Immunoassay Detects Salivary Anti-SSA/Ro-52 Autoantibodies in Seronegative Patients with Primary Sjögren's Syndrome.

The diagnostic work-up for Sjögren's syndrome is challenging and complex, including testing for serum autoantibodies to SSA/Ro and a labial salivary gland biopsy. Furthermore, the diagnosis is often delayed. In this study, we tested the hypothesis that anti-SSA/Ro autoantibodies are detectable in the saliva of patients with primary Sjögren's syndrome (pSS) because the disease affects the salivary glands, and these autoantibodies display greater discriminatory performance in saliva than in serum. SSA/Ro-52 Ags were used to develop what is, to our knowledge, a novel quantitative electrochemical-based immunoassay: the electric field-induced release and measurement (EFIRM) platform. The clinical utility was determined by measuring salivary anti-SSA/Ro-52 autoantibodies in patients with pSS and sicca (n = 34), patients without pSS with sicca (n = 35), and healthy subjects (n = 41). The statistical analysis of discrimination included the area under the receiver operating characteristic curve. Salivary anti-SSA/Ro-52 autoantibodies were measured in 94% (32 of 34) of patients with pSS with 85% (29 of 34) seropositivity. Four of the five seronegative patients with pSS had EFIRM-measurable anti-SSA/Ro-52 autoantibodies in saliva. Additionally, 60% (21 of 35) of the seronegative patients without pSS who had sicca had EFIRM-detectable SSA/Ro-52 autoantibodies in saliva, indicating the onset of autoimmune disease. Two of the 41 healthy control subjects had EFIRM-detectable SSA/Ro-52 autoantibodies in their saliva. Salivary SSA/Ro-52 autoantibodies significantly discriminated patients with pSS or patients with the initial stage of autoimmune disease from healthy subjects with an area under the receiver operating characteristic curve of 0.91. Our findings suggest that the proposed saliva SSA/Ro-52 immunoassay improves early and accurate diagnosis of seronegative patients with pSS and patients with early-onset autoimmune disease.

Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this antiresorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesions. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved proinflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.

SIRPα sequesters SHP-2 to promote IL-4 and IL-13 signaling and the alternative activation of macrophages.

The cytokines interleukin-4 (IL-4) and IL-13 bind to their shared receptor subunit IL-4Rα to direct the alternative activation of macrophages to promote immunosuppression and wound healing. Activated IL-4 and IL-13 receptors recruit the tyrosine phosphatases SHP-1 and SHP-2, which dephosphorylate and inhibit the IL-4Rα subunit. Here, we report that the immunoreceptor SIRPα spatially restricted SHP-2 to promote IL-4 and IL-13 signaling and the alternative activation of macrophages. This effect required the cytoplasmic ITIMs or ITSMs of SIRPα, which underwent tyrosine phosphorylation by Bruton's tyrosine kinase (Btk) that was activated in response to IL-4 and IL-13. This phosphorylation event resulted in the recruitment of SHP-2 to SIRPα and prevented it from binding to and inhibiting IL-4R and IL-13R. Binding of the ligand CD47 to the SIRPα extracellular domain promoted the Btk-mediated phosphorylation of the SIRPα cytoplasmic domain and hence SHP-2 sequestration. Conversely, loss of SIRPα enabled SHP-2 to bind to the γC and IL-13Rα1 subunits of IL-4R and IL-13R, respectively, and dephosphorylate IL-4Rα, dampening its signaling. Impaired wound healing in Sirpα−/− mice with experimental colitis correlated with a deficit of immunosuppressive macrophages in the colon, a condition that was corrected by transfusion of ex vivo­produced, alternatively activated SIRPαhigh macrophages. These studies reveal a previously unappreciated role for SIRPα in promoting IL-4 and IL-13 signaling and reveal the mechanistic basis by which SIRPα enhances the alternative activation of macrophages.

Role of pancreatic cancer-derived exosomes in salivary biomarker development.

Recent studies have demonstrated that discriminatory salivary biomarkers can be readily detected upon the development of systemic diseases such as pancreatic cancer, breast cancer, lung cancer, and ovarian cancer. However, the utility of salivary biomarkers for the detection of systemic diseases has been undermined due to the absence of the biological and mechanistic rationale as to why distal diseases from the oral cavity would lead to the development of discriminatory biomarkers in saliva. Here, we examine the hypothesis that pancreatic tumor-derived exosomes are mechanistically involved in the development of pancreatic cancer-discriminatory salivary transcriptomic biomarkers. We first developed a pancreatic cancer mouse model that yielded discriminatory salivary biomarkers by implanting the mouse pancreatic cancer cell line Panc02 into the pancreas of the syngeneic host C57BL/6. The role of pancreatic cancer-derived exosomes in the development of discriminatory salivary biomarkers was then tested by engineering a Panc02 cell line that is suppressed for exosome biogenesis, implanting into the C56BL/6 mouse, and examining whether the discriminatory salivary biomarker profile was ablated or disrupted. Suppression of exosome biogenesis results in the ablation of discriminatory salivary biomarker development. This study supports that tumor-derived exosomes provide a mechanism in the development of discriminatory biomarkers in saliva and distal systemic diseases.

DNA-mediated assembly of protein heterodimers on membrane surfaces.

We present a method based on self-assembling oligonucleotides to anchor proteins to a supported membrane surface. This anchoring method allows control of the surface density of multiple proteins. By incorporating additional recognition sequences into the DNA linkers, defined heterodimers can be produced upon the addition of a heterospecific DNA cross-linking strand. Characterization by fluorescence cross-correlation spectroscopy (FCCS) confirmed lateral mobility and the formation of specific heterodimers. We further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells.