; Donegan, Katherine; Fidler, Sarah; Ramjee, Gita; Hodson, Andrew; Dunn, David T; Porter, Kholoud; Kaleebu, Pontiano

    2012-01-01

    Objectives: To assess whether mean corpuscular volume (MCV) is useful in detecting non-adherence to AZTcontaining therapy. Design: Observational study within randomised controlled trial. Methods: We combined data from two treatment arms in SPARTAC, an RCT of short-course cART in primary HIV infection, classifying participants as responders (HIV-RNA decrease ≥1 log10 or reaching <400copies/ml) or nonresponders following cART initiation. We assessed the sensitivity and specificity of using different percentage increases in MCV for accurately differentiating between responders and non-responders. We further examined changes in MCV levels up to 24 weeks after protocol-indicated cART cessation. Results: Of 119 participants included in this analysis, 73 (61%) were women, 71 of whom were randomised in Africa. Ninety-eight (88%) and 84 (85%) were classified as responders at 4 and 12 weeks respectively following cART initiation. MCV increased by a mean 3% and 1% at week 4, and 14% and <1% at 12 weeks for responders and non-responders. A 2% MCV increase at 4 weeks had 62% sensitivity and specificity for identifying virological response. At 12 weeks, an 8% increase had 89% sensitivity and specificity. In responders, MCV remained lower for individuals in African compared to non-African sites throughout and rose from 85 vs 90 fL at cART start to 96 vs 103 fL at 12 weeks post-initiation then fell to 88 vs 93 fL and 86 vs 89 fL at 12 and 48 weeks post-cessation. Conclusion: In low-income countries, where HIV RNA may be unavailable, 12-weekly MCV measurements may be useful in monitoring adherence to AZT-containing regimens. PMID:22715353

  13. A transient three-plasmid expression system for the production of high titer retroviral vectors.

    PubMed

    Soneoka, Y; Cannon, P M; Ramsdale, E E; Griffiths, J C; Romano, G; Kingsman, S M; Kingsman, A J

    1995-02-25

    We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants. PMID:7899083

  14. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  15. Regional Anthropometry Changes in Antiretroviral-Naïve Persons Initiating a Zidovudine-Containing Regimen in Mbarara, Uganda

    PubMed Central

    Thompson, Vanessa; Medard, Bitekyerezo; Taseera, Kabanda; Chakera, Ali J.; Andia, Irene; Emenyonu, Nneka; Hunt, Peter W.; Martin, Jeffrey; Scherzer, Rebecca; Weiser, Sheri D.; Bangsberg, David R.

    2011-01-01

    Abstract Lipodystrophy is commonly reported in Africa after antiretroviral therapy (ART) is initiated, but few studies have objectively measured changes in body composition. Body composition was determined in 76 HIV-infected participants from Mbarara, Uganda after starting a thymidine-analog regimen, and annual change was determined using repeated measures analysis. We measured skinfolds (tricep, thigh, subscapular, and abdomen), circumferences (arm, hip, thigh, waist), and total lean and fat mass (using bioelectric impedance analysis). A cross-sectional sample of 49 HIV-uninfected participants was studied for comparison. At baseline, most body composition measures were lower in HIV-infected than uninfected participants, but waist circumference was similar. After 12 months on ART, there was little difference in body composition measures between HIV-infected and uninfected participants; median waist circumference appeared higher in HIV-infected participants (79 vs. 75 cm; p = 0.090). Among HIV-infected participants, increases were observed in total lean and fat mass, circumference, and skinfold measures; only the increase in tricep skinfold did not reach statistical significance (+1.05 mm; 95% confidence interval: −0.24, 2.34; p = 0.11). Regional anthropometry in peripheral and central body sites increased over 12 months after ART initiation in HIV-infected persons from southwestern Uganda, suggesting a restoration to health. Gains in the tricep skinfold, a reliable marker of subcutaneous fat, appeared blunted, which could indicate an inhibitory effect of zidovudine on peripheral subcutaneous fat recovery. PMID:21128866

  16. Foscarnet, zidovudine and dolutegravir combination efficacy and tolerability for late stage HIV salvage therapy: A case-series experience.

    PubMed

    Delory, Tristan; Papot, Emmanuelle; Rioux, Christophe; Charpentier, Charlotte; Auge-Courtoi, Claire; Michard, Florence; Peytavin, Gilles; Descamps, Diane; Matheron, Sophie; Yazdanpanah, Yazdan

    2016-07-01

    Salvage therapy including foscarnet (PFA), zidovudine (ZDV) and an optimized background ART (OBT) has been shown to be effective in patients with advanced HIV infection, and no therapeutic options. Dolutegravir (DTG) may offer a more active combination. Objective was to describe efficacy and tolerability of PFA-ZDV-DTG containing regimen. In our cohort, we identified patients who: (i) had plasma HIV-1 RNA load (pVL) >50 c/ml (>100 for HIV-2) on combination ART (cART); (ii) had at least 1 PI/r, 1 NRTI, 1 NNRTI (for HIV-1), and at least 1 raltegravir resistance mutations; (iii) were naive to DTG; and (iv) initiated on a PFA-ZDV-DTG containing-regimen with 48 weeks (W48) of follow-up. Out of 5 patients, 2 were infected with HIV-2. At PFA-ZDV-DTG initiation, CD4 cell count was (/mm(3) ) of 64, 40, 10, in HIV-1, and 37, 199, in HIV-2 infected patients; and pVL (log10 c/ml) of 4.8, 5.1, 4.4, in HIV-1, and 3.6, 4.2, in HIV-2 infected patients, respectively. Median OBT genotypic sensitivity score was 1.5 [1-2]. PFA was discontinued in one patient, due to an acute renal failure. At W48, one HIV-1 infected patient had a pVL <50 c/ml and two <200 c/ml; the two HIV-2 infected patients had pVL >100 c/ml. No lack of treatment adherence was observed. In treatment experienced HIV-infected patients, failing cART and without other therapeutic options, a PFA-ZDV-DTG combination therapy could be effective. Renal adverse events should be monitored. J. Med. Virol. 88:1204-1210, 2016. © 2015 Wiley Periodicals, Inc. PMID:26636432

  17. Zidovudine-induced nail hyper-pigmentation in 45-year-old women prescribed for HIV/tuberculosis co-infection

    PubMed Central

    Tandon, Vishal R.; Sadiq, Shamiya; Khajuria, Vijay; Mahajan, Annil; Sharma, Sudhaa; Gillani, Zahid

    2016-01-01

    Zidovudine is an important component of first-line antiretroviral treatment regimens used to manage HIV and tuberculosis (TB) co-infection. Nail pigmentation is documented both in adult as well as pediatric HIV patients, but to the best of our knowledge, it has not been reported in 45-year-old women of HIV/TB co-infection. Such an adverse drugs reactions (ADR), although is harmless and reversible, psychological aspects of such ADR may be immense to the extent that it can negatively affect the compliance and result in therapeutic failure. Thus, it is worth reporting. PMID:27134481

  18. Evidence for integration of retroviral vectors in a novel human repeat sequence

    SciTech Connect

    Kurdi-Haidar, B.; Friedmann, T.

    1994-09-01

    Retroviruses have become attractive vehicles for the introduction of foreign genes into mammalian cells not only for gene therapy but also to serve as anchor points for long-range mapping purposes. The information relating to retroviral integration in mammalian cells is derived mostly from studies of rodent genomes. The absence of information regarding integration sites of murine-based retroviral vectors in human cells has prompted us to investigate the characteristics of integration sites in the human genome. We have constructed a Moloney murine leukemia virus-based retroviral vector that carries the pUC8 origin of replication and the chloramphenicol resistance gene to allow the rescue of the flanking genomic sequences in plasmid form. We have infected human primary fibroblasts and myoblasts with this retroviral vector and isolated independently transduced clones. Genomic DNA was obtained from independent clones and the genomic fragment carrying the provirus-host sequence boundary was isolated after digestion of the genomic DNA, circularization, and transformation by electroporation of E. coli C cells to chloramphenicol resistance. Restriction map and nucleotide sequence analysis of the rescued plasmids showed that a number of the clones shared the same integration site within the human genome. We have used the nucleotide sequence information about the human DNA adjacent to the 3{prime}LTR to design a PCR-based assay diagnostic for this common integration site. Analysis revealed the presence of the same integration site in four out of twelve human primary fibroblast clones infected with this specific retroviral vector, and in one out of twelve human primary myoblast clones infected with a second retroviral vector. Further analysis revealed the common integration site to be a previously unreported primate repeat present in monkey and human genomes and absent from rodent, bovine and avian genomes.

  19. Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia

    PubMed Central

    Karlsson, Håkan; Bachmann, Silke; Schröder, Johannes; McArthur, Justin; Torrey, E. Fuller; Yolken, Robert H.

