Journal:

Article Title: Preclinical and Clinical Performance of the Efoora Test, a Rapid Test for Detection of Human Immunodeficiency Virus-Specific Antibodies

doi: 10.1128/JCM.43.5.2399-2406.2005

Figure Lengend Snippet: Efoora HIV rapid test preclinical testing locations and information

Article Snippet: Specimens tested at sites 2 and 4 were predominantly, if not exclusively, from patients whose infection originated in the United States and thus were probably subtype B. table ft1 table-wrap mode="anchored" t5 TABLE 2. caption a7 Site Date No. of specimens tested Reference test(s) a Specimen type(s) U.S. East Coast March 1999 1,931 Genetic Systems HIV-1/2, Bio-Rad WB Serum February 2000 291 Genetic Systems HIV-1/2, Genetic Systems rLAV EIA, Bio-Rad WB Fresh whole blood February 2001 528 Genetic Systems HIV-1/2, Bio-Rad WB Serum, plasma, whole blood U.S. Midwest June 1999 1,076 Genetic Systems HIV-1/2, Abbott HIV-1/2, Bio-Rad WB Serum, plasma Ivory Coast, West Africa June 1999 962 Enzygnost Plus, immunocapture HIV Serum, plasma South Africa March 2000 150 Genetic Systems HIV-1/2 Serum, plasma, whole blood Open in a separate window a rLAV, recombinant lymphadenopathy-associated virus (HIV-1).

Techniques:

Journal: Advanced Techniques in Diagnostic Microbiology

Article Title: Molecular Techniques for Blood and Blood Product Screening

doi: 10.1007/978-1-4614-3970-7_28

Figure Lengend Snippet: Licensed/approved clinical assays for infectious agents (Source: Center for Biologics Evaluation and Research, US Food and Drug Administration [ 48 ])

Article Snippet: ViroSeq HIV-1 Genotyping System with the 3700 Genetic Analyzer , HIV-1 genotyping , Plasma , Patient monitoring , Celera Diagnostics, Alameda, CA , 6/11/2003.

Techniques: HBsAg Assay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Clinical Proteomics, Virus, Stripping Membranes, Western Blot, Viral-load Assay, DNA Sequencing, Amplification, Biomarker Discovery, Infection, Reverse Transcription, Polymerase Chain Reaction, Quantitation Assay, T. Cruzi Assay

Journal: AIDS (London, England)

Article Title: Soluble toll-like receptor 2 is significantly elevated in HIV-1 infected breast milk and inhibits HIV-1 induced cellular activation, inflammation and infection.

doi: 10.1097/QAD.0000000000000381

Figure Lengend Snippet: Fig. 3. HIV-1 structural proteins significantly elevate soluble Toll-like receptor 2 in vitro and inhibition of HIV-induced interleukin-8 expression by soluble Toll-like receptor 2. (a) Quantification of sTLR2 by western blot analyses and optical densitometry and ELISA in supernatants from MCF-10A; P ¼ 0.014, 0.012, 0.039 and THP-1; P ¼ 0.0008, 0.006, 0.013 and 0.026 exposed to HIV-1 proteins (p17, p24 and gp41) and Pam3CSK4 for 20 h. (b–d) Inhibition of Pam3CSK4, p17, gp41-induced IL-8 expression by sTLR2. TZMbl-2 supernatant was collected, concentrated and sTLR2 concentration was evaluated (Supplementary Fig. 1, http://links.lww.com/QAD/A545) before being preincubated at various dilutions with Pam3CSK4, p17 and gp41 for 1 h at 378C and placed on TZMbl-2 cells. IL-8 mRNA analyses with various concentrations of sTLR2 after exposure to (b) Pam3CSK4 (P ¼ 0.022, P ¼ 0.005 and P ¼ 0.0158); (c) p17 (P ¼ 0.004, 0.004 and 0.003); (d) gp41 (P ¼ 0.0007, P ¼ 0.002 and P ¼ 0.009). Data set is representative of at least three different experiments completed in triplicate and nonparametric Mann–Whitney test was used to determine significance with corresponding P values considered statistically significant if P < 0.05, P < 0.01, P < 0.001. Error bars, SEM. A representative data set from three independent experiments completed in triplicate is shown.

