Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Work panel used and monoclonal antibody/dye information.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques:

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's non-activated CD4 + T cells show increased surface expression of CD46, which is dependent on pollen season. Isolated CD4 + T cells from 49 HDs and 58 patients with AEA were analyzed non-activated (NA). The surface expression of CD46 on NA CD4 + T cells was assessed in HDs, AEA in LPP and HPP (A) as well as in HDs vs. AEA based on disease severity in LPP (B) and HPP (C) . Data were measured by flow cytometry and are presented as median fluorescence intensity (MFI) in graphs (A–C) . Horizontal bars represent the median. Next, the surface expression of CD46 was analyzed in HDs and AEA CD4 + T cells under both non-activated (NA) conditions (D) and after stimulation with αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and a high dose of IL-2 (50 U/ml), depending on LPP and HPP (E) . Concentration of sCD46 was assessed in plasma samples using ELISA in 40 HDs and 40 AEA patients in both LPP and HPP (F) . Data are presented as a median +95% CI in graphs (D) , (E) and (F) . Statistical analysis was performed using the unpaired Mann–Whitney U -test for two-group comparisons, and the Kruskal–Wallis test with Dunn's correction for three or more comparisons within a single graph where appropriate; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; LPP, low pollen period; HPP, high pollen period; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Isolation, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's CD4 + T cells show increased expression of CD46 after stimulation in low pollen period. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) for 60 h. (A) Surface expression of CD46 on CD4 + T cells was compared in both HDs and AEA patients in dependence of LPP and HPP, as well as between AEA patients in dependence of LPP and HPP (B) . Serum ECP levels were correlated with percentage of CD46 + CD4 + T cells after αCD3/αCD46/IL-2 stimulation among HDs, AEA LPP and AEA HPP (C) . Surface expression of CD46 was measured by flow cytometry and is presented as median fluorescence intensity (MFI). Statistical analysis of CD46 expression was performed using Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all. Correlation analysis was performed using non-parametric Spearman correlation coefficient; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; LPP, low pollen period; HPP, high pollen period; ECP, eosinophilic cationic protein; MFI, median fluorescence intensity.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: CD4 + T cells differ in CD46 downregulation after calcitriol co-stimulation in both HDs and AEA patients. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured without stimuli (NA) or with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46) for 60 h alone or with calcitriol (1 × 10 −7 M). The surface expression of CD46 was analyzed before/after calcitriol stimulation in HDs (A) and AEA patients in LPP (B) . Subsequently, two different groups were identified based on CD46 expression after calcitriol stimulation based on following formula: x = MFI CD46 (αCD3/αCD46/IL-2/Calcitriol)—MFI CD46 (αCD3/αCD46/IL-2). When x < 0 = Group CD46D (Decrease) (A1,B1) , when x > 0 = Group CD46I (Increase) (A2,B2) . Representative histograms depicts surface CD46 expression on NA or activated CD4 + T cells in groups CD46D/CD46I in HDs (C) and AEA patients (D) . Statistical analysis was performed using Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; mAbs, monoclonal antibodies; LPP, low pollen period; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Expressing