    2001-01-01

    Schizophrenia is a serious brain disease of uncertain etiology. A role for retroviruses in the etiopathogenesis of some cases of schizophrenia has been postulated on the basis of clinical and epidemiological observations. We found sequences homologous to retroviral pol genes in the cell-free cerebrospinal fluids (CSFs) of 10 of 35 (29%) individuals with recent-onset schizophrenia or schizoaffective disorder. Retroviral sequences also were identified in the CSFs of 1 of 20 individuals with chronic schizophrenia. However, retroviral sequences were not identified in any of the CSFs obtained from 22 individuals with noninflammatory neurological diseases or from 30 individuals without evidence of neurological or psychiatric diseases (χ2 = 19.25, P < 0.001). The nucleotide sequences identified in the CSFs of the individuals with schizophrenia or schizoaffective disorder were related to those of the human endogenous retroviral (HERV)-W family of endogenous retroviruses and to other retroviruses in the murine leukemia virus genus. Transcription of RNA homologous to members of the HERV-W family of retroviruses also was found to be up-regulated differentially in the frontal cortex regions of brains obtained postmortem from individuals with schizophrenia, as compared with corresponding tissue from individuals without psychiatric diseases. The transcriptional activation of certain retroviral elements within the central nervous system may be associated with the development of schizophrenia in at least some individuals. The further characterization of retroviral elements within the central nervous system of individuals with schizophrenia might lead to improved methods for the diagnosis and management of this disorder. PMID:11296294

  20. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    PubMed Central

    2011-01-01

    Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy. PMID:21970612

  1. Quantum chemical modelling of reactivity and selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers

    NASA Astrophysics Data System (ADS)

    Topol, Igor A.; Nemukhin, Alexander V.; Burt, Stanley K.

    Interactions of 1,2-dithiolane species with zinc-containing sites, which mimic the zinc finger domains of retroviral and the cellular zinc finger proteins, have been investigated by quantum chemistry tools. According to the calculations, the immediate domains of zinc binding sites in the cellular and retroviral zinc fingers interact differently with such agents of the disulphide family. Thus, when approaching the model cellular-type domains, the molecules of 1,2-dithiolanes experience considerable potential barriers along the reaction path. However, these species react practically barrier-less with the model retroviral-type domains at the correlated DFT level. The results of the quantum chemical modelling provide firm support to the selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers. This can be of great practical importance for the design of therapeutics that accomplish functional inactivation of the zinc fingers of the human immunodeficiency virus (HIV-1) retroviral type nucleocapsid protein NCp7.

  2. Formulation and optimization of coated PLGA – Zidovudine nanoparticles using factorial design and in vitro in vivo evaluations to determine brain targeting efficiency

    PubMed Central

    Peter Christoper, G.V.; Vijaya Raghavan, C.; Siddharth, K.; Siva Selva Kumar, M.; Hari Prasad, R.

    2013-01-01

    In the current study zidovudine loaded PLGA nanoparticles were prepared, coated and further investigated for its effectiveness in brain targeting. IR and DSC studies were performed to determine the interaction between excipients used and to find out the nature of drug in the formulation. Formulations were prepared by adopting 23 factorial designs to evaluate the effects of process and formulation variables. The prepared formulations were subjected for in vitro and in vivo evaluations. In vitro evaluations showed particle size below 100 nm, entrapment efficiency of formulations ranges of 28–57%, process yield of 60–76% was achieved and drug release for the formulations were in the range of 50–85%. The drug release from the formulations was found to follow Higuchi release pattern, n–value obtained after Korsemeyer plot was in the range of 0.56–0.78. In vivo evaluations were performed in mice after intraperitoneal administration of zidovudine drug solution, uncoated and coated formulation. Formulation when coated with Tween 80 achieved a higher concentration in the brain than that of the drug in solution and of the uncoated formulation. Stability studies indicated that there was no degradation of the drug in the formulation after 90 days of preparation when stored in refrigerated condition. PMID:24648825

  3. Characterization of thermal and rheological properties of zidovudine, lamivudine and plasticizer blends with ethyl cellulose to assess their suitability for hot melt extrusion.

    PubMed

    Maru, Shital M; de Matas, Marcel; Kelly, Adrian; Paradkar, Anant

    2011-11-20

    The influence of antiretroviral drugs and plasticizers on the rheological and thermal characteristics of ethyl cellulose formulations intended for hot melt extrusion has been investigated. Antiretroviral drugs used were zidovudine and lamivudine, whilst plasticizers included triethylcitrate (TEC) and polyethylene glycol (PEG-6000). Physical mixtures containing ethyl cellulose with varying concentrations of drugs and plasticizers were characterized using differential scanning calorimetry (DSC) and parallel plate oscillatory rheometry. The viscosity of physical mixtures containing both drugs was lower than observed for pure ethyl cellulose, indicating that the drugs had a plasticizing effect. This was confirmed by lowering of the glass transition temperature (Tg) of ethyl cellulose. At the highest loading of 40% by weight, lamivudine appeared to become saturated within the polymer, causing an increase in viscosity and showing evidence of recrystallization upon cooling. Both TEC and PEG-6000 were found to lower the Tg of ethyl cellulose, although PEG-6000 recrystallized upon cooling which makes it unsuitable for use in the proposed controlled release formulations. Both plasticizers were also shown to reduce the viscosity of ethyl cellulose, more significantly so for TEC. The results indicate that ethyl cellulose formulations containing up to 40% by weight of zidovudine, not more than 30% by weight of lamivudine, with 5-10% by weight of TEC as the plasticizer are suitable for processing by hot melt extrusion. PMID:21925600

  4. Cancellers - Exploring the Possibility of Receptor Decoy Traps As a Superior Anti-Retroviral Strategy.

    PubMed

    Jeremiah, Sundararaj Stanley; Ohba, Kenji; Yamamoto, Naoki

    2016-01-01

    The global Human Immunodeficiency Virus (HIV) pandemic is still spreading due to the lack of ideal anti-retroviral measures and their availability. Till date, all attempts to produce an efficient vaccine have ended with unsatisfactory results. The highly active anti-retroviral therapy (HAART) is the only effective weapon currently available and is widely being used for curtailing the HIV pandemic. However, the HAART is also expected to fail in the near future due to the emergence and dissemination of antiviral resistance. This review sheds light on the reasons for the failure of the conventional anti-viral measures against HIV and the novel anti-retroviral strategies currently being developed. The various principles to be considered for the success of a novel anti-retroviral strategy are elaborately emphasized and an innovative concept is proposed on these lines. The proposed concept intends to use receptor decoy traps (RDT) called cancellers which are erythrocytes expressing the HIV entry receptors on their surface. If successfully developed, the cancellers would be capable of active targeting of the free HIV particles leading to the trapping of the viruses within the canceller, resulting in the neutralization of infectivity of the trapped virus. The possible ways of translating this concept into reality and the probable hurdles that can be encountered in the process are subsequently discussed. Also, the scope of cancellers in therapeutic and/or preventive strategies against HIV infection is envisaged upon their successful development. PMID:25882216

  5. Use of intron-disrupted polyadenylation sites to enhance expression and safety of retroviral vectors.

    PubMed

    Ismail, S I; Rohll, J B; Kingsman, S M; Kingsman, A J; Uden, M

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256-5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy. PMID:11119589

  6. Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors

    PubMed Central

    Ismail, Said I.; Rohll, Jonathan B.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy. PMID:11119589

  7. XPB mediated retroviral cDNA degradation coincides with entry to the nucleus

    SciTech Connect

    Yoder, Kristine E.; Roddick, William; Hoellerbauer, Pia; Fishel, Richard

    2011-02-20

    Retroviruses must integrate their cDNA to a host chromosome, but a significant fraction of retroviral cDNA is degraded before integration. XPB and XPD are part of the TFIIH complex which mediates basal transcription and DNA nucleotide excision repair. Retroviral infection increases when XPB or XPD are mutant. Here we show that inhibition of mRNA or protein synthesis does not affect HIV cDNA accumulation suggesting that TFIIH transcription activity is not required for degradation. Other host factors implicated in the stability of cDNA are not components of the XPB and XPD degradation pathway. Although an increase of retroviral cDNA in XPB or XPD mutant cells correlates with an increase of integrated provirus, the integration efficiency of pre-integration complexes is unaffected. Finally, HIV and MMLV cDNA degradation appears to coincide with nuclear import. These results suggest that TFIIH mediated cDNA degradation is a nuclear host defense against retroviral infection.

  8. Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In thi...

  9. Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes

    PubMed Central

    Bénit, Laurence; Dessen, Philippe; Heidmann, Thierry

    2001-01-01

    Phylogenetic analyses of retroviral elements, including endogenous retroviruses, have relied essentially on the retroviral pol gene expressing the highly conserved reverse transcriptase. This enzyme is essential for the life cycle of all retroid elements, but other genes are also endowed with conserved essential functions. Among them, the transmembrane (TM) subunit of the envelope gene is involved in virus entry through membrane fusion. It has also been reported to contain a domain, named the immunosuppressive domain, that has immunosuppressive properties most probably essential for virus spread within the host. This domain is conserved among a large series of retroviral elements, and we have therefore attempted to generate phylogenetic links between retroviral elements identified from databases following tentative alignments of the immunosuppressive domain and adjacent sequences. This allowed us to unravel a conserved organization among TM domains, also found in the Ebola and Marburg filoviruses, and to identify a large number of human endogenous retroviruses (HERVs) from sequence databases. The latter elements are part of previously identified families of HERVs, and some of them define new families. A general phylogenetic analysis based on the TM proteins of retroelements, and including those with no clearly identified immunosuppressive domain, could then be derived and compared with pol-based phylogenetic trees, providing a comprehensive survey of retroelements and definitive evidence for recombination events in the generation of both the endogenous and the present-day infectious retroviruses. PMID:11689652

  10. Genome adaptations of a tripartite motif protein for retroviral defense in cattle and sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tripartite motif (TRIM) genes encode proteins composed of RING, B-box, and coiled coil motif domains. Primate TRIM5' has been shown to be a primary determinant of retroviral host cell range restriction in primates. TRIM5 restriction was originally thought to be a primate-specific defense mechanism...

  11. Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging

    PubMed Central

    Johansson, Fredrik K.; Brodd, Josefin; Eklöf, Charlotta; Ferletta, Maria; Hesselager, Göran; Tiger, Carl-Fredrik; Uhrbom, Lene; Westermark, Bengt

    2004-01-01

    Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors. PMID:15273287

  12. Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging.

    PubMed

    Johansson, Fredrik K; Brodd, Josefin; Eklöf, Charlotta; Ferletta, Maria; Hesselager, Göran; Tiger, Carl-Fredrik; Uhrbom, Lene; Westermark, Bengt

    2004-08-01

    Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors. PMID:15273287

  13. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    PubMed Central

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  14. High-resolution structure of a retroviral protease folded as a monomer

    SciTech Connect

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-11-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C{sup α} deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  15. Rifabutin

    MedlinePlus

    ... warfarin (Coumadin), blood pressure or heart disease medication, diabetes medications, digoxin (Lanoxin), methadone, oral contraceptives, zidovudine (Retrovir), and vitamins. Rifabutin decreases the effectiveness of some oral contraceptives; ...

  16. [Highly Active AntiRetroviral Therapy and opportunistic protozoan infections].

    PubMed

    Pozio, E

    2004-06-01

    Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern

  17. Lamivudine and Zidovudine

    MedlinePlus

    ... Talk to your pharmacist or contact your local garbage/recycling department to learn about take-back programs in your community. See the FDA's Safe Disposal of Medicines website (http://goo.gl/c4Rm4p) for ...

  18. Lamivudine and Zidovudine

    MedlinePlus

    ... are in a class of medications called nucleoside reverse transcriptase inhibitors (NRTIs). They work by decreasing the ... staying asleep excessive tiredness depression stuffy nose cough hair loss Some side effects can be serious. The following ...

  19. Response to antiretroviral therapy (ART): comparing women with previous use of zidovudine monotherapy (ZDVm) in pregnancy with ART naïve women

    PubMed Central

    2014-01-01

    Background Short-term zidovudine monotherapy (ZDVm) remains an option for some pregnant HIV-positive women not requiring treatment for their own health but may affect treatment responses once antiretroviral therapy (ART) is subsequently started. Methods Data were obtained by linking two UK studies: the UK Collaborative HIV Cohort (UK CHIC) study and the National Study of HIV in Pregnancy and Childhood (NSHPC). Treatment responses were assessed for 2028 women initiating ART at least one year after HIV-diagnosis. Outcomes were compared using logistic regression, proportional hazards regression or linear regression. Results In adjusted analyses, ART-naïve (n = 1937) and ZDVm-experienced (n = 91) women had similar increases in CD4 count and a similar proportion achieving virological suppression; both groups had a low risk of AIDS. Conclusions In this setting, antenatal ZDVm exposure did not adversely impact on outcomes once ART was initiated for the woman’s health. PMID:24593018

  20. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  1. STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

    PubMed

    Obal, G; Trajtenberg, F; Carrión, F; Tomé, L; Larrieux, N; Zhang, X; Pritsch, O; Buschiazzo, A

    2015-07-01

    Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology. PMID:26044299

  2. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

    SciTech Connect

    Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine . E-mail: christine.hohenadl@vu-wien.ac.at

    2006-05-25

    To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.

  3. Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

    PubMed Central

    Spitzer, Dirk; Dittmar, Kurt E. J.; Rohde, Manfred; Hauser, Hansjörg; Wirth, Dagmar

    2003-01-01

    Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. PMID:12719600

  4. Expression of human. alpha. sub 1 -antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes

    SciTech Connect

    Kay, M.A.; Baley, P.; Rothenberg, S.; Leland, F; Fleming, L.; Ponder, K.P.; Liu, Tajen; Finegold, M.; Darlington, G.; Pokorny, W.; Woo, S.L.C. )

    1992-01-01

    The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. The authors have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here they demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. In two animals they have transplanted hepatocytes transduced with a retroviral vector containing the human {alpha}{sub 1}-antitrypsin cDNA under transcriptional control of the cytomegalovirus promotor. Both animals had significant human {alpha}{sub 1}-antitrypsin in the serum for 1 month. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstruction with the use of promoters of cellular genes that are active in the normal liver.

  5. Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

    PubMed

    Heider, Susanne; Kleinberger, Sandra; Kochan, Feliks; Dangerfield, John A; Metzner, Christoph

    2016-07-01

    Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. PMID:27170144

  6. Domesticated retroviral GAG gene in Drosophila: new functions for an old gene.

    PubMed

    Nefedova, L N; Kuzmin, I V; Makhnovskii, P A; Kim, A I

    2014-02-01

    The domestication of foreign genes is a powerful mechanism for new gene formation and genome evolution. It is known that domesticated retroviral gag genes in mammals not only take part in protecting against viral infection but also control cell division, apoptosis, function of the placenta, and other biological processes. In this study, we focused on the domesticated retroviral gag gene homolog (Grp) in the Drosophila melanogaster genome. According to the results of a bioinformatic analysis, the Grp gene product is primarily under purifying selection in Drosophilidae family. The Grp protein has been shown to be transmembrane. The Grp gene is expressed at the adult stage of D. melanogaster in gender-specific and tissue-specific manner. Also the Grp gene expression is increased in response to the gypsy retrovirus. A function of the protein as a component of the endosomic membrane is considered. PMID:24503082

  7. Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T.

    PubMed Central

    Burns, J. S.; Lemoine, L.; Lemoine, N. R.; Williams, E. D.; Wynford-Thomas, D.

    1989-01-01

    A recombinant murine retroviral vector encoding the SV40 virus large T antigen was used to infect stably an immortal line of differentiated rat thyroid epithelial cells, FRTL-5. Expression of SV40 T transformed these cells to anchorage independence and tumorigenicity but did not alter morphology or abolish tissue-specific functions and growth factor requirements. The resulting phenotype provides a model of well-differentiated human thyroid cancer. Images Figure 1 Figure 3 PMID:2544221

  8. Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials

    PubMed Central

    Cornelis, Guillaume; Vernochet, Cécile; Carradec, Quentin; Souquere, Sylvie; Mulot, Baptiste; Catzeflis, François; Nilsson, Maria A.; Menzies, Brandon R.; Renfree, Marilyn B.; Pierron, Gérard; Zeller, Ulrich; Heidmann, Odile; Dupressoir, Anne; Heidmann, Thierry

    2015-01-01

    Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials—which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya—also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell–cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto–maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses—displaying strong expression in the uterine glands where retroviral particles can be detected—plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades. PMID:25605903

  9. Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

    PubMed

    Slingsby, J H; Baban, D; Sutton, J; Esapa, M; Price, T; Kingsman, S M; Kingsman, A J; Slade, A

    2000-07-01

    A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells. PMID:10910141

  10. Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials.

    PubMed

    Cornelis, Guillaume; Vernochet, Cécile; Carradec, Quentin; Souquere, Sylvie; Mulot, Baptiste; Catzeflis, François; Nilsson, Maria A; Menzies, Brandon R; Renfree, Marilyn B; Pierron, Gérard; Zeller, Ulrich; Heidmann, Odile; Dupressoir, Anne; Heidmann, Thierry

    2015-02-01

    Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials-which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya-also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell-cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto-maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses-displaying strong expression in the uterine glands where retroviral particles can be detected-plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades. PMID:25605903

  11. Crystal structure of a monomeric retroviral protease solved by protein folding game players.

    PubMed

    Khatib, Firas; DiMaio, Frank; Cooper, Seth; Kazmierczyk, Maciej; Gilski, Miroslaw; Krzywda, Szymon; Zabranska, Helena; Pichova, Iva; Thompson, James; Popović, Zoran; Jaskolski, Mariusz; Baker, David

    2011-10-01

    Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs. PMID:21926992

  12. PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx

    PubMed Central

    Portilho, Débora M.; Arhel, Nathalie J.; Chelbi-Alix, Mounira K.; Nisole, Sébastien

    2015-01-01

    PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx. PMID:26566030

  13. Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy

    PubMed Central

    Davé, Utpal P.; Akagi, Keiko; Tripathi, Rati; Cleveland, Susan M.; Thompson, Mary A.; Yi, Ming; Stephens, Robert; Downing, James R.; Jenkins, Nancy A.; Copeland, Neal G.

    2009-01-01

    Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy. PMID:19461887

  14. Cryo-EM reveals a novel octameric integrase structure for β-retroviral intasome function

    PubMed Central

    Ballandras-Colas, Allison; Brown, Monica; Cook, Nicola J.; Dewdney, Tamaria G.; Demeler, Borries; Cherepanov, Peter; Lyumkis, Dmitry; Engelman, Alan N.