Article Snippet: HIV-1 components included p17 (Virogen, Mississauga, Canada), p24, nef, gp41 (Genway), gp120 (NIH AIDS Reference & Reagent Program) and ssRNA40 (Mobix, McMaster University).

Techniques: In Vitro, Inhibition, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY, IF-P

Journal: AIDS (London, England)

Article Title: Soluble toll-like receptor 2 is significantly elevated in HIV-1 infected breast milk and inhibits HIV-1 induced cellular activation, inflammation and infection.

doi: 10.1097/QAD.0000000000000381

Figure Lengend Snippet: Fig. 4. Soluble Toll-like receptor 2 physically interacts with p17, p24 and gp41. (a, b) Dot blots of positive controls (660 pmol Pam3CSK4 and 20 pmol sCD14), HIV-1 com- ponents (20 pmol p17, p24, gp41, gp120, ssRNA40 and nef), negative controls (1 ml PBS and 1 ml 1 mol/l urea) and membrane controls (1ng rsTLR2, and 1 :10 dilution of pooled HIV-uninfected breast milk (BM) on nitrocellulose membranes. The blots were incubated with mock-depleted HIV-1 unin- fected BM (a) or sTLR2-depleted BM (b). sTLR2 from pooled HIV-uninfected BM samples was immunodepleted using N-17 and nonspecific normal goat IgG antibodies as described previously [9]. The membranes were probed using anti-TLR2 goat IgG. (c, d) Immunoprecipitation of HIV-proteins with sTLR2. (c) sTLR2/HIV protein complexes were immunopreci- pitated with antip17 (a-p17) or antip24 (a-p24) antibodies and harvested with protein G-beads. Coimmunoprecipitated sTLR2 was separated by SDS-PAGE and revealed by WB with anti-TLR2. (d) sTLR2/gp41 protein complexes were immuno- precipitated with anti-TLR2 (a-TLR2) antibody and harvested with protein G-beads. Coimmunoprecipitated gp41 was separated by SDS-PAGE and revealed by WB with antigp41. In all panels, aliquots of cell lysate containing sTLR2 or recombinant gp41 were conducted in parallel for identification of specific protein bands.

Article Snippet: HIV-1 components included p17 (Virogen, Mississauga, Canada), p24, nef, gp41 (Genway), gp120 (NIH AIDS Reference & Reagent Program) and ssRNA40 (Mobix, McMaster University).

Techniques: Membrane, Incubation, Immunoprecipitation, SDS Page, Recombinant

Journal: Journal of Virology

Article Title: Selective Induction of Host Genes by MVA-B, a Candidate Vaccine against HIV/AIDS

doi: 10.1128/jvi.00749-10

Figure Lengend Snippet: FIG. 1. Infectivity and protein synthesis evaluation in IMDDC af- ter MVA or MVA-B infection. (A) HIV antigens and VACV protein expression in MVA- and MVA-B-infected IMDDC were detected by Western blot assay. At the times (hours) postinfection indicated above the lanes, equal amounts of proteins from extracts of cells mock in- fected (M) or infected with MVA or MVA-B (10 PFU/cell) were fractionated by SDS-PAGE, transferred to nitrocellulose, and incu- bated with antibodies against the HIV gag, HIV envelope (gp120), and specific VACV E3 early (p25) and A4 early-late (p39) proteins. eIF2 was used as a protein loading control. gpn represents the HIV-1 150- kDa fusion polyprotein Gag-Pol-Nef (B). IMDDC cultured on cover- slips treated as in panel A were fixed, permeabilized, and incubated with polyclonal gp120 antibody to show the HIV envelope protein, with antibody against the VACV E3 protein, and with To-Pro to show the DNA.