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: CD46D and CD46I groups of HDs and AEA patients differ in CD46 expression on both non-activated and stimulated CD4 + T cells. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured without stimuli (NA) or with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) and with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal). Surface expression of CD46 was measured in both groups CD46D/CD46I from HDs (A) , as well as from AEA patients (B) . Subsequently, percentage of CD46 downregulation after αCD3/αCD46/IL-2/Calcitriol stimulation was assessed in the group CD46D from HDs and AEA patients (C) . Identically, percentage of CD46 upregulation after αCD3/αCD46/IL-2/Calcitriol stimulation was assessed in the CD46I group from HDs and AEA patients (D) . The surface expression of CD46 was measured by flow cytometry and is presented as median fluorescence intensity (MFI) in graphs (A) and (B) . Graphs (C) and (D) are depicted as median + 95% CI. Statistical analysis was performed using the non-parametric Mann–Whitney U test; ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs (healthy donors), AEA, allergic eosinophilic asthma; NA, non-activated; mAbs, monoclonal antibodies; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Increased CD46 expression on CD4 + T cells from both HDs and AEA patients is independent of serum 25-OH vitamin D concentration. To clarify the relationship between the surface expression of CD46 on NA CD4 + T cells and serum levels of 25-OH vitamin D, these parameters were correlated in both HDs ( n = 49) (A) and AEA patients in LPP ( n = 58) (B) , as well as in HPP (C) . Next, serum levels of 25-OH vitamin D were compared between HDs and AEA (D) as well as in dependence on asthma severity (E) . The serum concentration of 25-OH vitamin D was also compared after dividing HDs and AEA patients into CD46D/CD46I groups (F) . Correlation analysis was performed using the non-parametric Spearman correlation coefficient, comparison of serum 25-OH vitamin D levels between HDs and AEA was performed using the non-parametric Mann–Whitney U -test, while the Kruskal–Wallis test with Dunn's correction was used for multiple comparisons all vs. all; ns (not significant), * p ≤ 0.05. HDs, healthy donors; AEA, allergic eosinophilic asthma; NA, non-activated; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol co-stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol co-stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Concentration Assay, Comparison, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patients in low pollen period show increased CD25 expression on stimulated CD4 + T cells, which is further promoted by calcitriol. CD4 + T cells from 49 HDs and 58 patients with AEA were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. (A) Surface expression of CD25 (IL-2 receptor α chain) and proliferation (B) were analyzed by flow cytometry before/after calcitriol stimulation with αCD3/αCD46/IL-2 in HDs and AEA patients in LPP and HPP groups using the non-parametric Mann–Whitney U -test. The data are presented as median intensity fluorescence (MFI), where horizontal bars indicate the median. Based on the CD46 dynamics after calcitriol stimulation, HDs and AEA patients were divided into groups CD46D/CD46I and surface expression of CD25 was analyzed separately. (C) To better understand the effect of calcitriol on CD25 expression in CD4 + T cells between HDs and AEA patients, we expressed the results as the percentage of upregulation after stimulation. Next, sIL-2RA levels were assessed in cell culture SNs, with HDs and AEA LPP patients divided into CD46D/CD46I groups (E) and are presented as a median +95% CI. Data from panels (C) and (E) were analyzed using the Wilcoxon-sign rank test and data from graphs (D) and (F) using the Kruskal–Wallis test with Dunn's correction; ns (not significant), * p ≤ 0.05. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; LPP, low pollen period; sIL-2RA, soluble IL-2 receptor α chain; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; MFI, median fluorescence intensity; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Expressing, Cell Culture, Flow Cytometry, MANN-WHITNEY, Fluorescence

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: Stimulated CD4 + T cells from CD46D group produce significantly more IFN-γ and IL-10 in HDs and react to calcitriol co-stimulation with downregulation of IFN-γ and upregulation of IL-10. CD4 + T cells from 49 HDs were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/αCD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. The concentrations of IFN-γ (A) and IL-10 (B) were assessed using ELISA and analyzed in accordance with the division of HDs into CD46D/CD46I groups. The results are presented as the median +95% CI. Similarly, the percentages of IFN-γ + CD4 + T cells (C) and IL-10 + CD4 + T cells (D) obtained from flow cytometry were analyzed, with the horizontal bar representing the median. Data were analyzed using the Kruskal–Wallis test with Dunn's correction for multiple comparisons all vs. all. (E) The timeline showing IFN-γ and IL-10 production by αCD3/αCD46/IL-2-stimulated CD4 + T cells at 0, 12, 36, 60 and 84 h of incubation is based on data from three healthy donors and is presented as the median + 95% confidence interval. (F) Representative dot-plots depict differences in IFN-γ and IL-10 production by activated CD4 + T cells respecting the division into the groups CD46D/CD46I. The data from flow cytometry were obtained from CD4 + T cells treated with Brefeldin A during the final 4 h of stimulation and were gated from 90.000 CD4 + T cells. The numbers in the top right corner show percentage of CD4 + T cells in each quadrant. The data are representatives of 107 individual samples performed in 18 series. HDs, healthy donors; mAbs, monoclonal antibodies; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Expressing