    2016-01-01

    Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration. PMID:26887496

  15. TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling

    PubMed Central

    Lascano, Josefina; Uchil, Pradeep D.; Mothes, Walther

    2015-01-01

    ABSTRACT Host restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling. IMPORTANCE The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to

  16. Positive selection of Iris, a retroviral envelope-derived host gene in Drosophila melanogaster.

    PubMed

    Malik, Harmit S; Henikoff, Steven

    2005-10-01

    Eukaryotic genomes can usurp enzymatic functions encoded by mobile elements for their own use. A particularly interesting kind of acquisition involves the domestication of retroviral envelope genes, which confer infectious membrane-fusion ability to retroviruses. So far, these examples have been limited to vertebrate genomes, including primates where the domesticated envelope is under purifying selection to assist placental function. Here, we show that in Drosophila genomes, a previously unannotated gene (CG4715, renamed Iris) was domesticated from a novel, active Kanga lineage of insect retroviruses at least 25 million years ago, and has since been maintained as a host gene that is expressed in all adult tissues. Iris and the envelope genes from Kanga retroviruses are homologous to those found in insect baculoviruses and gypsy and roo insect retroviruses. Two separate envelope domestications from the Kanga and roo retroviruses have taken place, in fruit fly and mosquito genomes, respectively. Whereas retroviral envelopes are proteolytically cleaved into the ligand-interaction and membrane-fusion domains, Iris appears to lack this cleavage site. In the takahashii/suzukii species groups of Drosophila, we find that Iris has tandemly duplicated to give rise to two genes (Iris-A and Iris-B). Iris-B has significantly diverged from the Iris-A lineage, primarily because of the "invention" of an intron de novo in what was previously exonic sequence. Unlike domesticated retroviral envelope genes in mammals, we find that Iris has been subject to strong positive selection between Drosophila species. The rapid, adaptive evolution of Iris is sufficient to unambiguously distinguish the phylogenies of three closely related sibling species of Drosophila (D. simulans, D. sechellia, and D. mauritiana), a discriminative power previously described only for a putative "speciation gene." Iris represents the first instance of a retroviral envelope-derived host gene outside vertebrates

  17. TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction

    PubMed Central

    Imam, Sabrina; Talley, Sarah; Nelson, Rachel S.; Dharan, Adarsh; O'Connor, Christopher; Hope, Thomas J.

    2016-01-01

    ABSTRACT TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins. IMPORTANCE Restriction factors are a class of

  18. Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.

    PubMed

    Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel; Rivière, Isabelle

    2015-04-01

    The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice-grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks' yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

  19. A phase I trial of retroviral BRCA1sv gene therapy in ovarian cancer.

    PubMed

    Tait, D L; Obermiller, P S; Redlin-Frazier, S; Jensen, R A; Welcsh, P; Dann, J; King, M C; Johnson, D H; Holt, J T

    1997-11-01

    Gene transfer of BRCA1sv (a normal splice variant of BRCA1) into ovarian cancer cells produces growth inhibition in vitro and tumor suppression in nude mouse xenografts. As an initial step toward gene replacement therapy for ovarian cancer, we conducted a Phase I trial to assess the pharmacokinetics and toxicity of i.p. BRCA1sv retroviral vector therapy. Following placement of an indwelling Port-a-Cath in patients, a dose escalation study was performed of four daily i.p. infusions spanning doses from 3 to 300 ml (i.e., 10(10) viral particles) at half-log intervals (23 cycles in 12 patients). Gene transfer and expression were documented by PCR, Southern blot, reverse transcription-PCR, and nuclease protection assays. Pharmacokinetics were assessed by PCR and Southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of 12 patients developed an acute sterile peritonitis, which spontaneously resolved within 48 h. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to 4 months. Eight patients showed stable disease for 4-16 weeks, and three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). The vector-related complication of peritonitis was observed in three patients but resolved quickly as in preclinical mouse studies. Ovarian cancer may provide an important model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by i.p. therapy. PMID:9815585

  20. VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer

    PubMed Central

    2013-01-01

    Background The function of the non-coding portion of the human genome remains one of the most important questions of our time. Its vast complexity is exemplified by the recent identification of an unusual and notable component of the transcriptome - very long intergenic non-coding RNAs, termed vlincRNAs. Results Here we identify 2,147 vlincRNAs covering 10 percent of our genome. We show they are present not only in cancerous cells, but also in primary cells and normal human tissues, and are controlled by canonical promoters. Furthermore, vlincRNA promoters frequently originate from within endogenous retroviral sequences. Strikingly, the number of vlincRNAs expressed from endogenous retroviral promoters strongly correlates with pluripotency or the degree of malignant transformation. These results suggest a previously unknown connection between the pluripotent state and cancer via retroviral repeat-driven expression of vlincRNAs. Finally, we show that vlincRNAs can be syntenically conserved in humans and mouse and their depletion using RNAi can cause apoptosis in cancerous cells. Conclusions These intriguing observations suggest that vlincRNAs could create a framework that combines many existing short ESTs and lincRNAs into a landscape of very long transcripts functioning in the regulation of gene expression in the nucleus. Certain types of vlincRNAs participate at specific stages of normal development and, based on analysis of a limited set of cancerous and primary cell lines, they appear to be co-opted by cancer-associated transcriptional programs. This provides additional understanding of transcriptome regulation during the malignant state, and could lead to additional targets and options for its reversal. PMID:23876380

  1. Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

    PubMed Central

    Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel

    2015-01-01

    The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

  2. High-resolution structure of a retroviral protease folded as a monomer.

    PubMed

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-11-01

    Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS. PMID:22101816

  3. Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection

    SciTech Connect

    Smith, L.J.; Benchimol, S.

    1988-02-01

    An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.

  4. Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.

    PubMed

    Coren, Lori V; Jain, Sumiti; Trivett, Matthew T; Ohlen, Claes; Ott, David E

    2015-03-01

    Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins. PMID:25757546

  5. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    PubMed

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  6. Evaluation of in-vitro cytotoxicity and cellular uptake efficiency of zidovudine-loaded solid lipid nanoparticles modified with Aloe Vera in glioma cells.

    PubMed

    K S, Joshy; Sharma, Chandra P; Kalarikkal, Nandakumar; Sandeep, K; Thomas, Sabu; Pothen, Laly A

    2016-09-01

    Zidovudine loaded solid lipid nanoparticles of stearic acid modified with Aloe Vera (AV) have been prepared via simple emulsion solvent evaporation method which showed excellent stability at room temperature and refrigerated condition. The nanoparticles were examined by Fourier transform infrared spectroscopy (FT-IR), which revealed the overlap of the AV absorption peak with the absorption peak of modified stearic acid nanoparticles. The inclusion of AV to stearic acid decreased the crystallinity and improved the hydrophilicity of lipid nanoparticles and thereby improved the drug loading efficacy of lipid nanoparticles. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging revealed that, the average particle size of unmodified (bare) nanoparticles was 45.66±12.22nm and modified solid lipid nanoparticles showed an average size of 265.61±80.44nm. Solid lipid nanoparticles with well-defined morphology were tested in vitro for their possible application in drug delivery. Cell culture studies using C6 glioma cells on the nanoparticles showed enhanced growth and proliferation of cells without exhibiting any toxicity. In addition, normal cell morphology and improved uptake were observed by fluorescence microscopy images of rhodamine labeled modified solid lipid nanoparticles compared with unmodified nanoparticles. The cellular uptake study suggested that these nanoparticles could be a promising drug delivery system to enhance the uptake of antiviral drug by brain cells and it could be a suitable drug carrier system for the treatment of HIV. PMID:27207037

  7. Evaluation of zidovudine encapsulated ethylcellulose microspheres prepared by water-in-oil-in-oil (w/o/o) double emulsion solvent diffusion technique.

    PubMed

    Das, Malay Kumar; Rao, Kalakuntala Rama

    2006-01-01

    The preparation of zidovudine-loaded ethylcellulose microspheres by w/o/o double emulsion solvent diffusion method with high entrapment capacity and sustained release is described. A mixed solvent system (MSS) consisting of acetonitrile and dichloromethane in a 1:1 ratio and light liquid paraffin was selected as primary and secondary oil phases, respectively. Span 80 was used as the secondary surfactant for stabilizing the external oil phase. Spherical free flowing microspheres were obtained. The prepared microspheres were characterized by entrapment efficiency, in vitro release behavior, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The drug-loaded microspheres showed 32 - 55% entrapment capacity. The in vitro release profile could be altered significantly by changing various processing and formulation parameters to give sustained release of drug from the microspheres. The DSC thermograms confirmed the absence of any drug-polymer interaction. SEM studies showed that the microspheres were spherical and porous in nature. The in vitro release profiles from microspheres of different polymer-drug ratios were best fitted to Higuchi model with high correlation coefficient and the n value obtained from Korsmeyer-Peppas model was ranged between 0.23 - 0.54. The drug release was found to be diffusion controlled mechanism. PMID:17514878

  8. 3'-end processing and kinetics of 5'-end joining during retroviral integration in vivo.

