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Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research.

Maccari G, Genoni A, Sansonno S, Toniolo A - Sci Rep (2016)

Bottom Line: Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects.When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings.However, their epitopes may align with a few human proteins at low expected values.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanotechnology and Innovation, Italian Institute of Technology, Pisa, Italy.

ABSTRACT
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.

No MeSH data available.


Related in: MedlinePlus

Indirect immunofluorescence of the human AV3 cell line: staining of enterovirus-infected cells with MAbs 9D5 and 5D-8.1 (green; counterstaining, Evans blue).Uninfected cells (top panel), cells acutely infected with CV-B4 (middle panel), cells persistently infected with CV-B1pc (lower panel).At the concentration of 5 μg/ml, MAb 9D5 did not produce fluorescence in uninfected cells (a). 5D-8.1 produced fine granular cytoplasmic fluorescence in uninfected cells at the concentration of 1 μg/ml (b). Background staining disappeared when this antibody was used at concentrations ≤1 μg/ml (c). AV3 cells 4 hrs post-infection with CV-B4: staining by 5D-8.1 (d,e), or 9D5 (f).AV3 cells undergoing persistent infection by the CV-B1pc strain. 5D-8.1: dotted cytoplasmic fluorescence (g). 9D5: VP1 staining frequently seen in cells showing mitotic bars (h) or dividing (i). Original magnification: 20× (a–e) or 100× (f–i).
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f4: Indirect immunofluorescence of the human AV3 cell line: staining of enterovirus-infected cells with MAbs 9D5 and 5D-8.1 (green; counterstaining, Evans blue).Uninfected cells (top panel), cells acutely infected with CV-B4 (middle panel), cells persistently infected with CV-B1pc (lower panel).At the concentration of 5 μg/ml, MAb 9D5 did not produce fluorescence in uninfected cells (a). 5D-8.1 produced fine granular cytoplasmic fluorescence in uninfected cells at the concentration of 1 μg/ml (b). Background staining disappeared when this antibody was used at concentrations ≤1 μg/ml (c). AV3 cells 4 hrs post-infection with CV-B4: staining by 5D-8.1 (d,e), or 9D5 (f).AV3 cells undergoing persistent infection by the CV-B1pc strain. 5D-8.1: dotted cytoplasmic fluorescence (g). 9D5: VP1 staining frequently seen in cells showing mitotic bars (h) or dividing (i). Original magnification: 20× (a–e) or 100× (f–i).

Mentions: In uninfected cells (AV3 and LLC-MK2 lines) MAb 9D5 did not produce fluorescence even at the concentration of 5 μg/ml (Fig. 4a), while 5D-8.1 yielded fine perinuclear and cytoplasmic fluorescence when used at the concentration of 1 μg/ml (Fig. 4b), but not at concentrations ≤1 μg/ml (Fig. 4c). The two investigated MAbs produced dotted cytoplasmic fluorescence in human and monkey cells acutely infected with CV-B4 (Fig. 4d–f). Fine dotted fluorescence was also seen in AV3 cells undergoing persistent infection by the CV-B1pc strain isolated from a case of pancreatic carcinoma (Fig. 4g–i). In persistent infection, VP1 was expressed frequently in cells showing mitotic bars or dividing (Fig. 4h,i). The slow infectious process was not accompanied by manifest CPE.


Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research.

Maccari G, Genoni A, Sansonno S, Toniolo A - Sci Rep (2016)

Indirect immunofluorescence of the human AV3 cell line: staining of enterovirus-infected cells with MAbs 9D5 and 5D-8.1 (green; counterstaining, Evans blue).Uninfected cells (top panel), cells acutely infected with CV-B4 (middle panel), cells persistently infected with CV-B1pc (lower panel).At the concentration of 5 μg/ml, MAb 9D5 did not produce fluorescence in uninfected cells (a). 5D-8.1 produced fine granular cytoplasmic fluorescence in uninfected cells at the concentration of 1 μg/ml (b). Background staining disappeared when this antibody was used at concentrations ≤1 μg/ml (c). AV3 cells 4 hrs post-infection with CV-B4: staining by 5D-8.1 (d,e), or 9D5 (f).AV3 cells undergoing persistent infection by the CV-B1pc strain. 5D-8.1: dotted cytoplasmic fluorescence (g). 9D5: VP1 staining frequently seen in cells showing mitotic bars (h) or dividing (i). Original magnification: 20× (a–e) or 100× (f–i).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835795&req=5

f4: Indirect immunofluorescence of the human AV3 cell line: staining of enterovirus-infected cells with MAbs 9D5 and 5D-8.1 (green; counterstaining, Evans blue).Uninfected cells (top panel), cells acutely infected with CV-B4 (middle panel), cells persistently infected with CV-B1pc (lower panel).At the concentration of 5 μg/ml, MAb 9D5 did not produce fluorescence in uninfected cells (a). 5D-8.1 produced fine granular cytoplasmic fluorescence in uninfected cells at the concentration of 1 μg/ml (b). Background staining disappeared when this antibody was used at concentrations ≤1 μg/ml (c). AV3 cells 4 hrs post-infection with CV-B4: staining by 5D-8.1 (d,e), or 9D5 (f).AV3 cells undergoing persistent infection by the CV-B1pc strain. 5D-8.1: dotted cytoplasmic fluorescence (g). 9D5: VP1 staining frequently seen in cells showing mitotic bars (h) or dividing (i). Original magnification: 20× (a–e) or 100× (f–i).
Mentions: In uninfected cells (AV3 and LLC-MK2 lines) MAb 9D5 did not produce fluorescence even at the concentration of 5 μg/ml (Fig. 4a), while 5D-8.1 yielded fine perinuclear and cytoplasmic fluorescence when used at the concentration of 1 μg/ml (Fig. 4b), but not at concentrations ≤1 μg/ml (Fig. 4c). The two investigated MAbs produced dotted cytoplasmic fluorescence in human and monkey cells acutely infected with CV-B4 (Fig. 4d–f). Fine dotted fluorescence was also seen in AV3 cells undergoing persistent infection by the CV-B1pc strain isolated from a case of pancreatic carcinoma (Fig. 4g–i). In persistent infection, VP1 was expressed frequently in cells showing mitotic bars or dividing (Fig. 4h,i). The slow infectious process was not accompanied by manifest CPE.

Bottom Line: Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects.When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings.However, their epitopes may align with a few human proteins at low expected values.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanotechnology and Innovation, Italian Institute of Technology, Pisa, Italy.

ABSTRACT
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.

No MeSH data available.


Related in: MedlinePlus