Journal: One Health

Article Title: Human 3D liver spheroids support productive infection of a novel tick-borne phenuivirus

doi: 10.1016/j.onehlt.2026.101321

Figure Lengend Snippet: Assembly and characterization of human 3D liver spheroids via DNA origami NAC-linkers. (A) Schematic of 3D liver spheroid self-assembly from primary human hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells using NAC-linkers. (B) Atomic force microscopy image of NAC-linkers. Scale bars, 200 nm. (C) 1% agarose gel electrophoresis confirming cholesterol-modified NAC-linkers assembly (lanes: DNA marker, M13mp18 scaffold, and NAC-linkers). (D) Bright-field image of a mature spheroid. (E) Hematoxylin and eosin (H&E) staining of a spheroid section. (F) Immunofluorescence staining of cell type markers in human 3D liver spheroids: albumin (ALB, hepatocytes), CD31 (endothelial cells), and CD68 (Kupffer cells). Scale bars, 200 μm.

Article Snippet: Primary human hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells (IxCell Biotechnology) were mixed at specific ratios and co-incubated with NAC-Linker A and B (Puheng Biomedicine, NAC001) to facilitate NAC structure formation on the cell surfaces.

Techniques: Microscopy, Agarose Gel Electrophoresis, Modification, Marker, Staining, Immunofluorescence

Journal: One Health

Article Title: Human 3D liver spheroids support productive infection of a novel tick-borne phenuivirus

doi: 10.1016/j.onehlt.2026.101321

Figure Lengend Snippet: Adaptation and pathogenesis of MKWV in human 3D liver spheroids. (A) Schematic of serial passaging of the HLJ1 strain in spheroids, yielding the adapted NAC-Org5 strain. (B, C) Viral RNA copies (B) and TCID₅₀ titers (C) across passages (P1-P5). (D) Bright-field image of spheroids infected with passage 5 (P5) virus, showing structural disruption. Scale bar, 100 μm. (E) Quantification of spheroid diameter post-infection. (F) Transmission electron micrographs of virions within cytoplasmic vesicles of infected spheroids. Scale bars: 1 μm (left), 200 nm (right). (G) Representative images and quantification of nuclei showing infection-induced cell death. Scale bar, 200 μm. (H) Western blot detecting cleaved caspase-3 in spheroids at 48 and 72 h post-infection (hpi). (I) Multiplex immunofluorescence showing NAC-Org5 tropism for CD31 + endothelial cells and CD68 + Kupffer cells, with weaker detection in ALB + hepatocytes. Scale bar, 200 μm. (J) Functional assessment of infected spheroids: ATP (viability), ALT/AST/LDH (damage), ALB/urea (synthetic function). (K) RT-qPCR analysis of pro-inflammatory cytokine mRNA expression, normalized to β-actin. Data are mean ± SD ( n = 5 biological replicates). * p < 0.05, ** p < 0.01.

Article Snippet: Primary human hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells (IxCell Biotechnology) were mixed at specific ratios and co-incubated with NAC-Linker A and B (Puheng Biomedicine, NAC001) to facilitate NAC structure formation on the cell surfaces.

Techniques: Passaging, Infection, Virus, Disruption, Transmission Assay, Western Blot, Multiplex Assay, Immunofluorescence, Functional Assay, Quantitative RT-PCR, Expressing

Journal: One Health

Article Title: Human 3D liver spheroids support productive infection of a novel tick-borne phenuivirus

doi: 10.1016/j.onehlt.2026.101321

Figure Lengend Snippet: Pathogenicity of the NAC-Org5 strain in murine models. (A) Experimental schematic for intracranial (3-day-old) and intraperitoneal (3-week-old) inoculation of BALB/c mice. (B, C) Survival (B) and weight change (C) of suckling mice after NAC-Org5 infection. (D) Viral load in tissues and blood of suckling mice at 7 dpi. (E, F) Survival (E) and weight change (F) of 3-week-old mice. (G) Viral load in tissues and blood of 3-week-old mice at 7 dpi. Data are from 3 independent experiments. (H) Representative H& E -stained liver sections from 3-week-old mice at 7 and 15 dpi, showing inflammatory infiltrates and hepatocyte necrosis that resolves by 15 dpi. Scale bar, 100 μm. *** p < 0.001.

Article Snippet: Primary human hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells (IxCell Biotechnology) were mixed at specific ratios and co-incubated with NAC-Linker A and B (Puheng Biomedicine, NAC001) to facilitate NAC structure formation on the cell surfaces.

Techniques: Infection, Staining

Journal: JHEP Reports

Article Title: The dual role of TIGIT in regulatory and effector T cells in chronic liver disease