    PubMed Central

    Roe, T; Chow, S A; Brown, P O

    1997-01-01

    Retroviral replication depends on integration of viral DNA into a host cell chromosome. Integration proceeds in three steps: 3'-end processing, the endonucleolytic removal of the two terminal nucleotides from each 3' end of the viral DNA; strand transfer, the joining of the 3' ends of viral DNA to host DNA; and 5'-end joining (or gap repair), the joining of the 5' ends of viral DNA to host DNA. The 5'-end joining step has never been investigated, either for retroviral integration or for any other transposition process. We have developed an assay for 5'-end joining in vivo and have examined the kinetics of 5'-end joining for Moloney murine leukemia virus (MLV). The interval between 3'-end and 5'-end joining is estimated to be less than 1 h. This assay will be a useful tool for examining whether viral or host components mediate 5'-end joining. MLV integrates its DNA only after its host cell has completed mitosis. We show that the extent of 3'-end processing is the same in unsynchronized and aphidicolin-arrested cells. 3'-end processing therefore does not depend on mitosis. PMID:8995657

  9. Retroviral Retention Activates a Syk-Dependent HemITAM in Human Tetherin

    PubMed Central

    Galão, Rui Pedro; Pickering, Suzanne; Curnock, Rachel; Neil, Stuart J.D.

    2014-01-01

    Summary Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosine kinase (Syk), which is required for downstream NF-κB activation, indicating that the tetherin cytoplasmic tail resembles the hemi-immunoreceptor tyrosine-based activation motifs (hemITAMs) found in C-type lectin pattern recognition receptors. Retroviral-induced tetherin signaling is coupled to the cortical actin cytoskeleton via the Rac-GAP-containing protein RICH2 (ARHGAP44), and a naturally occurring tetherin polymorphism with reduced RICH2 binding exhibits decreased phosphorylation and NF-κB activation. Thus, upon virion retention, this linkage to the actin cytoskeleton likely triggers tetherin phosphorylation and subsequent signal transduction to induce an antiviral state. PMID:25211072

  10. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta

    PubMed Central

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has “captured” a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  11. The Development of Metabolic Risk Factors After the Initiation of the Second Line Anti- Retroviral Therapy

    PubMed Central

    Mittal, Apoorva; Achappa, Basavaprabhu; Madi, Deepak; Chowta, Mukta N; Ramapuram, John T; Rao, Satish; Unnikrishnan, B; Mahalingam, Soundarya

    2013-01-01

    Background and objective: A Highly Active Anti-Retroviral Therapy (HAART) is accompanied with several metabolic effects like adipose redistribution and insulin resistance. In this study, we evaluated the association between a HAART and lipodystrophy. Methods: A cross sectional study, whose subjects were Human Immunodeficiency Virus (HIV) infected patients, was conducted at a tertiary care hospital in south India. Among these, 27 were on protease inhibitors for at-least 6 months and 13 were drug naive patients. The assessments of lipodystrophy, fasting blood sugar and the fasting lipid profile were done and these parameters were compared in the two groups. Results: The analysis of the data which was collected, showed an elevation in the total cholesterol levels in the individuals who were on the protease inhibitors versus the drug naive patients. There was a significant elevation in the Low Density Lipoprotein (LDL) cholesterol levels and a decrease in High Density Lipoprotein (HDL) cholesterol levels in the individuals who were on protease inhibitors. It was also observed that the HDL cholesterol levels decreased with an increase in the duration of the therapy. The LDL cholesterol levels increased with the duration of the therapy. Conclusion: The human immunodeficiency virus infection is itself related to the metabolic complications which are aggravated on the use of second line anti retroviral therapy. Therefore, after initiating the treatment with protease inhibitors, a periodic evaluation of the serum lipid levels and the blood sugar profile should be done as a standard care. PMID:23542168

  12. Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

    PubMed Central

    Van Beveren, C; Rands, E; Chattopadhyay, S K; Lowy, D R; Verma, I M

    1982-01-01

    The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed. Images PMID:6281466

  13. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

    PubMed

    Jones, Nathan D; Lopez, Miguel A; Hanne, Jeungphill; Peake, Mitchell B; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  14. Passive Immunotherapy for Retroviral Disease: Influence of Major Histocompatibility Complex Type and T-Cell Responsiveness

    NASA Astrophysics Data System (ADS)

    Hasenkrug, Kim J.; Brooks, Diane M.; Chesebro, Bruce

    1995-11-01

    Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4^+ and CD8^+ T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.

  15. Endogenous non-retroviral RNA virus elements evidence a novel type of antiviral immunity.

    PubMed

    Honda, Tomoyuki; Tomonaga, Keizo

    2016-01-01

    Vertebrate genomes contain many virus-related sequences derived from both retroviruses and non-retroviral RNA and DNA viruses. Such non-retroviral RNA sequences are possibly produced by reverse-transcription and integration of viral mRNAs of ancient RNA viruses using retrotransposon machineries. We refer to this process as transcript reversion. During an ancient bornavirus infection, transcript reversion may have left bornavirus-related sequences, known as endogenous bornavirus-like nucleoproteins (EBLNs), in the genome. We have recently demonstrated that all Homo sapiens EBLNs are expressed in at least one tissue. Because species with EBLNs appear relatively protected against infection by a current bornavirus, Borna disease virus, it is speculated that EBLNs play some roles in antiviral immunity, as seen with some endogenous retroviruses. EBLNs can function as dominant negative forms of viral proteins, small RNAs targeting viral sequences, or DNA or RNA elements modulating the gene expression. Growing evidence reveals that various RNA viruses are reverse-transcribed and integrated into the genome of infected cells, suggesting transcript reversion generally occurs during ongoing infection. Considering this, transcript reversion-mediated interference with related viruses may be a novel type of antiviral immunity in vertebrates. Understanding the biological significance of transcript reversion will provide novel insights into host defenses against viral infections. PMID:27510928

  16. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    NASA Astrophysics Data System (ADS)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  17. Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification

    SciTech Connect

    Greenberg, S.J.; Ehrlich, G.D.; Abbott, M.A.; Hurwitz, B.J.; Waldmann, T.A.; Poiesz, B.J. )

    1989-04-01

    Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3{prime} gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.

  18. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    PubMed Central

    Jones, Nathan D.; Lopez Jr, Miguel A.; Hanne, Jeungphill; Peake, Mitchell B.; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E.

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3′-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3′-hydroxyls into the target DNA separated by 4–6 bp. Host DNA repair restores the resulting 5′-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  19. New insights into retroviral Gag–Gag and Gag–membrane interactions

    PubMed Central

    Maldonado, José O.; Martin, Jessica L.; Mueller, Joachim D.; Zhang, Wei; Mansky, Louis M.

    2014-01-01

    A critical aspect of viral replication is the assembly of virus particles, which are subsequently released as progeny virus. While a great deal of attention has been focused on better understanding this phase of the viral life cycle, many aspects of the molecular details remain poorly understood. This is certainly true for retroviruses, including that of the human immunodeficiency virus type 1 (HIV-1; a lentivirus) as well as for human T-cell leukemia virus type 1 (HTLV-1; a deltaretrovirus). This review discusses the retroviral Gag protein and its interactions with itself, the plasma membrane and the role of lipids in targeting Gag to virus assembly sites. Recent progress using sophisticated biophysical approaches to investigate – in a comparative manner – retroviral Gag–Gag and Gag–membrane interactions are discussed. Differences among retroviruses in Gag–Gag and Gag–membrane interactions imply dissimilar molecular aspects of the viral assembly pathway, including the interactions of Gag with lipids at the membrane. PMID:25009535

  20. Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells

    PubMed Central

    García-Hernández, B.; Castellanos, A.; López, A.; Orfao, A.; Sánchez-García, I.

    1997-01-01

    A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis. PMID:9371830

  1. Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

    PubMed Central

    Matsubara, T; Beeman, R W; Shike, H; Besansky, N J; Mukabayire, O; Higgs, S; James, A A; Burns, J C

    1996-01-01

    The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo. Images Fig. 2 Fig. 4 Fig. 5 PMID:8650240

  2. Improved self-inactivating retroviral vectors derived from spleen necrosis virus.

    PubMed Central

    Olson, P; Nelson, S; Dornburg, R

    1994-01-01

    Self-inactivating (SIN) retroviral vectors contain a deletion spanning most of the right long terminal repeat's (LTR's) U3 region. Reverse transcription copies this deletion to both LTRs. As a result, there is no transcription from the 5' LTR, preventing further replication. Many previously developed SIN vectors, however, had reduced titers or were genetically unstable. Earlier, we reported that certain SIN vectors derived from spleen necrosis virus (SNV) experienced reconstitution of the U3-deleted LTR at high frequencies. This reconstitution occurred on the DNA level and appeared to be dependent on defined vector sequences. To study this phenomenon in more detail, we developed an almost completely U3-free retroviral vector. The promoter and enhancer of the left LTR were replaced with those of the cytomegalovirus immediate-early genes. This promoter swap did not impair the level of transcription or alter its start site. Our data indicate that SNV contains a strong initiator which resembles that of human immunodeficiency virus. We show that the vectors replicate with efficiencies similar to those of vectors possessing two wild-type LTRs. U3-deleted vectors carrying the hygromycin B phosphotransferase gene did not observably undergo LTR reconstitution, even when replicated in helper cells containing SNV-LTR sequences. However, vectors carrying the neomycin resistance gene did undergo LTR reconstitution with the use of homologous helper cell LTR sequences as template. This supports our earlier finding that sequences within the neomycin resistance gene can trigger recombination. Images PMID:7933088

  3. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

    PubMed

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  4. IL-12-conditioning improves retrovirally-mediated transduction efficiency of CD8+ T cells

    PubMed Central

    Andrijauskaite, Kristina; Suriano, Samantha; Cloud, Colleen A.; Li, Mingli; Kesarwani, Pravin; Stefanik, Leah S.; Moxley, Kelly M.; Salem, Mohamed L; Garrett-Mayer, Elizabeth; Paulos, Chrystal M.; Mehrotra, Shikhar; Kochenderfer, James N.; Cole, David J.; Rubinstein, Mark P.