Journal: Frontiers in Allergy

Article Title: Low CD46 expression on activated CD4 + T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors

doi: 10.3389/falgy.2024.1462579

Figure Lengend Snippet: AEA patient's CD4 + T cells from CD46D group show increased production of IFN-γ after stimulation, which can be downregulated by calcitriol and simultaneously produce more IL-10. CD4 + T cells from 49 HDs and 58 AEA patients were cultured with a mixture of αCD3 (10 μg/ml), αCD46 (5 μg/ml) mAbs and high dose of IL-2 (50 U/ml) (αCD3/ α CD46/IL-2) or with calcitriol (1 × 10 −7 M) (αCD3/αCD46/IL-2/Cal) for 60 h. Concentrations of IFN-γ and IL-10 were measured in cell culture SNs using ELISA. IFN-γ production was compared between AEA LPP and AEA HPP groups (A) as well as in groups CD46D (B) and CD46I (C) . An identical analysis was performed with production of IL-10 (D–F) . To simplify the evaluation of the calcitriol's effect in both HDs and AEA patients, with respect to the CD46D/CD46I group division, results are presented as the percentage of downregulation in IFN-γ (G) and percentage of upregulation in IL-10 (H) after αCD3/αCD46/IL-2/Cal stimulation. Results are presented as the median + 95% CI. Paired data were analyzed using Wilcoxon matched-pairs signed rank test (black color) and unpaired data were analyzed unpaired Mann–Whitney U -test (grey color); ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. HDs, healthy donors; AEA, allergic eosinophilic asthma; mAbs, monoclonal antibodies; SNs, supernatant; CD46D, group where CD46 expression on CD4 + T cells decrease after calcitriol stimulation; CD46I, group where CD46 expression on CD4 + T cells increase after calcitriol stimulation; CI, confidence interval.

Article Snippet: CD46 , AF700 , MEM-258 , A7-342-T100 , Exbio.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Expressing

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of porcine cell lines with regard to their CD46 pig expression. (A) Phenotypical characterization of porcine cell lines by immunofluorescence staining using a CD46 pig -specific mab (green, MCA2310GA) and DAPI (blue). Asterisks (*) indicate cell lines subjected to conventional RT-PCR for subsequent sequencing. (B) Immunofluorescence staining of APPV P100 (porcine APPV-specific antiserum, red), CD46 pig (green, MCA2310GA), and DAPI (blue) at 72 h after infection of NPTr cells. (C) Strategy used for genetic characterization and manipulation of the CD46 pig gene locus. The CD46 pig -encoding mRNA was amplified by two RT-PCRs (101/710 and 604/1192) for subsequent cloning and sequencing. Absence of a CD46 pig -encoding mRNA in porcine lymphoma cell line 38A 1 D was confirmed by RT-PCRs targeting the individual CD46 pig domains (primer pairs: 192/353, 387/543, 571/710, 766/937). Positions of signal peptide (SP), complement control proteins 1 to 4 (ccp1-4), serine, threonine, proline-rich region (STP), and transmembrane domain (TM) encoded by the mRNA are depicted. In addition, positions of guide RNAs (gRNA CD46-2 and -7) used for construction of CD46 pig knockout cells are indicated (for details see ).

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing, Infection, Amplification, Cloning, Control, Knock-Out

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of genetically engineered CD46 pig knockout cells. (A) Phenotypical characterization of CD46 pig knockout cells by immunofluorescence staining using a mab against CD46 pig (green, MCA2310GA) and DAPI (blue). Immunofluorescence staining of CD46 pig (green) from wild-type (WT) cell lines served as a control and is shown in . (B) CRISPR/Cas9 induced genome alterations on both alleles characterized by sequencing of plasmids containing PCR amplicons flanking target sites of the guide RNAs (primers 101fw/710rev). Consensus nucleotide sequences and deduced amino acid sequences of the regions encoding the C terminus of SP and the N terminus of ccp1 are shown. For comparison, nucleotide and deduced CD46 pig amino acid sequences of WT as determined for SPEV and PK15 cells are given in the top row. The border between SP/ccp1 and position of gRNAs including respective protospacer adjacent motifs (PAM, boxed) are indicated. For selected engineered CD46 pig knockout cell lines (ΔCD46) the corresponding sequences including deletions (Δ nt) and insertions (+ nt) are shown below the WT CD46 sequence.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Immunofluorescence, Staining, Control, CRISPR, Sequencing, Comparison

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Primers used in this study

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Sequencing, Plasmid Preparation

