Journal: Biomedicines

Article Title: Lymphocytic Myocarditis in Children with Parvovirus B19 Infection: Pathological and Molecular Insights

doi: 10.3390/biomedicines12081909

Figure Lengend Snippet: Correlation of CD3+ T cell infiltration and B19V DNA copies in children with acute and chronic myocarditis. ( A ) Comparison of CD3+ cell count in acute myocarditis without infection, other virus infections, and B19V infection between two age groups (0–2 years and 3–16 years). ( B ) Comparison of CD3+ cell count in chronic myocarditis without infection, other virus infections, and B19V infection between two age groups (0–2 years and 3–16 years). ( C ) Correlation of CD3+ T cell infiltration with viral DNA load in the myocardium (blue) and BC (red). ( D ) Correlation of CD3+ T cell infiltration with viral load in the myocardium (blue) and plasma (red). CD3+ cell count is presented as the number of cells per mm 2 . B19V DNA load is given as the number of copies per µg of DNA (heart or BC) and per ml of plasma. Data are presented as mean ± SEM; ns, not significant; * p < 0.05, and ** p < 0.01.

Article Snippet: For immunohistological detection of cardiac immune cells, a monoclonal rabbit anti-CD3 antibody (clone SP7, 1:500, Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal mouse anti-human CD68 antibody (clone PG-M1, 1:50) and a monoclonal mouse anti-human HLA-DR alpha-chain antibody (clone TAL.1B5, 1:50), both from DAKO, Hamburg, Germany, were used.

Techniques: Comparison, Cell Counting, Infection, Virus, Clinical Proteomics

Journal: Biomedicines

Article Title: Lymphocytic Myocarditis in Children with Parvovirus B19 Infection: Pathological and Molecular Insights

doi: 10.3390/biomedicines12081909

Figure Lengend Snippet: Histological/immunohistological presentation of fatal myocarditis in a 12-month-old patient ( A ) with cardiac B19V infection. ( A , B ) HE staining of heart tissue shows acute myocarditis characterized by myocyte necrosis and extensive inflammatory infiltrate. ( C – F ) Immunohistochemical staining (brown cells) reveals the presence of many CD3+ T cells ( C ), some CD20+ B cells ( D ), and numerous CD68+ macrophages ( E ), with many of them expressing MHCII ( F ). ( G , H ) Detection of B19V DNA (black signals) via radioactive ISH in endothelial cells of cardiac vessels.

Article Snippet: For immunohistological detection of cardiac immune cells, a monoclonal rabbit anti-CD3 antibody (clone SP7, 1:500, Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal mouse anti-human CD68 antibody (clone PG-M1, 1:50) and a monoclonal mouse anti-human HLA-DR alpha-chain antibody (clone TAL.1B5, 1:50), both from DAKO, Hamburg, Germany, were used.

Techniques: Infection, Staining, Immunohistochemical staining, Expressing

Journal: Biomedicines

Article Title: Lymphocytic Myocarditis in Children with Parvovirus B19 Infection: Pathological and Molecular Insights

doi: 10.3390/biomedicines12081909

Figure Lengend Snippet: Morphological presentation of fatal myocarditis in a 17-month-old patient B after cardiac B19V infection. ( A ) Masson’s trichrome staining of heart tissue shows acute myocarditis with myocyte necrosis, many CD3+ T cells ( B ) and CD68+ macrophages ( C ) comparable to findings in patient A. ( D ) Detection of B19V DNA (black signals) via radioactive ISH in the endothelium of a cardiac vessel.

Article Snippet: For immunohistological detection of cardiac immune cells, a monoclonal rabbit anti-CD3 antibody (clone SP7, 1:500, Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal mouse anti-human CD68 antibody (clone PG-M1, 1:50) and a monoclonal mouse anti-human HLA-DR alpha-chain antibody (clone TAL.1B5, 1:50), both from DAKO, Hamburg, Germany, were used.