    2016-01-01

    The ability to genetically modify T cells is a critical component to many immunotherapeutic strategies and research studies. However, the success of these approaches is often limited by transduction efficiency. Since retroviral vectors require cell division for integration, transduction efficiency is dependent on the appropriate activation and culture conditions for T cells. Naïve CD8+ T cells which are quiescent must be first activated to induce cell division to allow genetic modification. To optimize this process, we activated mouse T cells with a panel of different cytokines, including IL-2, IL-4, IL-6, IL-7, IL-12, IL-15 and IL-23, known to act on T cells. After activation, cytokines were removed, and activated T cells were retrovirally transduced. We found that IL-12 pre-conditioning of mouse T cells greatly enhanced transduction efficiency while preserving function and expansion potential. We also observed a similar transduction enhancing effect of IL-12 pre-conditioning on human T cells. These findings provide a simple method to improve the transduction efficiencies of CD8+ T cells. PMID:26182912

  5. Endogenous retroviral long terminal repeats within the HLA-DQ locus.

    PubMed Central

    Kambhu, S; Falldorf, P; Lee, J S

    1990-01-01

    Two endogenous retroviral long terminal repeats (LTRs) were found in the human major histocompatibility complex locus HLA-DQ. The solo LTRs, unlinked to retrovirus structural genes, are located approximately 5 kilobases apart from each other and in the same transcriptional orientation, which is opposite to that for the HLA-DQB1 gene. These elements exhibit greater than 90% homology to the LTRs of the human endogenous retrovirus HERV-K10. The conservation of putative regulatory elements found within the LTRs and their position relative to the HLA-DQB1 gene suggest that these elements may confer distinct regulatory properties on genes in the HLA-DQ region. Polymorphic variation between different HLA haplotypes for the presence of the LTRs at this location and of the molecular architecture within this subregion is supported by polymerase chain reaction and Southern blot analysis. Comparisons of chromosomes with and without the LTRs in this region will provide a unique opportunity in the human genome to analyze transposition or integration of retroviral sequences. Images PMID:2114643

  6. Endogenous non-retroviral RNA virus elements evidence a novel type of antiviral immunity

    PubMed Central

    Honda, Tomoyuki; Tomonaga, Keizo

    2016-01-01

    ABSTRACT Vertebrate genomes contain many virus-related sequences derived from both retroviruses and non-retroviral RNA and DNA viruses. Such non-retroviral RNA sequences are possibly produced by reverse-transcription and integration of viral mRNAs of ancient RNA viruses using retrotransposon machineries. We refer to this process as transcript reversion. During an ancient bornavirus infection, transcript reversion may have left bornavirus-related sequences, known as endogenous bornavirus-like nucleoproteins (EBLNs), in the genome. We have recently demonstrated that all Homo sapiens EBLNs are expressed in at least one tissue. Because species with EBLNs appear relatively protected against infection by a current bornavirus, Borna disease virus, it is speculated that EBLNs play some roles in antiviral immunity, as seen with some endogenous retroviruses. EBLNs can function as dominant negative forms of viral proteins, small RNAs targeting viral sequences, or DNA or RNA elements modulating the gene expression. Growing evidence reveals that various RNA viruses are reverse-transcribed and integrated into the genome of infected cells, suggesting transcript reversion generally occurs during ongoing infection. Considering this, transcript reversion-mediated interference with related viruses may be a novel type of antiviral immunity in vertebrates. Understanding the biological significance of transcript reversion will provide novel insights into host defenses against viral infections. PMID:27510928

  7. Detection of retroviral super-infection from non-invasive samples.

    PubMed

    Goffe, Adeelia S; Blasse, Anja; Mundry, Roger; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

    2012-01-01

    While much attention has been focused on the molecular epidemiology of retroviruses in wild primate populations, the correlated question of the frequency and nature of super-infection events, i.e., the simultaneous infection of the same individual host with several strains of the same virus, has remained largely neglected. In particular, methods possibly allowing the investigation of super-infection from samples collected non-invasively (such as faeces) have never been properly compared. Here, we fill in this gap by assessing the costs and benefits of end-point dilution PCR (EPD-PCR) and multiple bulk-PCR cloning, as applied to a case study focusing on simian foamy virus super-infection in wild chimpanzees (Pan troglodytes). We show that, although considered to be the gold standard, EPD-PCR can lead to massive consumption of biological material when only low copy numbers of the target are expected. This constitutes a serious drawback in a field in which rarity of biological material is a fundamental constraint. In addition, we demonstrate that EPD-PCR results (single/multiple infection; founder strains) can be well predicted from multiple bulk-PCR clone experiments, by applying simple statistical and network analyses to sequence alignments. We therefore recommend the implementation of the latter method when the focus is put on retroviral super-infection and only low retroviral loads are encountered. PMID:22590569

  8. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

    PubMed Central

    Bertran, J; Miller, J L; Yang, Y; Fenimore-Justman, A; Rueda, F; Vanin, E F; Nienhuis, A W

    1996-01-01

    Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. PMID:8794313

  9. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    SciTech Connect

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. )

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  10. In vitro dissolution of generic immediate-release solid oral dosage forms containing BCS class I drugs: comparative assessment of metronidazole, zidovudine, and amoxicillin versus relevant comparator pharmaceutical products in South Africa and India.

    PubMed

    Reddy, Nallagundla H S; Patnala, Srinivas; Löbenberg, Raimar; Kanfer, Isadore

    2014-10-01

    Biowaivers are recommended for immediate-release solid oral dosage forms using dissolution testing as a surrogate for in vivo bioequivalence studies. Several guidance are currently available (the World Health Organization (WHO), the US FDA, and the EMEA) where the conditions are described. In this study, definitions, criteria, and methodologies according to the WHO have been applied. The dissolution performances of immediate-release metronidazole, zidovudine, and amoxicillin products purchased in South African and Indian markets were compared to the relevant comparator pharmaceutical product (CPP)/reference product. The dissolution performances were studied using US Pharmacopeia (USP) apparatus 2 (paddle) set at 75 rpm in each of three dissolution media (pH1.2, 4.5, and 6.8). Concentrations of metronidazole, zidovudine, and amoxicillin in each dissolution media were determined by HPLC. Of the 11 metronidazole products tested, only 8 could be considered as very rapidly dissolving products as defined by the WHO, whereas 2 of those products could be considered as rapidly dissolving products but did not comply with the f 2 acceptance criteria in pH 6.8. All 11 zidovudine products were very rapidly dissolving, whereas in the case of the 14 amoxicillin products tested, none of those products met any of the WHO criteria. This study indicates that not all generic products containing the same biopharmaceutics classification system (BCS) I drug and in similar strength and dosage form are necessarily in vitro equivalent. Hence, there is a need for ongoing market surveillance to determine whether marketed generic products containing BCS I drugs meet the release requirements to confirm their in vitro bioequivalence to the respective reference product. PMID:24848760

  11. Development of an optimized dose for coformulation of zidovudine with drugs that select for the K65R mutation using a population pharmacokinetic and enzyme kinetic simulation model.

    PubMed

    Hurwitz, Selwyn J; Asif, Ghazia; Kivel, Nancy M; Schinazi, Raymond F

    2008-12-01

    In vitro selection studies and data from large genotype databases from clinical studies have demonstrated that tenofovir disoproxil fumarate and abacavir sulfate select for the K65R mutation in the human immunodeficiency virus type 1 polymerase region. Furthermore, other novel non-thymine nucleoside reverse transcriptase (RT) inhibitors also select for this mutation in vitro. Studies performed in vitro and in humans suggest that viruses containing the K65R mutation remained susceptible to zidovudine (ZDV) and other thymine nucleoside antiretroviral agents. Therefore, ZDV could be coformulated with these agents as a "resistance repellent" agent for the K65R mutation. The approved ZDV oral dose is 300 mg twice a day (b.i.d.) and is commonly associated with bone marrow toxicity thought to be secondary to ZDV-5'-monophosphate (ZDV-MP) accumulation. A simulation study was performed in silico to optimize the ZDV dose for b.i.d. administration with K65R-selecting antiretroviral agents in virtual subjects using the population pharmacokinetic and cellular enzyme kinetic parameters of ZDV. These simulations predicted that a reduction in the ZDV dose from 300 to 200 mg b.i.d. should produce similar amounts of ZDV-5'-triphosphate (ZDV-TP) associated with antiviral efficacy (>97% overlap) and reduced plasma ZDV and cellular amounts of ZDV-MP associated with toxicity. The simulations also predicted reduced peak and trough amounts of cellular ZDV-TP after treatment with 600 mg ZDV once a day (q.d.) rather than 300 or 200 mg ZDV b.i.d., indicating that q.d. dosing with ZDV should be avoided. These in silico predictions suggest that 200 mg ZDV b.i.d. is an efficacious and safe dose that could delay the emergence of the K65R mutation. PMID:18838591

  12. Poly(ethylene oxide/propylene oxide) copolymer thermo-reversible gelling system for the enhancement of intranasal zidovudine delivery to the brain.

