Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural beta-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue beta-peptide, beta-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (beta-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and beta-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.
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PMID:Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells. 1451 18

Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against human immunodeficiency virus type 1 (HIV-1). However, the antiviral mechanism of prostratin is not well defined. In the present study, we report that prostratin exhibits potent antiviral activity against different strains of HIV-1 (subtypes B and D), a clinical HIV isolate (L1), HIV-2 (ROD and EHO) and SIV (MAC251) with EC50-values ranging from 0.02-0.09 microg/ml. Prostratin was equally active against HIV strains resistant to the polyanionic binding inhibitor dextran sulphate, the fusion inhibitor T-20 (enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs). In contrast, prostratin lost 4.4- and 6.8-fold of its effect against the HIV strains resistant to AMD3100 and the quaternary ammonium salt QAS10+, respectively. As shown by time-of-addition experiments, prostratin needs to be present at the time of viral adsorption to exert its antiviral activity. We selected an HIV strain (NL4.3/PROS) resistant to prostratin in MT-4 cells. The sensitivity of NL4.3/PROS towards prostratin, dextran sulphate and QAS10+ was reduced by 3.2, 4.1 and >50-fold, respectively. However, NL4.3/ PROS was still sensitive to AMD3100, T-20, NRTIs (zidovudine and nevirapine) and a PI (ritonavir). Recombination of the gp160-gene of the NL4.3/PROS strain in a NL4.3 wild-type molecular clone fully rescued its phenotypic resistance. DNA sequencing of the NL4.3/PROS strain revealed mutations throughout the gp120 gene previously associated with resistance towards other HIV entry inhibitors. We concluded that prostratin inhibits the entry step of the replication cycle of HIV by interacting with a cellular target necessary for viral entry.
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PMID:Potent and selective inhibition of HIV and SIV by prostratin interacting with viral entry. 1496 38

Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.
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PMID:Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool. 1502 9

Influenza virus infection can cause severe complications in human immunodeficiency virus type-1 (HIV-1)-infected individuals leading to an increased risk of complications and death compared to that seen in uninfected individuals. We assessed the capacity of influenza virus (Flu) to modulate transcription of the HIV-1 long terminal repeat (LTR) in human CD4+ T cells. We found that Flu is able to promote expression of both the transiently transfected and stably integrated HIV-1 LTR-driven reporter gene. Experiments performed with Arthrobacter-derived neuraminidase and ammonium chloride revealed that Flu-dependent activation of HIV-1 transcription required an intimate contact between Flu and the target cell and efficient entry of Flu inside human CD4+ T cells. Amplification of a Flu-specific mRNA by RT-PCR indicated that human T cells were indeed productively infected with Flu. Virus preparations rendered noninfectious after UV irradiation could no longer upregulate HIV-1 LTR activity. Furthermore, experiments conducted with wild type and NF-kappaB-mutated HIV-1 LTR-directed reporter vectors suggested that the positive action of Flu on HIV-1 LTR activity was mediated through the induction of NF-kappaB. Our data show that fully competent Flu can lead to NF-kappaB-dependent activation of HIV-1 transcription in CD4+ T cells.
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PMID:Influenza virus activates human immunodeficiency virus type-1 gene expression in human CD4-expressing T cells through an NF-kappaB-dependent pathway. 1563 53

We identified a postentry restriction, termed Lv2, which determines the cellular tropism of two related human immunodeficiency virus type 2 (HIV-2) isolates and is dependent on the sequence of the capsid (CA) and envelope (Env) proteins. To explain the reliance on both CA and Env, we proposed that restrictive Envs deliver susceptible capsids to a compartment where Lv2 is active whereas nonrestrictive Envs deliver capsids into a compartment where Lv2 is either absent or less active. To test this model, we used compounds that affect endocytic pathways (ammonium chloride, bafilomycin A1, hypertonic sucrose) or lipid rafts (methyl-beta-cyclodextrin) to treat restrictive cells and show that restricted virus can be rescued from Lv2 if a lipid-raft-dependent, pH-independent endocytic pathway is inhibited. Furthermore, viral entry into HeLa/CD4 cells containing a tailless CD4 receptor, located outside lipid rafts, was fully permissive. Finally, we show that a variety of primary HIV-1 and HIV-2 viruses are susceptible to Lv2. Thus, we show that the route of entry, determined by the viral envelope, can influence cellular tropism by avoiding intracellular blocks to infection.
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PMID:An envelope-determined, pH-independent endocytic route of viral entry determines the susceptibility of human immunodeficiency virus type 1 (HIV-1) and HIV-2 to Lv2 restriction. 1601 4

