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  • 1
    Language: English
    In: Current HIV Research, 2010, Vol.8(7), p.554-563
    Description: The cytidine deaminase APOBEC3G has been identified as an antiviral host factor that combats HIV-1. The protein was found to be present in HIV-1 target cells such as macrophages and dendritic cells. The antiviral state of these cells has been partially attributed to a G→A hypermutation of the HIV genome caused by APOBEC3G during reverse transcription. However, the viral infectivity factor (Vif) counteracts this antiviral mechanism by inducing the inactivation of APOBEC3G. In this study, we tested the effect of APOBEC3G expression on the HIV-1 infection of cells derived from purified CD34+ cells that have been transduced with lentiviral vectors containing APOBEC3G and/or shRNAs directed against APOBEC3G and then have been differentiated before infection with HIV-1. In cell lines, the infection was strongly inhibited after upregulation of APOBEC3G. The infection could then be rescued after transducing these cells with shRNAs targeting APOBEC3G. In cells derived from purified CD34+ cells a strong inhibition of the HIV-1 infection was observed in both a Vif defective HIV-1 virus and the corresponding wild-type HIV-1 virus with Vif. Our data implies that when APOBEC3G is expressed high enough, it can escape the inhibition from Vif, thereby exerting its antiviral activity.
    Keywords: Apobec3g ; Cd34+ Cells ; Macrophages ; Lentiviral Vectors ; Hiv-1 ; Vif ; Hiv-1 Infection ; Apobec3g Expression ; Cytidine Deaminase ; Dendritic Cell ; Ga Hypermutation ; Hiv Genome ; Vif Defective Hiv-1 Virus ; Wild-Type Hiv-1 Virus ; Immune System ; Dendritic Cells ; T Cells ; Hypermutations ; Cellular Endonuclease ; Monocytes ; Nucleocapsid Protein ; Hek-293t ; Hela ; Tzm-Bl Cells ; Dulbecco'S Modified Eagle'S Medium ; Dmem ; Fetal Bovine Serum ; Glutamine ; Penicillin ; Streptomycin ; Stem Cell Transplantation ; Intraglobine ; Pulmozyme ; Pbs ; Mercaptoethanol ; Sodium Pyruvate ; Flow Cytometry ; Immunofluorescent Antibodies ; Fitc Antibody ; Reverse Transcription ; Plasmid ; Calcium Phosphate ; Western Blot ; Anti-Gapdh Antibody ; P24 Elisa
    ISSN: 1570-162X
    E-ISSN: 1873-4251
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  • 2
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(5), p.e97478
    Description: HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PLoS ONE, 2010, Vol.5(1), p.e8968
    Description: Due to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time. ; pSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions. ; Adult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes).
    Keywords: Research Article ; Cell Biology ; Dermatology ; Developmental Biology -- Cell Differentiation ; Developmental Biology -- Stem Cells ; Ecology -- Conservation And Restoration Ecology
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: PLoS ONE, 01 January 2018, Vol.13(1), p.e0190669
    Description: The standardized assessments of HIV-specific immune responses are of main interest in the preclinical and clinical stage of HIV-1 vaccine development. In this regard, HIV-1 Env-pseudotyped viruses play a central role for the evaluation of neutralizing antibody profiles and are produced according...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: PLoS ONE, 2012, Vol.7(3), p.e32568
    Description: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. ; In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different co-incubation experiments were performed with competing ligands of the respective receptor. ; This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.
    Keywords: Research Article ; Biology ; Materials Science ; Medicine ; Biotechnology ; Pharmacology ; Biochemistry
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: PLoS ONE, 2010, Vol.5(12), p.e14213
    Description: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. ; In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. ; With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.
    Keywords: Research Article ; Biotechnology ; Neurological Disorders ; Pharmacology -- Drug Development
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(8), p.e105401
    Description: Technical progress has simplified tasks in lab diagnosis and improved quality of test results. Errors occurring during the pre-analytical phase have more negative impact on the quality of test results than errors encountered during the total analytical process. Different infrastructures of...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: Cryobiology, October 2013, Vol.67(2), pp.193-200
    Description: The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions.
    Keywords: Cryopreservation ; Optimal Sample Storage ; Pbmc Recovery ; Pbmc Functionality ; Elispot ; Biology
    ISSN: 0011-2240
    E-ISSN: 1090-2392
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  • 9
    Language: English
    In: Toxicology in Vitro, December 2011, Vol.25(8), pp.1557-1567
    Description: ► We compared the cytotoxicity of TP and TP-PM on Jurkat and HT29 cells. ► TP and TP-PM could induce an inhibition of cell growth and proliferation in both tumor cell lines. ► TP and TP-PM induced apoptosis and caused activation of caspase 3/7. ► TP-PM induced in tested cell lines stronger effects than TP. Triptolide (TP), a diterpenoid triepoxide purified from the Chinese herb Hook F is characterized by strong anti-tumor effects on various cancer cells. Except its anti-tumor effects, TP also shows multiple pharmacological side activities, such as anti-inflammatory, immune-suppressive and male anti-fertility. In order to reduce these side effects, especially the immuno-suppressive activity when used to cure cancer, a novel polymeric micelle system containing TP (TP-PM) was constructed. The immune-modulation effects of TP-PM have been evaluated by previous studies. In this study, we compared the cytotoxicity of TP and TP-PM on Jurkat and HT29 cells. Therefore, we determined the cell viability, membrane integrity, cell proliferation, apoptosis, and caspase 3/7 activity after exposure to TP and TP-PM. The results demonstrated that actually low concentrated TP and TP-PM could induce an inhibition of cell growth and proliferation as well as membrane damage in both tumor cell lines. TP and TP-PM induced apoptosis and caused activation of caspase 3/7 even at low concentrations. Both formulations destroyed the membrane of Jurkat cells, nevertheless, TP-PM showed stronger pernicious effects. In general, TP-PM induced in both tested cell lines stronger effects than TP. Therefore, polymeric micelles can be considered as promising carriers for TP in cancer therapy.
    Keywords: Cytotoxicity ; Triptolide ; Polymeric Micelles ; In Vitro ; Pharmacy, Therapeutics, & Pharmacology ; Chemistry ; Public Health
    ISSN: 0887-2333
    E-ISSN: 1879-3177
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  • 10
    Language: English
    In: PLoS ONE, May 14, 2014, Vol.9(5)
    Description: HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.
    Keywords: Antigenic Determinants – Analysis ; Antigenic Determinants – Health Aspects ; Antigenic Determinants – Technology Application ; Infection – Analysis ; Infection – Health Aspects ; Infection – Technology Application ; Proteins – Analysis ; Proteins – Health Aspects ; Proteins – Technology Application ; Monoclonal Antibodies – Analysis ; Monoclonal Antibodies – Health Aspects ; Monoclonal Antibodies – Technology Application ; HIV – Analysis ; HIV – Health Aspects ; HIV – Technology Application
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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