    PubMed

    Ved, Parag M; Kim, Kwonho

    2011-06-15

    The purpose of this study was to investigate the olfactory transfer of zidovudine (ZDV) after intranasal (IN) administration and to assess the effect of thermoreversible gelling system on its absorption and brain uptake. The nasal formulation was prepared by dissolving ZDV in pH 5.5 phosphate buffer solution comprising of 20% polyethylene oxide/propylene oxide (Poloxamer 407, PLX) as thermoreversible gelling agent and 0.1% n-tridecyl-β-D-maltoside (TDM) as permeation enhancer. This formulation exhibited a sufficient stability and an optimum gelation profile at 27-30 °C. The in vitro permeation studies across the freshly excised rabbit nasal mucosa showed a 53% increase in the permeability of ZDV from the formulation. For in vivo evaluation, the drug concentrations in the plasma, cerebrospinal fluid (CSF) and six different regions of the brain tissues, i.e. olfactory bulb (OB), olfactory tract (OT), anterior, middle and posterior segments of cerebrum (CB), and cerebellum (CL) were determined by LC/MS method following IV and IN administration in rabbits at a dose of 1mg/kg. The IN administration of Poloxamer 407 and TDM based formulation showed a systemic bioavailability of 29.4% while exhibiting a 4 times slower absorption process (t(max) = 20 min) than control solution (t(max) = 5 min). The CSF and brain ZDV levels achieved after IN administration of the gelling formulation were approximately 4.7-56 times greater than those attained after IV injection. The pharmacokinetic and brain distribution studies revealed that a polar antiviral compound, ZDV could preferentially transfer into the CSF and brain tissue via an alternative pathway, possibly olfactory route after intranasal administration. PMID:21356294

  13. Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

    PubMed

    Mirambeau, Gilles; Lyonnais, Sébastien; Gorelick, Robert J

    2010-01-01

    Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture. PMID:21045549

  14. [Hybrid nanocarriers for controlled delivery of antitumour and retroviral drugs delivery].

    PubMed

    Horcajada, Patricia; Serre, Christian; Férey, Gérard; Couvreur, Patrick; Gref, Ruxandra

    2010-01-01

    The efficient delivery of drugs in the body requires the use of non-toxic nanocarriers. Most of the existing materials show poor drug loading and/or rapid release of the proportion of the drug that is simply adsorbed (or anchored) at the external surface of the nanocarrier. The new porous hybrid solids, with the ability to tune their structures and porosities are well suited to serve as nanocarriers for delivery and imaging applications. Here we show that specific non-toxic porous iron(III) - based metal - organic frameworks with engineered cores and surfaces, as well as imaging properties, function as superior nanocarriers for efficient controlled delivery of antitumour and retroviral drugs against cancer and AIDS. They also potentially associate therapeutics and diagnostics, and open the way for theranostics, or -personalized patient treatments. double dagger. PMID:20819715

  15. Feline mediastinal lymphoma: a retrospective study of signalment, retroviral status, response to chemotherapy and prognostic indicators.

    PubMed

    Fabrizio, Francesca; Calam, Amy E; Dobson, Jane M; Middleton, Stephanie A; Murphy, Sue; Taylor, Samantha S; Schwartz, Anita; Stell, Anneliese J

    2014-08-01

    Historically, feline mediastinal lymphoma has been associated with young age, positive feline leukaemia virus (FeLV) status, Siamese breed and short survival times. Recent studies following widespread FeLV vaccination in the UK are lacking. The aim of this retrospective multi-institutional study was to re-evaluate the signalment, retroviral status, response to chemotherapy, survival and prognostic indicators in feline mediastinal lymphoma cases in the post-vaccination era. Records of cats with clinical signs associated with a mediastinal mass and cytologically/histologically confirmed lymphoma were reviewed from five UK referral centres (1998-2010). Treatment response, survival and prognostic indicators were assessed in treated cats with follow-up data. Fifty-five cases were reviewed. The median age was 3 years (range, 0.5-12 years); 12 cats (21.8%) were Siamese; and the male to female ratio was 3.2:1.0. Five cats were FeLV-positive and two were feline immunodeficiency-positive. Chemotherapy response and survival was evaluated in 38 cats. Overall response was 94.7%; complete (CR) and partial response (PR) rates did not differ significantly between protocols: COP (cyclophosphamide, vincristine, prednisone) (n = 26, CR 61.5%, PR 34.0%); Madison-Wisconsin (MW) (n = 12, CR 66.7%, PR 25.0%). Overall median survival was 373 days (range, 20-2015 days) (COP 484 days [range, 20-980 days]; MW 211 days [range, 24-2015 days] [P = 0.892]). Cats achieving CR survived longer (980 days vs 42 days for PR; P = 0.032). Age, breed, sex, location (mediastinal vs mediastinal plus other sites), retroviral status and glucocorticoid pretreatment did not affect response or survival. Feline mediastinal lymphoma cases frequently responded to chemotherapy with durable survival times, particularly in cats achieving CR. The prevalence of FeLV-antigenaemic cats was low; males and young Siamese cats appeared to be over-represented. PMID:24366846

  16. Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

    NASA Technical Reports Server (NTRS)

    Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

    2002-01-01

    Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no

  17. Development of retroviral vectors for insertional mutagenesis in medaka haploid cells.

    PubMed

    Lin, Fan; Liu, Qizhi; Yuan, Yongming; Hong, Yunhan

    2015-12-01

    Insertional mutagenesis (IM) by retrovirus (RV) is a high-throughput approach for interrogating gene functions in model species. Haploid cell provides a unique system for genetic screening by IM and prosperous progress has been achieved in mammal cells. However, little was known in lower vertebrate cells. Here, we report development of retroviral vectors (rvSAchCVgfp, rvSAchCVpf and rvSAchSTpf) and establishment of IM library in medaka haploid cells. Each vector contains a modified gene trapping (GT) cassette, which could extend the mutated cell population including GT insertions not in-frame or in weakly expressed genes. Virus titration determined by flow cytometry showed that rvSAchSTpf possessed the highest supernatant virus titer (1.5×10(5)TU/ml) in medaka haploid cell, while rvSAchCVpf produced the lowest titer (2.8×10(4)TU/ml). However, quantification of proviral DNAs in transduced cells by droplet digital PCR (ddPCR) demonstrated that the "real titer" may be similar among the three vectors. Furthermore, an IM library was established by FACS of haploid cells transduced with rvSAchCVgfp at a MOI of 0.1. A single copy RV integration in the majority of cells was confirmed by ddPCR in the library. Notably, there was a significant decrease of haploid cell percentage after FACS, suggesting potential trapping for survival/growth essential genes. Our results demonstrated successful development of retroviral vectors for IM in medaka haploid cells, serving for haploid genetic screening of host factors for virus infection and genes underlying certain cellular processes in fish model. PMID:26192464

  18. Murine retroviral vector producer cells survival and toxicity in the peritoneal cavity of dogs.

    PubMed

    Link, C J; Moorman, D W; Ackerman, M; Levy, J P; Seregina, T

    2000-01-01

    Retroviral vector producer cells (VPC) can effectively transfer genes in vivo. To develop a safe method to target gene delivery into intraperitoneal tumors, we have examined the toxicity of intraperitoneal (i.p.) infusion of retroviral VPC in a xenogeneic canine model. Mongrel dogs were injected intraperitoneally (i.p.) with 2 x 10(9) murine LTKOSN.2 VPC. The animals did not demonstrate acute toxicity and tolerated the i.p. infusion of the cells without difficulty. Starting 7 days after i.p. injection, the dogs received intravenous injections of ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. The treatment dogs underwent peritoneal washings on days 3, 7 and 14 after their initial infusion of cells to study the persistence of the VPC. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes and renal function remained normal throughout the study. Although, transient mild elevations occurred of serum alkaline phosphate, the remaining hepatic enzymes remained normal. Histologic examination of tissues from animals sacrificed after the i.p. administration of the VPC revealed no tissue destruction of the normal peritoneal lining. The dogs mounted an antibody response to the murine VPC that was first observed 7 days post injection. PCR analysis of selected tissues after GCV administration did not reveal persistent vector sequences. These results demonstrated that the injection of xenogeneic VPC is not accompanied by significant adverse effects over a 1 month period following administration into the canine peritoneal cavity. These data support the potential clinical application of the VPC in Phase I clinical trials in humans. PMID:11212841

  19. Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  20. Multiple Groups of Novel Retroviral Genomes in Pigs and Related Species

    PubMed Central

    Patience, Clive; Switzer, William M.; Takeuchi, Yasuhiro; Griffiths, David J.; Goward, Melanie E.; Heneine, Walid; Stoye, Jonathan P.; Weiss, Robin A.

