Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus

Figures

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  Figure 1. Peptide sequences of Coxsackievirus B1 (CVB1)‐2A protease (2Apro) immunized in this study (red bold characters). The region of the sequence chosen for immunization shows low sequence homology with cogent regions of other enteroviruses. cox, Coxsackievirus; ec, echovirus; he, human enterovirus; svdvu, swine vesicular virus; bobev, bovine enterovirus.

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  Figure 2. Enzyme‐linked immunosorbent assay analysis of interactions between antiserum against Coxsackievirus B1 2A protease (CVB1‐2Apro; ET2112) and non‐immune serum and CVB1‐ or CVB3‐2Apro peptides (Figure 1). Mean ± standard deviation, n = 4, *P < 0.05 values between antiserum ET2112/CVB1‐2Apro ‐coated plate (●) and antiserum ET2112/CVB3‐2Apro‐ coated plate (○) data, + P < 0.05 versus non‐immune serum.

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  Figure 3. Polyclonal antibody ET2112 recognizes Coxsackievirus B1 2A protease (CVB1‐2A pro ) expressed in Vero cells. 2Apro is stained positive in scattered Vero cells infected by CVB1 at (a) multiplicity of infection (MOI) 0.1 and (b) 3.0 incubated for 24 h. (c) No positive Vero cells for 2Apro in mock manipulated for 24 h. (d) Enterovirus capsid protein 1 (VP1) was stained positive in Vero cells infected by CVB1 at (d) 0.1 MOI and (e) 3.0 MOI and incubated for 24 h. (f) No positive Vero cells for VP1 in mock manipulated cells for 24 h. Scale bar, 20 μm. (g–p) Double immunostaining for 2Apro (blue) and VP1(red) in Vero cells incubated with or without CVB1 at 3.0 MOI for 24 h. Cytosol of Vero cells are stained positive for (g) 2Apro (blue) and for (h) VP1 (red). (i) Merged image of (g) and (h) shows that 2Apro and VP1 is located in the same cell stained magenta. (j–l) Magnified view of double immunostaining for 2Apro and VP1 in Vero cells show that (j) 2Apro (blue, arrows) and for (k) VP1 (red, arrowheads) locates in the same cell. Nucleus is shown by asterisk. (m) Magnified image of white box [m] in figure l shows that VP1 (red, arrowhead) is localized around/near the nucleus (asterisk)…

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  Figure 4. Absorption test of Coxsackievirus 2A proteinase (2Apro) antibody by immunized peptide. (a) Immunohistochemical staining for enterovirus (EV) 2A pro in the islet of fulminant type 1 diabetes (arrows). (b) After absorption of antiserum, immunostaining of enterovirus was completely abolished. (c) Immunohistochemical staining of the islet on serial section of (a) for enterovirus capsid protein 1 (VP1; arrows) using 5D8/1 is less dense compared with (a) 2Apro staining. Scale bar, 20 μm.

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  Figure 5. Double immunostaining of fulminant type 1 diabetes pancreas for 2A proteinase (2Apro; antiserum ET2112), enterovirus capsid protein 1 (VP1; antibody: 5D8/1) and nucleus (TO‐PRO‐3 iodide; TP3). (a) Immunostaining of the pancreatic islet of fulminant type 1 diabetes for 2Apro using antiserum ET2112 (blue). Scale bar, 40 µm. (b) Immunostaining of the pancreatic islet of fulminant type 1 diabetes for VP1 using antiserum 5D8/1 (red). Scale bar, 40 µm. (c) Merged image of (a) and (b) shows that most VP1‐positive islet cells are also positive for 2Apro (magenta). Magnified photo (inset) shows that VP1 and 2Apro are colocalized in the central part of islet cells (magenta), in addition to 2Apro (blue) being stained in the peripheral cytosolic compartment of islet cells. Scale bar, 40 μm unless otherwise mentioned. (d) Staining of the pancreatic islet cell nuclei of fulminant type 1 diabetes with TP3. (e) Staining of the pancreatic pseudo‐atrophied islet of fulminant type 1 diabetes for 2Apro using antiserum ET2112. (f) Immunostaining of the pancreatic pseudo‐atrophied islet of fulminant type 1 diabetes for VP1 using antiserum 5D8/1. (g) Merged image of (e) and (f) shows that 2Apro and VP1…

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  Figure 6. (a) Immunostaining for 2A proteinase (2Apro) using antiserum ET2112 in the formaldehyde‐fixed paraffin‐embedded pancreas obtained from a fulminant type 1 diabetes patient shows dense immunostaining of islets cells. No positive staining for 2Apro using antiserum ET2112 was observed in (b) formaldehyde‐fixed paraffin‐embedded pancreas, (c) duodenum, (d) stomach, (e) colon, (f) liver and (g) thyroid gland of non‐diabetes autopsied individuals.