Techniques: Infection, Staining

Journal: Biomedicines

Article Title: Lymphocytic Myocarditis in Children with Parvovirus B19 Infection: Pathological and Molecular Insights

doi: 10.3390/biomedicines12081909

Figure Lengend Snippet: B19V replication in B cells of the spleen. Splenic tissue from patient B was immunohistochemically stained for CD20+ B lymphocytes ( A , C , E ) and CD3+ T lymphocytes ( B , D , F ) (visualized in brown). Consecutive radioactive ISH clearly shows the localization of B19V DNA in B cells (black signal) at different magnifications ( A – E ).

Article Snippet: For immunohistological detection of cardiac immune cells, a monoclonal rabbit anti-CD3 antibody (clone SP7, 1:500, Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal mouse anti-human CD68 antibody (clone PG-M1, 1:50) and a monoclonal mouse anti-human HLA-DR alpha-chain antibody (clone TAL.1B5, 1:50), both from DAKO, Hamburg, Germany, were used.

Techniques: Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: IL-10 modRNA transcript produces functional protein in vitro (A–D) IL-10 modRNA produced IL-10 protein in mouse embryonic fibroblasts (MEFs), C2C12 mouse muscle myoblasts, human embryonic kidney 293 (HEK293) cells, and Jurkat cells (human T cells) at different time points by IL-10 ELISA. MEF (2 × 10 4 ), C2C12 (1 × 10 5 ), and 293 cells (1 × 10 5 ) were transfected with 100 ng modRNA. Jurkat cells (5 × 10 5 ) were transfected with 200 ng modRNA. All transfection experiments were performed in 96-well flat plate using Lipofectamine 3000 reagent. (E) Jurkat cells expressed IL-10 protein when transfected with IL-10 modRNA. Jurkat cells (12 × 10 6 ) were transfected without or with 5,000 ng IL-10 modRNA in 6-well plate. Cells were harvested one day after transfection, and 20 μg protein extract was analyzed using western blot assay. β-Actin was used as loading control. (F and G) Jurkat cells treated with IL-10 modRNA secreted functional IL-10 protein, which inhibited IL-2 and IFN-γ production in vitro . Jurkat cells (3 × 10 5 cells per well) transfected with 200 ng IL-10 modRNA were seeded in the top chambers of Transwell 96-well plates. Splenocytes (SPL; 3 × 10 5 cells per well) were seeded in bottom reservoir and stimulated with coated anti-CD3 (2 μg/mL) and soluble anti-CD28 (2 μg/mL). Supernatants in bottom reservoir were collected at different time points and analyzed using IL-2 and IFN-γ ELISA. Statistical data are represented as mean ± SD. The IL-10 modRNA group was compared with the buffer group at different time points (∗∗p < 0.005 and ∗∗∗p < 0.0005, Student’s t test).

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Functional Assay, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: Degree of lymphocytic infiltration into facial allograft (A) Macroscopic and histological changes (indicated by hematoxylin and eosin staining) in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Facial OMC allografts from allotransplanted mice receiving single 10 μg dose of IL-10 modRNA or saline buffer on POD 3 were analyzed. Infiltrating lymphocytes into skin and muscle layers of facial OMC allografts are shown by dense violet staining. Histological Banff classification of facial OMC allografts between buffer and IL-10 modRNA groups is as shown in the right of (A). Each group contains four mice. Scale bars, 50 μm. (B) Immunohistochemistry in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Infiltrating CD3 + T cells or FoxP3 + cells into skin and muscle layers of facial OMC allografts are shown by red staining. Because CD3 is expressed in the cell surface of T cells, the staining displays a circle red. FoxP3 is a critical transcription factor and marker for mouse CD4 + CD25 + natural Tregs, and the staining displays a dense red. Scale bars, 50 μm.

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Staining, Saline, Immunohistochemistry, Marker