Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas

The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process

Predicted location of the putative mutation causing posterior polymorphous corneal dystrophy on 20p11.23.

<p>Filled triangles show 5% significance level of disease mutation location on chromosome 20 in cM; a 60 kb region between markers D20S182 and M189K21.</p

Summary of posterior polymorphous corneal dystrophy study families and subjects.

<p>Number of phenotyped and genotyped affected family members, unaffected first degree relatives and spouses included in this study. Presence of shared haplotype across 20p12.1- 20q12 spanning over 23 Mb as well as the core mini-haplotype at 20p12.1-20p11.23 in affected individuals, previous molecular genetic analysis and geographical origin within the Czech Republic of the eldest family member known to be affected is also shown.</p><p>N = No, Y = yes.</p><p>Linkage analysis for families 1 and 2 was reported in Gwilliam <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045495#pone.0045495-Gwilliam1" target="_blank">[11]</a>. Results of previous candidate gene screening in families 15–19 has been reported in Liskova <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045495#pone.0045495-Liskova2" target="_blank">[23]</a>.</p

Estimated age for posterior polymorphous corneal dystrophy linked to 20p12.1-20p11.23 in Czech patients.

<p>Maximum probability represented by 5% significance interval shown as filled triangles was found at 90 generations (range 64 to 133), i.e. 1800 years assuming a 20-year generation time.</p

Geographical origin of families with posterior polymorphous corneal dystrophy within the southwestern part of the Czech Republic.

<p>The geographical origin of the eldest members of each family is indicated by *. Twelve of the families, of which ten were genotyped and shown to share common haplotype, can be traced to a region of 13 km radius around the town of Klatovy. Two other genotyped families with the common full haplotype spanning over 23 Mb originate from an approximate 40 km radius from Klatovy, however knowledge of the place of origin only extended to three generations in both families.</p