Mab003

Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R&D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R&D Systems, respectively). As controls, groups of neonates were directly injected i.p. on P3 with PBS or the same mouse monoclonal isotype control or anti-BMP9 and anti-BMP10 Abs (15 mg/kg). Neonates were euthanized by CO₂ asphyxiation on P6 and non-heparinized blood was collected. Neonates were enucleated and eyes fixed in 4% paraformaldehyde for 20 min on ice and retinas were isolated.

The GC cells were seeded in a T75 tissue culture flask and grown to 40–50% confluence (depending on the growth rate of the cell lines). The growth medium was then replaced with serum‐free medium to further culture for 24 h, and the supernatants were harvested for future assays. For neutralization experiments, the neutralizing antibody against TIMP1, PAI1 or IGFBP1 was preincubated at 4 °C with 1 mL supernatant for 6 h, then used for HUVECs coculture in tube formation experiments. The neutralizing antibodies against TIMP1 (AF970‐SP; R&D Systems, Minneapolis, MN, USA), PAI1 (AF1786‐SP; R&D Systems), IGFBP1 (MAB675‐SP; R&D Systems) and control antibody (MAB003; R&D Systems) were 0.2, 1.5, 20 and 5 µg·mL⁻¹, respectively.
The conditioned media (CMs) used in the experiments were: (a) rIL35, serum‐free medium with different concentrations of recombinant IL35 (Cloud‐Clone Corp., RPC008Hu01); (b) supernatants from HGC27 cells that were cultured with rIL35 for 24 h; (c) supernatants from the pc‐IL12A, pc‐EBI3 and pc‐Ctrl HGC27 cells, respectively; (d) supernatants from the sh‐IL12A, sh‐EBI3 and pYr‐ctrl AGS cells, respectively; (e) supernatants from pc‐IL12A HGC27 cells pretreated with PAI1, IGFBP1 or control neutralizing antibodies for 6 h, respectively; and (f) supernatants from sh‐IL12A AGS cells pretreated with TIMP1 or control neutralizing antibodies for 6 h, respectively.

Infected cells from the OSGC at the time when 50% of virus particles are released (BT₅₀) were fixed with 4% PFA for 20 min at room temperature, washed, and permeabilized with 70% ethanol. Subsequently, cells were washed twice in PBS and incubated for 1 h in the dark and at room temperature with anti-Flavivirus group antigen (MAB10216, clone D1-4G2-4-15, 1:200 dilution; Millipore, Germany) or mouse IgG2a isotype control (MAB003, 1:50 dilution; R&D Systems) in PBS containing 0.1% bovine serum albumin (BSA). Afterward, the cells were washed three times with PBS–0.1% BSA and incubated for 1 h with goat anti-mouse IgG2a conjugated with Alexa Fluor 488 (1:250 dilution; Life Technologies, Inc., The Netherlands) in PBS–0.1% BSA at room temperature and in the dark. After 1 h, cells were washed three times and mounted with ProLong Diamond Antifade mountant with DAPI (4′,6-diamidino-2-phenylindole [Life Technologies, Inc., USA]). Zika virus-infected cells were identified by use of a Zeiss LSM 700 confocal laser scanning microscope fitted on an Axio observer Z1 inverted microscope (Zeiss). All images were processed using Zen 2010 software (Zeiss). Per sample, 5 high-power fields were photographed and scored blindly by three individuals to determine the percentages of infected and noninfected cells.

The pET30a vector (in our laboratory) was used as an expression vector for N. cyriacigeorgica GUH-2 HBHA in E. coli. The plasmid pK18mobsacB (in our laboratory) was used to construct the hbha deletion mutant. Anti-TLR4 (NB100-56727, Novus Biological, USA), anti-TLR2 (NB100-56726, Novus Biological, USA), and IgG_2A isotype control (MAB003, R&D, USA) were used in the present study. The following antibodies were purchased from Cell Signaling Technology (Danvers, USA): anti-p-Jnk (4668), anti-p-ERK1/2 (4370), anti-p-p38 (4511), anti-p-p65 (3033), and anti-β-actin (7074). The following pharmacological inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA): p38 (SB203580), ERK1/2 (PD98059), JNK (SP600125), and NF-κB (BAY 11-7082). The endotoxin removal kit was purchased from GenScript (Nanjing, China). Human and mouse TNF-α, IL-6, and IL-10 ELISA kits were used in this study (BD, USA). DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) and Alexa Fluor® 488 donkey anti-mouse IgG were purchased from ThermoFisher Scientific (Carlsbad, CA, USA). The adjuvant was purchased from Biodrago (Suzhou, China).

Modification of anti-claudin-4 mAb with p-SCN-Bn-DTPA and subsequent radiolabelling with indium-111 were conducted following methods described by Brom et al. [16 ]. In brief, to a solution of anti-claudin-4 MAB4219 (200 μg) or mouse IgG_2A (200 μg, MAB003, R&D Systems) in 0.1 M NaHCO₃ (pH 9, 100 μl, Chelex treated) was added 20 M equivalents of p-SCN-Bn-DTPA (245.6 μM) in anhydrous dimethyl sulphoxide. The reaction mixture was incubated at room temperature for 30 min with gentle shaking (450 rpm), and the excess p-SCN-Bn-DTPA was removed by Sephadex-G50 size exclusion chromatography (Sigma-Aldrich). The affinity of the DTPA-conjugated MAB4219 for claudin-4 was evaluated by flow cytometry in Panc-1 and HT1080 cells and compared to that of the unmodified antibody.
Indium-111 in 0.02 M hydrochloric acid (sourced from Mallinckrodt Pharmaceuticals) was added to a 2-mg/ml solution of the DTPA-modified antibody to achieve a ratio of at least 1 MBq to 1 μg. The reaction mixtures were incubated at room temperature for 1 h, and the radiolabelling efficiency was determined by iTLC using an eluent of 0.1 M sodium citrate buffer (pH 5.5). The crude reaction mixture was purified by Sephadex-G50 size exclusion chromatography, eluting with 100-μl fractions of phosphate-buffered saline (pH 7.4).