Article Snippet: Protein lysates (100 g) were fractionated by 14% or 8% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes, and incubated with antiE3L (obtained from B. L. Jacobs, University of Arizona), anti-A39K (55), anti-WR (56), anti-gpn (21), anti-gp120 (21), anti-eIF-2 (Santa Cruz, Santa Cruz, CA), or anti-MICA (R&D Systems) antibodies, followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Infection, Expressing, Western Blot, SDS Page, Control, Cell Culture, Incubation

Journal: Journal of Virology

Article Title: Selective Induction of Host Genes by MVA-B, a Candidate Vaccine against HIV/AIDS

doi: 10.1128/jvi.00749-10

Figure Lengend Snippet: FIG. 5. MICA protein upregulation after MVA-B infection of human THP-1 monocytes and role of gp120. (A) MVA-B infection is associated with increased levels of soluble MICA compared to those in uninfected or MVA-infected THP-1 cells. The data are representative of three independent experiments. O.D, optical density. (B) MVA-B infection leads to an increased susceptibility to NK cell lysis compared to that of uninfected and MVA-infected THP-1 cells. Primary human NK cells maintained in IL-2 were cocultured at the indicated E/T ratios with mock-infected THP-1 cells or THP-1 cells infected with either MVA or MVA-B. (C) HIV envelope increases MICA protein levels. At 6 hpi, equal amounts of proteins from cell extracts of THP-1 cells mock infected (M) or infected with MVA or MVA-B (10 PFU/cell) were fractionated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies to MICA or eIF2 (used as a protein loading control).

Article Snippet: Protein lysates (100 g) were fractionated by 14% or 8% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes, and incubated with antiE3L (obtained from B. L. Jacobs, University of Arizona), anti-A39K (55), anti-WR (56), anti-gpn (21), anti-gp120 (21), anti-eIF-2 (Santa Cruz, Santa Cruz, CA), or anti-MICA (R&D Systems) antibodies, followed by secondary antibodies (mouse and rabbit peroxidase conjugates).

Techniques: Infection, Lysis, SDS Page, Incubation, Control

Journal: Infection and Immunity

Article Title: Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3β

doi: 10.1128/iai.01329-08

Figure Lengend Snippet: FIG. 1. ET induces maturation of MDDCs. MDDCs were pretreated with 10 g/ml polymyxin B for 45 min and were then treated with either 100 ng/ml LPS (no pretreatment with polymyxin B), 300 M dcAMP, 100 ng/ml PA plus 100 ng/ml EF (ET), or no treatment (NT) (A), or 50 M forskolin, 20 ng/ml of CT, or no treatment (B). Cells were harvested 48 h after treatment and incubated with anti-DC-SIGN–FITC, anti-CD83– APC, and anti-CD86–FITC (panel A only) and then subjected to flow cytometric analysis. The data depicted represent the results for one of six independent experiments conducted on the MDDCs generated from six different donors. The histograms depict data for the unstained sample (shaded histogram), data for the stained sample (solid black line), and the geometric mean fluorescence under each treatment condition.

Article Snippet: MDDCs were treated as indicated in the figure legends for 48 h. To measure cell surface marker expression, MDDCs were harvested by scraping and washing with Dulbecco’s PBS and were then incubated on ice for 1 h with the following fluorophore-conjugated antibodies: anti-DC-SIGN–fluorescein isothiocyanate (FITC) (R&D Systems, Minneapolis, MN), anti-CD14–phycoerythrin (Caltag, Burlingame, CA), anti-CD83– APC (Caltag), anti-HLA-DR–APC (Caltag), anti-CD86–FITC (Caltag), and anti-CCR7–phycoerythrin (R&D Systems) diluted in media per manufacturer’s suggestions.

Techniques: Incubation, Generated, Staining

Journal: Infection and Immunity

Article Title: Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3β

doi: 10.1128/iai.01329-08

Figure Lengend Snippet: FIG. 3. Effects of LT on ET-mediated DC maturation and chemotaxis. (A and B) MDDCs were pretreated with 10 g/ml polymyxin B for 45 min and were then treated either with 100 ng/ml PA plus 100 ng/ml EF (ET), 100 ng/ml PA plus 100 ng/ml LF (LT), 100 ng/ml PA plus 100 ng/ml EF plus 100 ng/ml LF (ET/LT), or no treatment (NT) (A) or with 100 ng/ml PA plus 100 ng/ml EF (ET), 100 ng/ml PA plus 100 ng/ml EF plus 100 ng/ml LF (ET/LT), 100 ng/ml PA plus 100 ng/ml EF plus 100 ng/ml LF(H719C) (ET/mLT), or no treatment (NT) (B). Cells were harvested 48 h after treatment and incubated with anti-DC-SIGN–FITC, anti-CD83–APC, and anti-CD86–FITC and then subjected to flow cytometric analysis. The data depicted represent the results for one of five independent experiments conducted on MDDCs generated from five different donors. The histograms depict data for the unstained sample (shaded histogram), data for the stained sample (solid black line), and the geometric mean fluorescence under each treatment condition. (C) MDDCs were pretreated with 10 g/ml polymyxin B for 45 min and then treated with either 100 ng/ml PA plus 100 ng/ml EF (ET), 100 ng/ml PA plus 100 ng/ml LF (LT), 100 ng/ml PA plus 100 ng/ml EF plus 100 ng/ml LF (ET/LT), or 100 ng/ml PA plus 100 ng/ml EF plus 100 ng/ml LF(H719C) (ET/mLT). Following 48 h of treatment, the migration of MDDCs toward MIP-3 was measured using a transwell assay employing uncoated inserts (5-m pore). Data are shown as the increase in migration compared with that of immature (NT) MDDCs for each donor and are the average values for five donors SD. Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. **, P 0.05, compared to NT. (D) Cells were pretreated with 10 g/ml polymyxin B for 45 min and then treated for 2 h with 500 ng/ml PA plus 500 ng/ml EF (ET), 500 ng/ml PA plus 500 ng/ml LF (LT), or 500 ng/ml PA plus 500 ng/ml EF plus 500 ng/ml LF (ET/LT) or were left untreated (NT). WCEs (20 g/sample) were analyzed by Western blotting using antibodies specific for MEK2 (N terminus) or -tubulin.

Article Snippet: MDDCs were treated as indicated in the figure legends for 48 h. To measure cell surface marker expression, MDDCs were harvested by scraping and washing with Dulbecco’s PBS and were then incubated on ice for 1 h with the following fluorophore-conjugated antibodies: anti-DC-SIGN–fluorescein isothiocyanate (FITC) (R&D Systems, Minneapolis, MN), anti-CD14–phycoerythrin (Caltag, Burlingame, CA), anti-CD83– APC (Caltag), anti-HLA-DR–APC (Caltag), anti-CD86–FITC (Caltag), and anti-CCR7–phycoerythrin (R&D Systems) diluted in media per manufacturer’s suggestions.

Techniques: Chemotaxis Assay, Incubation, Generated, Staining, Migration, Transwell Assay, Comparison, Western Blot