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Relevance of CD46 pig for the entry of porcine pestiviruses. Wild-type (WT) SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2) were infected with APPV P17 , APPV P100 , BuPV, and CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, and Paderborn at an MOI of 1, respectively. Immunofluorescence staining was performed at 72 h p.i. using porcine APPV-specific antiserum, a porcine BuPV-specific antiserum, and a mab against CSFV, respectively. A strong reduction of APPV infection is evident on all SPEVΔCD46 cell lines in comparison to that on SPEV cells. PK15 cells display significantly lower permissivity to APPV P100 compared to that of SPEV cells. Non-culture-adapted APPV P17 obtained from early passage revealed the same CD46 pig dependency as the culture-adapted variant (APPV P100 ). With regard to infections with CSFV and BuPV, there are no differences in permissivity between the WT and the CD46 pig knockout cell lines.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Immunofluorescence, Staining, Comparison, Variant Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Production of infectious particles and RNA replication of porcine pestiviruses in dependence on CD46 pig . Wild-type (WT) SPEV and PK15, as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2), were infected with APPV P100 , CSFV Alfort-Tübingen (AlfT), and BuPV at an MOI of 1, respectively. (A) Supernatants were harvested 72 h p.i. to determine virus titers by using endpoint dilution assays in quadruplicates and in three repetitions. (B) Cells were collected at 72 h p.i. for RNA preparation and subsequent RT-PCR analysis. TaqMan based qRT-PCR assays were used for detection of CSFV and APPV genomes, whereas a SYBR green-based real-time RT-PCR was performed for detection of BuPV genomes. 30 ng total RNA was used per reaction. Samples collected from three individual experiments were tested in duplicates. Mean values with standard deviations are shown. APPV genome copy numbers obtained from WT cells are significantly higher compared to those from CD46 pig knockout cells (***, P < 0.0001, highly significant; *, P < 0.01, significant). CSFV and BuPV genome levels obtained from WT cells did not show significant differences compared to genome loads detected in infected knockout cells.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Virus, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Impact of CD46 pig at early time points of porcine pestivirus infections. (A) Immunofluorescence analysis of CSFV- and BuPV-infected cells. Wild-type (WT) PK15 and PK15ΔCD46 clone 2 cells were infected with CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, Paderborn, and BuPV at an MOI of 1. Infections with different CSFV strains and BuPV showed no dependency on CD46 pig even very early after infection (16 h p.i.). (B) Fluorescence in situ hybridization (FISH) analysis of APPV-infected cells. WT SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clone 2 and PK15ΔCD46 clone 2) were infected with cell culture-adapted APPV P100 at an MOI of 0.5. Scale bars indicate 100 µm for lower magnification and 50 µm for higher magnification. A strong reduction of APPV P100 infection is evident on both CD46 pig knockout cell lines in comparison to that on WT cells at early time point of infection (16 h p.i.). APPV P100 genomes were observed only on single CD46 pig knockout cells within the infected wells. APPV P100 infection of CD46 pig -expressing WT SPEV cells at a later time point (72 h p.i.) and noninfected SPEV cells (NIC) served as controls.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Immunofluorescence, Infection, Fluorescence, In Situ Hybridization, Knock-Out, Cell Culture, Comparison, Expressing

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Comparison of E2 envelope protein sequences of pestiviruses. (A) Phylogenetic tree (maximum likelihood) based on E2 amino acid sequences of known pestivirus species (APPV: AUL76967 ; bat: AFK85014 , AYV99177 ; rodent: ATP66856 , ATP66857 , YP009109567; pangolin: QIE06437 ; LindaV: YP009407716; whale: MK910228 ; BuPV: YP008992092; BDV: AAC16444 ; Aydin: YP006860588; ovine Italy: MG770617 ; giraffe: NP620053; pronghorn: YP009026415; BVDV-1: Q01499 ; BVDV-2: YP009513240; BVDV-3: AB871953 ; CSFV: YP009508222). APPV and CSFV sequence (bold) are the same as shown in the alignment. (B) Alignment (ClustalW) of APPV (isolate L277) and CSFV (Alfort 187) E2 amino acid sequences. Highlighted is the CSFV sequence analogous to the motif in the E2 of BVDV folding into a hairpin that might serve as ligand to the CD46 bov receptor . The positions of two nonsynonymous mutations (N751K and D752N) which occurred during cell culture adaptation of APPV are highlighted by a box.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Comparison, Sequencing, Cell Culture

Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control