doi: 10.1016/j.jhepr.2025.101405

Figure Lengend Snippet: Hepatic recruitment and residency markers are upregulated on TIGIT + Tregs. TIGIT + FoxP3 + cells reside around hepatocytes in AIH livers and the TIGIT ligand-CD155 is expressed on inflamed hepatocytes. Ex vivo Tregs from patient peripheral blood and explant livers were phenotyped using flow cytometry. Details of samples used for flow cytometry experiments are in unless stated otherwise. Each data point represents values for an individual patient. (A) Representative histogram (CXCR3 and CD69) or contour plot (CD29/VLA-4) showing the expression of hepatic recruitment and residency markers by live CD3 + CD4 + CD25 + CD127 low TIGIT + Tregs as determined by flow cytometry. In the histograms, grey represents isotype, black represents positive stain. (B) Paired analysis comparing expression of the hepatic recruitment markers CXCR3, VLA-4, and tissue residency marker CD69 by blood and liver TIGIT + and TIGIT - Tregs using flow cytometry. Statistical analysis was performed using mixed-effects analysis, with the Geisser–Greenhouse correction and Šídák’s multiple comparisons test, with individual variances for each comparison. p values are displayed for statistically significant comparisons only ( p <0.05). (C) Immunohistochemistry staining of AIH explant liver sections and non-cirrhotic donor liver sections for TIGIT (brown). (C–E) The right panels represent a magnified view of the area shown on the left (red box). (D) Immunohistochemistry staining for CD155 (brown) on AIH and non-cirrhotic donor explant liver. (E) Representative confocal micrograph showing immunofluorescence staining for TIGIT (red) and FoxP3 (green) in an AIH explant section. Blue = DAPI. (F–G) Quantification of (F) CD155 intensity and (G) TIGIT frequency from immunohistochemistry staining of AIH explant liver and non-cirrhotic donor liver using eight randomly selected fields of view analysed using QuPath. Error bars represent standard error of mean (SEM). Statistical analysis was performed using an unpaired t test. p values are displayed for each statistical comparison made. AIH, autoimmune hepatitis; CDB, chronic liver disease blood; CLD, chronic liver diseases; DiL, disease liver; DoL, donor liver; HCB, healthy control blood; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Tregs, regulatory T cells.

Article Snippet: Primary human hepatocytes (PHHs; Thermo Fisher Scientific, Waltham, Massachusettes, USA) were thawed in thawing media (#CM7500, Thermo Fisher Scientific, Waltham, Massachusettes, USA), washed, and plated in a collagen-coated 96-well flat-bottomed plate in hepatocyte plating media (#CM3000, Thermo Fisher Scientific, Waltham, Massachusettes, USA).

Techniques: Ex Vivo, Flow Cytometry, Expressing, Staining, Marker, Comparison, Immunohistochemistry, Immunofluorescence, Control

Journal: JHEP Reports

Article Title: The dual role of TIGIT in regulatory and effector T cells in chronic liver disease

doi: 10.1016/j.jhepr.2025.101405

Figure Lengend Snippet: TIGIT interactions on TIGIT-expressing CD8 + T cells inhibit hepatocyte apoptosis. (A) Representative histogram of granzyme B and perforin expression, gated on live CD3 + CD8 + TIGIT + T cells. Grey represents isotype, black represents positive stain. (B) Paired comparison of granzyme B and perforin expression by TIGIT + and TIGIT - CD8 + T cells. Statistical tests were conducted using mixed-effects analysis, with the Geisser–Greenhouse correction and Tukey’s multiple comparisons test, with individual variances computed for each comparison. Details of samples used for flow cytometry are in . p values are displayed for statistically significant comparisons only ( p <0.05). (C) PBMCs from AIH patient blood were used for flow cytometry staining for naive T cell markers, CD45RA and CCR7, gated on live CD3 + CD8 + TIGIT + or TIGIT - T cells. (D) Paired comparison of antigen-experienced marker CD40L expression on TIGIT + and TIGIT - CD8 + T cells. Statistical analysis was performed using a mixed-effects analysis, with the Geisser–Greenhouse correction and Šídák’s multiple comparisons test, with individual variances for each comparison. (E) ICC staining of PHHs in co-culture with either TIGIT - or TIGIT + (magenta) labelled CD8 + T cells (CellTracker™ Red/CTR, orange). T cells were isolated from blood derived from patients with AIH using FACS and rested overnight before labelling and co-culture with PHHs. (F) Hepatocyte cell death after 24 h in co-culture with TIGIT - or TIGIT + CD8 + T cells or TIGIT - or TIGIT + Tconv cells in the presence or absence of a TIGIT neutralising antibody (monoclonal; 5 μg/ml) Data were collected from two individual biological repeats. Statistical analysis was performed using one-way ANOVA, with p values shown for statistically significant comparisons only ( p <0.05). Error bars represent the standard error of the mean (SEM). (G) Time-lapse images showing hepatocyte cell death. Images were taken every 30 min of PHH and TIGIT - CD8 + T cells labelled with CellTrace Red (CTR) in culture. The red arrow points towards an immune cell, and the white arrow points towards an apoptosing hepatocyte. The grey image shows the acquisition of phase gradient. (H) Representative images of ICC staining showing the expression of granzyme B (white) on either TIGIT - CD8 + T cells or TIGIT + CD8 T cells (CTR, magenta) and a magnified image of the interaction between a TIGIT - CD8 + T cell (CTR, magenta) and hepatocytes (CellTracker™ Green/CTG, yellow) or the lack of interaction between a TIGIT + CD8 + T cell and hepatocytes. AIH, autoimmune hepatitis; AIHB, AIH patient blood; CDB, chronic liver disease blood; DiL, disease liver; DoL, donor liver; HCB, healthy control blood; ICC, immunocytochemistry; PBMCs, peripheral blood mononuclear cells; PHHs, primary human hepatocytes; Tconv, conventional T cells; TIGIT, T cell immunoreceptor with Ig and ITIM domains.

Article Snippet: Primary human hepatocytes (PHHs; Thermo Fisher Scientific, Waltham, Massachusettes, USA) were thawed in thawing media (#CM7500, Thermo Fisher Scientific, Waltham, Massachusettes, USA), washed, and plated in a collagen-coated 96-well flat-bottomed plate in hepatocyte plating media (#CM3000, Thermo Fisher Scientific, Waltham, Massachusettes, USA).

Techniques: Expressing, Staining, Comparison, Flow Cytometry, Marker, Co-Culture Assay, Isolation, Derivative Assay, Control, Immunocytochemistry