The presentation of exogenous protein antigens in a major histocompatibility complex class I-restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination.
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PMID:Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo. 1615 87

Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.
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PMID:Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway. 1628 48

Rutin deca(H-) sulfate sodium (RDS) is one of the most important drug candidates, which possesses very good activity as inhibitor of the complement system of warm-blooded animals and human immunodeficiency virus (HIV). In order to understand RDS metabolism and disposition, an ion-pairing coupled with solid-phase extraction technique (IP-SPE) was developed to extract RDS from rat plasma sample. Tetrabutyl ammonium bromide (TBAB) buffer (0.2 M, pH 8.0) was used as the ion-pairing extraction reagent and LC-18 was used as SPE sorbent. In addition, an ion-pairing HPLC method was established for the specific determination of RDS. A reversed phase C8 column was used for the separation of RDS and nitrendipine (internal standard). The mobile phase was composed of 10 mM phosphate buffer solution containing 25 mM TBAB-acetonitrile (52:48, v/v, pH 7.5). The calibration curve was linear from 0.3 to 30 nmol/mL. The analytical recovery from rat plasma was found to be 97.9+/-4.1% (n = 15). LOD and LOQ for RDS in plasma were calculated to be 0.12 nmol/mL and 0.30+/-0.024 nmol/mL (R.S.D. = 8.2%, n = 5), respectively. The intra- and inter-day precision was less than 9.2%. The assay was applied to a preliminary pharmacokinetic study in three male rats after those received a single intravenous bolus via caudal vein of 12 micromol/kg RDS.
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PMID:Determination of rutin deca(H-) sulfate sodium in rat plasma using ion-pairing liquid chromatography after ion-pairing solid-phase extraction. 1651 96

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.
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PMID:Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction. 1691 49

The intracellular metabolism of nucleoside reverse transcriptase inhibitors (NRTI) in mononuclear cells has been thoroughly studied, but that in red blood cells (RBC) has been disregarded. However, the phosphorylation of other analogous nucleosides (in particular, ribavirin) has been described previously. In this study, we investigated for the first time the phosphorylation of NRTI in human RBC. The presence of intracellular zidovudine (AZT) monophosphate, AZT triphosphate, lamivudine (3TC) triphosphate, and tenofovir (TFV) diphosphate, as well as endogenous dATP, dGTP, and dTTP, in RBC collected from human immunodeficiency virus-infected patients was examined. We observed evidence of a selective phosphorylation of 3TC, TFV, and endogenous purine deoxynucleosides to generate their triphosphate moieties. Conversely, no trace of AZT phosphate metabolites was found, and only faint dTTP signals were visible. A comparison of intracellular TFV diphosphate and 3TC triphosphate levels in RBC and peripheral blood mononuclear cells (PBMC) further highlighted the specificity of NRTI metabolism in each cell type. These findings raise the issue of RBC involvement in drug-drug interaction, drug pharmacokinetics, and drug-induced toxicity. Moreover, the typical preparation of PBMC samples by gradient density centrifugation does not prevent their contamination with RBC. We demonstrated that the presence of RBC within PBMC hampers an accurate determination of intracellular TFV diphosphate and dATP levels in clinical PBMC samples. Thus, we recommend removing RBC during PBMC preparation by using an ammonium chloride solution to enhance both the accuracy and the precision of intracellular drug monitoring.
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PMID:Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells. 1743 52


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