Background Several previous studies from our laboratory had indicated that the salivary gland epithelial cells (SGEC) of primary Sjögren's syndrome (SS) patients manifest cell autonomous (intrinsic) activation, which is demonstrated in non-neoplastic cell lines derived from patients (SS-SGEC). As for other autoimmune diseases, it is widely proposed that infectious factors may incite SS. In fact, several viral agents have been proposed to be involved in the development of SS, including EBV, retroviruses (such as HTLV-1 and endogenous retrotransposons) and the Coxsackie virus. Recently, the sequence and antigen of HDV were detected in the salivary gland tissues of approximately half of SS patients studied [Weller et al., 2016], however, in the absence of detectable hepatitis B virus (HBV) surface antigen and antibodies to HBV or HDV. Therefore, we hypothesized that HDV may be causally related with the cell-autonomous aberrations observed in the SS-SGEC lines and may trigger the inflammatory and autoimmune reactions that characterize SS.
Objectives Herein, using non-neoplastic SGEC lines derived from SS patients, we sought to address directly whether the SGEC of these patients manifest evidence for infection by the HDV virus, despite the fact that patients studied did not display any evidence of infection by HBV or HDV viruses.
Methods For that purpose, we purified total RNA from SGEC lines from non-SS controls (n=7) and SS patients (n=7). Reverse transcription of RNA isolated from salivary gland epithelial cells (SGEC) was performed using random hexamer primers as per the manufacturer's specifications. We performed PCR for the identification of HDV RNA sequences with three sets of primers from different regions of the viral sequence. RNA extracted from HDV-positive sera was used as positive control in HDV-specific PCR.
Results All SGEC lines from SS patients and non-SS controls tested were found positive for HDV RNA, as tested by the first two sets of primers (two sets of primers for first and nested PCR with expected PCR products 329bp and 234bp, respectively). Nevertheless, when we selected primers for another region of the HDV genome (set of primers with expected PCR products 375bp) all RNA samples from SGEC lines tested were found negative for HDV RNA, whereas the HDV RNA control specimen was positive.
Conclusions We conclude that SS-SGEC are not actually infected by the HDV virus, but are probably carriers of an HDV-like sequence. In fact, the putative HDV virus-like sequence has been previously shown be highly homologous to the RNA binding-protein cytoplasmic polyadenylation element-binding protein-3 (CPEB3), which belongs to a family of genes regulating messenger RNA polyadenylation and is structurally and biochemically related to the human HDV ribozymes (Salehi-Ashtiani et al, 2006). We are currently in the process of further analyzing the nature of this HDV-like sequence, through cloning and Sanger sequencing of the RT-PCR products, as well as performing of HDV-specific immunostaining studies in both SGEC lines and salivary gland tissues.
Disclosure of Interest None declared