Journal: eLife

Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

doi: 10.7554/eLife.79990

Figure Lengend Snippet: HIV-1 pseudoviruses were added to langerin-expressing 293T cells. Yeast mannan (10 µg/ml) was added to block the lectin-dependent binding of pseudoviruses. A langerin mutant lacking calcium binding ability (lectin (-)) was used as the negative control. ( A ) Immunoblots of pseudoviruses attached to the cell surface. Unbound and attached viruses were collected from the supernatant of the culture medium and TBS-EDTA buffer, respectively. Samples of unbound viruses were diluted 50-fold to adjust the band intensities. The expression levels of SNAP-tagged langerin show that the numbers of transfected cells were approximately the same in each experiment. Pr55 gag and p24 indicate unprocessed and fully-processed capsid proteins, respectively. ( B ) Immunoblots of internalized pseudoviruses. Birbeck granules were isolated by precipitation using streptavidin-agarose, and intracellular viruses and langerin were detected by their respective antibodies. Tubulins in the whole-cell lysates were detected for loading controls. ( C ) Quantification of internalized viruses using p24 ELISA. Horizontal lines indicate the mean. NS and Asterisk indicate no significant difference and statistically significant differences (p=0.07 (MRGD); 9.4×10 –5 (MRGK); 9.9×10 –9 (ARGK); and 5.5×10 –9 (lectin(-))) calculated using Bonferroni-corrected Student’s t -tests (N=4), respectively. Figure 5—source data 1. Original blot image of (right, anti-p24). Figure 5—source data 2. Annotated blot image of (right, anti-p24). Figure 5—source data 3. Original blot image of (right, anti-langerin). Figure 5—source data 4. Annotated blot image of (right, anti-langerin). Figure 5—source data 5. Original blot image of (left, anti-p24) and (anti-p24). Figure 5—source data 6. Annotated blot image of (left, anti-p24) and (anti-p24). Figure 5—source data 7. Original blot image of (anti-langerin). Figure 5—source data 8. Original blot image of (anti-tubulin). Figure 5—source data 9. Annotated blot images of (anti-langerin and anti-tubulin).

Article Snippet: Viruses, langerin, and tubulin were detected by immunoblotting using HIV-1 gag p24 antibody (1:1000 dilution, MAB7360, mouse monoclonal, R&D systems, Minneapolis, MN), human CD207 antibody (1:1000 dilution, 11841–1-AP, rabbit polyclonal, Proteintech Group Inc, Rosemont, IL), and α-tubulin antibody (1:3,000 dilution, B-512, mouse monoclonal, Sigma-Aldrich), respectively.

Techniques: Expressing, Blocking Assay, Binding Assay, Mutagenesis, Negative Control, Western Blot, Transfection, Isolation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

doi: 10.7554/eLife.79990

Figure Lengend Snippet: ( A ) Surface labeling of langerin. Langerin-expressing cells were surface-labeled using biotin N-hydroxysulfosuccinimide ester, and labeled langerin were immunoprecipitated using streptavidin agarose. ‘Int’ and ‘Sur’ indicate intracellular and surface langerin, respectively. The intracellular: surface ratio of langerin was approximately 5:1 and this ratio was not significantly affected by mutations. ( B ) Quantification of unbound viruses using p24 ELISA. Medium supernatants were diluted 1000-fold before loading into the ELISA plate. Concentration of viruses in the medium supernatant were nearly equal among the wild type and the mutants. ( C ) Anti-langerin immunoblot of stably-expressing cell line. The expression level of langerin in the stable cell line was approximately 30% of that of transiently-expressing cells. Given that the transformation efficiency of transiently-expressing cells was 40%, the actual expression level of langerin per cell is estimated to be ~12%. ( D ) Electron microscopy of the stable cell line. Birbeck granule formation was induced by addition of yeast mannan. Short and isolated Birbeck granules were observed (arrow) ( E ) Quantification of Birbeck granule formation. ‘Transient’ corresponds to WT mannan (+) in . 74 Birbeck granules in 20 stably-expressing cells were measured. p=2.0 × 10 –30 (individual length), 2.5×10 –12 (individual length), and 9.7×10 –14 (number). Figure 5—figure supplement 1—source data 1. Original blot image of . Figure 5—figure supplement 1—source data 2. Annotated blot image of . Figure 5—figure supplement 1—source data 3. Original blot image of (anti-langerin). Figure 5—figure supplement 1—source data 4. Original blot image of (anti-tubulin).

Article Snippet: Viruses, langerin, and tubulin were detected by immunoblotting using HIV-1 gag p24 antibody (1:1000 dilution, MAB7360, mouse monoclonal, R&D systems, Minneapolis, MN), human CD207 antibody (1:1000 dilution, 11841–1-AP, rabbit polyclonal, Proteintech Group Inc, Rosemont, IL), and α-tubulin antibody (1:3,000 dilution, B-512, mouse monoclonal, Sigma-Aldrich), respectively.

Techniques: Labeling, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Stable Transfection, Transformation Assay, Electron Microscopy, Isolation