Journal: Annals of Neurology

Article Title: Ocrelizumab Concentration Is a Good Predictor of SARS‐CoV ‐2 Vaccination Response in Patients with Multiple Sclerosis

doi: 10.1002/ana.26534

Figure Lengend Snippet: Ocrelizumab concentration and B‐cells in relation to SARS‐CoV‐2 anti‐RBD antibody titer. These scatterplots show ocrelizumab concentration (μg/ml) (A) and B‐cells (CD19+ cells/μl) (B) at T = 0 in relation to SARS‐CoV‐2 anti‐RBD antibody titer (AU/ml) at T = 70. The T = 0 is baseline at the first vaccination, T = 70 is 70 days after the first vaccination (28 days after the second vaccination). SARS‐CoV‐2 = severe acute respiratory syndrome‐coronavirus 2.

Article Snippet: At T = 0 and T = 42, serum samples were collected and stored to measure ocrelizumab concentration using an enzyme‐linked immunosorbent assay (ELISA) developed by Sanquin Diagnostic Services, Amsterdam, The Netherlands.

Techniques: Concentration Assay

Journal: Annals of Neurology

Article Title: Ocrelizumab Concentration Is a Good Predictor of SARS‐CoV ‐2 Vaccination Response in Patients with Multiple Sclerosis

doi: 10.1002/ana.26534

Figure Lengend Snippet: ROC curves for ocrelizumab concentration, B‐cell count, and the combined model at T = 0. Shows the ROC curves for ocrelizumab concentration (μg/ml) at T = 0 (A), B‐cells (CD19+ cells/μl) at T = 0 (B), and the combined model of A en B (C) as independent variables in which the presence of SARS‐CoV‐2 IgG anti‐RDB response at T = 70 (based on the threshold of 4.0 AU/ml) was the dependent variable. ROC curves were constructed using logistic regression models. T = 0 is baseline at the first vaccination, and T = 70 is 70 days after the first vaccination (28 days after the second vaccination). AUC = area under the curve; OCR = ocrelizumab; ROC = receiver operating characteristic; SARS‐CoV‐2 = severe acute respiratory syndrome‐coronavirus 2.

Article Snippet: At T = 0 and T = 42, serum samples were collected and stored to measure ocrelizumab concentration using an enzyme‐linked immunosorbent assay (ELISA) developed by Sanquin Diagnostic Services, Amsterdam, The Netherlands.

Techniques: Concentration Assay, Cell Counting, Construct

Journal: Molecular and Cellular Biology

Article Title: Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL

doi: 10.1128/mcb.24.21.9317-9326.2004

Figure Lengend Snippet: FIG. 3. SIMPL modulates p65 and not c-Jun- or C/EBP-dependent transcription. (A to D) HEK 293 cells were transfected with 0.5 g of the indicated constructs, 20 ng of the promoterless sea pansy luciferase construct, and 0.2 g of either (A and C) a firefly luciferase construct under the control of the IL-8 gene promoter or (B and D) an artificial promoter composed of three tandem repeats of the NF-B response element from the human immunodeficiency virus long terminal repeat. Twenty-four hours later, cultures were harvested and processed for luciferase activity as described in Materials and Methods. In all experiments, cell lysates normalized for protein content were subjected to Western analysis to confirm expression of the Flag-tagged SIMPL construct and the p65 construct. (E) HEK cells were transfected with 0.5 g of the indicated constructs. Twenty-four hours later, cultures were harvested, RNA was isolated, and RNase protection assays were performed as described in Materials and Methods. (F) The autoradiogram used to generate the figure in panel E was quantitated by densitometry, and the ratio of the values obtained (IL-8/GAPDH) was replotted as a histogram.

Article Snippet: IKK , IKK , and p65 antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Construct, Luciferase, Control, Virus, Activity Assay, Western Blot, Expressing, Isolation

Journal: Molecular and Cellular Biology

Article Title: Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL

doi: 10.1128/mcb.24.21.9317-9326.2004

Figure Lengend Snippet: FIG. 4. SIMPL-p65 complexes form in response to TNF-. (A and B) HEK 293 cells were transfected with an expression vector encoding p65 and an expression vector encoding either wild-type SIMPL or SIMPLNLS. Twenty-four hours later, the indicated cultures were treated with recombinant human TNF- and were harvested 15 min later. Cell lysates were prepared, and p65 antisera were used to generate im- munocomplexes. (A) Immunocomplexed materials or (B) cell lysates used to generate the immunocomplexes were analyzed by Western analysis for SIMPL or p65. (C) Duplicate sets of HEK 293 cell cultures were plated; 24 h later, one set was not treated and the second set was treated with rhuTNF for 15 min; all cultures were then harvested. Cell lysates were prepared, and immunocomplexes were generated with SIMPL antisera. Immunocomplexes (-SIMPL) were subjected to SDS-PAGE, and West- ern blots were prepared and probed with p65 antisera.

Article Snippet: IKK , IKK , and p65 antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Expressing, Plasmid Preparation, Recombinant, Western Blot, Generated, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL

doi: 10.1128/mcb.24.21.9317-9326.2004

Figure Lengend Snippet: FIG. 5. Loss of SIMPL does not prevent nuclear localization of p65. (A) Duplicate sets of C3H10T1/2 mouse embryo fibroblasts were transfected with 0.5 g of the indicated constructs. Twenty-four hours later, half of the cultures were treated with TNF- (), and all cultures were fixed and washed with PBS 15 min later. Cultures were processed for indirect immunofluorescence by using antiserum that recognizes p65. (B) C3H10T1/Z mouse embryo fibroblasts were transfected with a construct encoding Flag-SIMPL. Forty-eight hours later, cultures were treated with recombinant human TNF- (10 ng/ml) for 15 min. Cytoplasmic and nuclear fractions were prepared, and equal amounts of cellular protein were subjected to SDS-PAGE. A Western blot was prepared and probed with p65 or Flag-specific antiserum.

Article Snippet: IKK , IKK , and p65 antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Construct, Recombinant, SDS Page, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL

doi: 10.1128/mcb.24.21.9317-9326.2004

Figure Lengend Snippet: FIG. 6. SIMPL functions as a p65 coactivator. (A) HEK 293 cells were transfected with 1.0 g of the indicated constructs, 5 ng of a p65-Gal4 construct, and 200 ng of a Gal4-luciferase construct. Cultures were allowed to incubate for 24 to 36 h, at which time cells were harvested and lysates were made and assayed for luciferase activity. (B) Wild-type (first three bars) or RelA / (last three bars) MEFs were transfected with 250 ng of the indicated vectors plus 200 ng of an IL-8-luciferase construct and 20 ng of the promoterless sea pansy luciferase construct. Twenty-four hours later, cultures were harvested and luciferase activities were measured. (C) HEK 293 cells were transfected with empty vector (control) or a construct expressing antisense SIMPL. Twenty-four hours later, cultures were stimulated with recombinant human-TNF- (10 ng/ml), and cultures were harvested at the indicated times. Cell lysates, normalized by protein concentration, were subjected to SDS-PAGE, Western blots were prepared and probed with IB and GAPDH antisera. Films were scanned and quantitated by using TotalLab (Nonlinear Dynamics). (D) Model of TNF control of NF-B-dependent gene expression.

Article Snippet: IKK , IKK , and p65 antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, Control, Expressing, Recombinant, Protein Concentration, SDS Page, Western Blot, Gene Expression