**4.3. Antigenicity of Herp for anti-dsDNA Ab production in mice**

Immunization of normal BALB/c mice with Herp elicited anti-dsDNA Abs and caused glomerular IgG deposition [62]. However, urinary protein level did not increase and overt nephritis did not develop. The pathological changes in the kidneys in Herp-immunized BALB/c mice went no further than silent lupus nephritis.

Nucleosomes, which are major autoantigens in SLE, are exposed at the apoptotic cell surface [66,67]. Anti-nucleosome Abs are present in SLE at a rate of more than 50% and they have been linked to lupus nephritis [68]. Nucleosomes and histones are present in glomerular deposits [69]. Nucleosomes bind to glomerular endothelial cells and serve as targets for antinucleosome Abs [70]. Therefore, a portion of anti-nucleosome Abs may be involved in lupus nephritis [71]. However, even oligonucleosomes are much less effective than Herp in inducing anti-nucleosome Abs as well as anti-dsDNA Abs [62]. Therefore, to reproduce overt lupus nephritis, BALB/c mice were immunized with Herp followed by immunization with oligonucleosomes. In this procedure, both anti-dsDNA Ab and anti-nucleosome Abproducing clones induced by Herp may be able to recognize oligonucleosomes easily. The production of anti-dsDNA Abs and glomerular IgG deposition were observed in all mice. In addition, overt nephritis with significant proteinuria occurred in one mouse (Figure 2). Although further investigations are in progress to define the mechanisms, it was speculated that (i) the Herp-induced anti-dsDNA Abs efficiently bound to nucleosomes and formed pathogenic immune complexes, and (ii) affinity maturation and epitope spreading of Herpinduced anti-dsDNA Abs occurred by nucleosomes.

**Figure 2.** Overt nephritis in a BALB/c mouse immunized with Herp followed by immunization with oligonucleosomes. Left: Periodic acid Schiff (PAS) staining. Right: Immunofluorescence staining with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG Ab.

[Methods] Five 6-week-old female BALB/c mice were immunized intraperitoneally with 100 g of Herp on days 0 and 10 and 50 g of Herp on day 20, followed by immunization with 10 g of oligonucleosomes on days 30, 40, and 50. Preparation of Herp and oligonucleosomes was described previously [62]. Fresh-frozen tissue sections 4 m thick were fixed in 100% acetone for 10 min at 4°C and blocked with 5% normal goat serum and 3% BSA in PBS overnight at 4°C. Sections were stained with FITC-conjugated goat F(ab')2 anti-mouse IgG Ab (KPL) for 1 h at room temperature.