    2001-01-01

    In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group γ1, four novel groups of gamma retrovirus (γ2 to γ5) and four novel groups of beta retrovirus (β1 to β4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace × Duroc F1 hybrid pig ranged from 2 (β2 and γ5) to approximately 50 (γ1). The γ1, γ2, and β4 genomes were transcribed into RNA in adult kidney tissue. Apart from γ1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, β2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of γ1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes. PMID:11222700

  1. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  2. Sites of retroviral DNA integration: From basic research to clinical applications.

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  3. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    PubMed

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-01

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin. PMID:11593038

  4. Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

    PubMed Central

    Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

    2015-01-01

    Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

  5. Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

    PubMed Central

    Hazuda, D; Felock, P; Hastings, J; Pramanik, B; Wolfe, A; Goodarzi, G; Vora, A; Brackmann, K; Grandgenett, D

    1997-01-01

    In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration. PMID:8985421

  6. Spectrum of imaging appearances of intracranial cryptococcal infection in HIV/AIDS patients in the anti-retroviral therapy era.

    PubMed

    Offiah, Curtis E; Naseer, Aisha

    2016-01-01

    Cryptococcus neoformans infection is the most common fungal infection of the central nervous system (CNS) in advanced human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) patients, but remains a relatively uncommon CNS infection in both the immunocompromised and immunocompetent patient population, rendering it a somewhat elusive and frequently overlooked diagnosis. The morbidity and mortality associated with CNS cryptococcal infection can be significantly reduced by early recognition of the imaging appearances by the radiologist in order to focus and expedite clinical management and treatment. The emergence and evolution of anti-retroviral therapy have also impacted significantly on the imaging appearances, morbidity, and mortality of this neuro-infection. The constellation of varied imaging appearances associated with cryptococcal CNS infection in the HIV and AIDS population in the era of highly active anti-retroviral therapy (HAART) will be presented in this review. PMID:26564776

  7. Early upper digestive tract side effects of zidovudine with tenofovir plus emtricitabine in West African adults with high CD4 counts

    PubMed Central

    Ouattara, Eric; Danel, Christine; Moh, Raoul; Gabillard, Delphine; Peytavin, Gilles; Konan, Romuald; Carrou, Jérome Le; Bohoussou, Franck; Eholie, Serge P; Anglaret, Xavier

    2013-01-01

    Introduction Tenofovir (TDF) with emtricitabine (FTC) and zidovudine (ZDV) is a recognized alternate first-line antiretroviral (ART) regimen for patients who cannot start treatment with non-nucleoside reverse transcriptase inhibitors (NNRTIs). Clinical studies comparing TDF+FTC+ZDV to other regimens are lacking. Methods Participants in a trial of early ART in Côte d'Ivoire (Temprano ANRS 12136) started treatment with TDF/FTC plus either efavirenz (EFV) or ZDV (HIV-1+2 dually infected patients and women refusing contraception or previously treated with nevirapine). We compared rates of upper digestive serious adverse events (sAEs) between TDF/FTC+EFV and TDF/FTC+ZDV patients during the first six months of treatment. sAEs were defined as either grade 3–4 AEs or persistent grade 1–2 AEs leading to drug discontinuation. Results A total of 197 patients (76% women, median CD4 count 395/mm3) started therapy with TDF/FTC, 126 with EFV and 71 with ZDV. During the first six months of ART, 94 patients had digestive AEs (nausea/vomiting) of any grade (EFV 36/126, 29%; ZDV 58/71, 82%, p<0.0001), including 20 sAEs (EFV 3/126, 5%; ZDV 17/71, 24%, p<0.0001). In-patients on TDF/FTC+ZDV with digestive AEs, the median time to the first symptom was two days (IQR: 1–4). Plasma ZDV (Cmax) distributions and pill ZDV dosages were normal. Patients with digestive AEs had higher haemoglobin levels and tended to have higher body mass indices and more frequent past histories of cotrimoxazole (CTX) prophylaxis. Conclusions We observed an unexpectedly high rate of digestive sAEs in West African adults, mostly women, who started a 3-nuc ART with TDF/FTC+ZDV in Côte d'Ivoire. These adults were participating in a trial of early ART and had much higher CD4 counts than those who currently routinely start ART in sub-Saharan Africa. They all received CTX concomitantly with ZDV. We suggest that further early prescriptions of TDF+XTC+ZDV should be carefully monitored and that whenever possible

  8. Anaemia and zidovudine-containing antiretroviral therapy in paediatric antiretroviral programmes in the IeDEA Paediatric West African Database to evaluate AIDS

    PubMed Central

    Renner, Lorna A; Dicko, Fatoumata; Kouéta, Fla; Malateste, Karen; Gueye, Ramatoulaye D; Aka, Edmond; Eboua, Tanoh K; Azondékon, Alain; Okomo, Uduok; Touré, Pety; Ekouévi, Didier; Leroy, Valeriane

    2013-01-01

    Introduction There is a risk of anaemia among HIV-infected children on antiretroviral therapy (ART) containing zidovudine (ZDV) recommended in first-line regimens in the WHO guidelines. We estimated the risk of severe anaemia after initiation of a ZDV-containing regimen in HIV-infected children included in the IeDEA West African database. Methods Standardized collection of data from HIV-infected children (positive PCR<18 months or positive serology ≥18 months) followed up in HIV programmes was included in the regional IeDEA West Africa collaboration. Ten clinical centres from seven countries contributed (Benin, Burkina Faso, Côte d'Ivoire, Gambia, Ghana, Mali and Senegal) to this collection. Inclusion criteria were age <16 years and starting ART. We explored the data quality of haemoglobin documentation over time and the incidence and predictors of severe anaemia (Hb<7g/dL) per 100 child-years of follow-up over the duration of first-line antiretroviral therapy. Results As of December 2009, among the 2933 children included in the collaboration, 45% were girls, median age was five years; median CD4 cell percentage was 13%; median weight-for-age z-score was −2.7; and 1772 (60.4%) had a first-line ZDV-containing regimen. At baseline, 70% of the children with a first-line ZDV-containing regimen had a haemoglobin measure available versus 76% in those not on ZDV (p≤0.01): the prevalence of severe anaemia was 3.0% (n=38) in the ZDV group versus 10.2% (n=89) in those without (p<0. 01). Over the first-line follow-up, 58.9% of the children had ≥1 measure of haemoglobin available in those exposed to ZDV versus 60.4% of those not (p=0.45). Severe anaemia occurred in 92 children with an incidence of 2.47 per 100 child-years of follow-up in those on a ZDV-containing regimen versus 4.25 in those not (p≤0.01). Adjusted for age at ART initiation and first-line regimen, a weight-for-age z-score ≤−3 was a strong predictor associated with a 5.59 times risk of severe

  9. Retroviral insertional mutagenesis identifies genes that collaborate with NUP98‐HOXD13 during leukemic transformation

    PubMed Central

    Slape, Christopher; Hartung, Helge; Lin, Ying‐Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D

    2007-01-01

    The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We previously demonstrated that expression of the NUP98‐HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighbouring retroviral insertion events. We identified numerous common insertion sites located near protein‐coding genes, and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98‐HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were upregulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein‐coding loci. Our data provides new insights into NHD13 mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms. PMID:17545593

  10. Structural and functional studies of murine Mbo I repeat LTR (MRL) retroviral genes on the Y chromosome

    SciTech Connect

    Ch'ang, L.Y.; Hoyt, P.R.; Wang, T.H.; Kanagala, R.; Henley, D.C.; Yang, D.M.; Yang, W.K. )

    1991-03-15

    The mouse genome harbors approximately 200 copies of MRL retroviral elements (or MuRRs) that are preferentially expressed in the reproductive system. The MRL retroviral gene family is wildly distributed in the genus Mus. About 10% of the elements are located on the Y chromosome and the abundance is probably due to gene amplification. Multiple copies of Y chromosome-specific MRL retroviral sequences are present only in the genome of M spretus and M. musculus. Structural and sequence analyses revealed a truncation of the male-specific MRL elements isolated from a BALB/c mouse DNA library. Consequently, two-thirds of an intact LTR was retained at the 5{prime} end and the 3{prime} structure was disrupted immediately downstream of the pol gene with a concomitant loss of the 3{prime} LTR. Southern analysis of male and female mouse DNA confirmed that sequences adjacent to the 3{prime} breakpoint were Y chromosome specific. These sequences are length polymorphic in nature and appear to be co-amplified with MRL retroviral genes on the Y chromosome. A collinear cDNA of 9.5 kb containing fused MRL and Y chromosome sequences was also isolated from a testis library. The LTR of male-specific MRL elements was unable to drive the expression of the bacterial CAT gene in cultured mouse NIH/3T3 and mink CCL64 cells. However, when its enhancer domain was linked to an SV40 promoter, the CAT gene was expressed at a significant level. Differential binding activities to male-specific MRL were found in nuclear extracts of the liver, kidney, and testis.