Distinctive profile of monomeric and polymeric anti-SSA/Ro52 immunoglobulin A1 isoforms in saliva of patients with primary Sjögren's syndrome and Sicca.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is the second most common chronic autoimmune connective tissue disease. Autoantibodies, immunoglobulin (IgG) anti-SSA/Ro, in serum is a key diagnostic feature of pSS. Since pSS is a disease of the salivary gland, we investigated anti-SSA/Ro52 in saliva. METHODS: Using a novel electrochemical detection platform, Electric Field-Induced Release and Measurement, we measured IgG/M/A, IgG, IgA, IgA isotypes (IgA1 and IgA2) and IgA1 subclasses (polymeric and monomeric IgA1) to anti-SSA/Ro52 in saliva supernatant of 34 pSS, 35 dry eyes and dry mouth (patients with Sicca) and 41 health controls. RESULTS: Saliva IgG/M/A, IgG, IgA, IgA isotypes and IgA1 subclasses to anti-SSA/Ro52 differed significantly between pSS, non-pSS Sicca and healthy subjects. Elevated monomeric IgA1 was observed in patients with non-pSS Sicca while elevated polymeric IgA1 (pIgA1) was observed in patients with pSS. Salivary polymeric but not monomeric IgA1 (mIgA1) isoform correlated with focus score (r2=0.467, p=0.001) CONCLUSIONS: Salivary anti-Ro52 polymeric IgA1 isoform is associated with glandular inflammation in pSS, while salivary monomeric IgA1 is associated with Sicca. Whether IgA1 isotope switching plays a role in the progression of the Sicca to pSS warrants further investigation.

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.

Direct Detection of 4-Dimensions of SARS-CoV-2: Infection (vRNA), Infectivity (Antigen), Binding Antibody, and Functional Neutralizing Antibody in Saliva.

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (EFIRM) technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA-approved tests. The vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID50/mL, respectively. The vRNA assay differentiated between acutely infected (n=10) and infection-naïve patients (n=33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n=35), vaccinated with prior infection (n=13), and vaccinated infection-naïve patients (n=13) from control (n=81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with an LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n=31) and pre-pandemic controls (n=60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%. Our multiparameter assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics.

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.

Machine learning prediction of COVID-19 severity levels from salivaomics data.

The clinical spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the strain of coronavirus that caused the COVID-19 pandemic, is broad, extending from asymptomatic infection to severe immunopulmonary reactions that, if not categorized properly, may be life-threatening. Researchers rate COVID-19 patients on a scale from 1 to 8 according to the severity level of COVID-19, 1 being healthy and 8 being extremely sick, based on a multitude of factors including number of clinic visits, days since the first sign of symptoms, and more. However, there are two issues with the current state of severity level designation. Firstly, there exists variation among researchers in determining these patient scores, which may lead to improper treatment. Secondly, researchers use a variety of metrics to determine patient severity level, including metrics involving plasma collection that require invasive procedures. This project aims to remedy both issues by introducing a machine learning framework that unifies severity level designations based on noninvasive saliva biomarkers. Our results show that we can successfully use machine learning on salivaomics data to predict the severity level of COVID-19 patients, indicating the presence of viral load using saliva biomarkers.

Variability in Severe Acute Respiratory Syndrome Coronavirus 2 IgG Antibody Affinity to Omicron and Delta Variants in Convalescent and Community mRNA-Vaccinated Individuals.

The emergence of the omicron and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters, and whether existing mRNA vaccines for the original SARS-CoV-2 strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1-specific salivary Abs to the omicron and delta variants using a cohort of mRNA-vaccinated (n = 109) and convalescent (n = 19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5, 26.7, and 14.7% in measurable signal to the omicron strain and 13.4, 2.4, and -6.4% mean reduction to the delta variant for convalescent, Pfizer-, and Moderna-vaccinated groups, respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves.

Variability in SARS-Cov-2 IgG Antibody Affinity To Omicron and Delta Variants in Convalescent and Community mRNA Vaccinated Individuals.

The emergence of Omicron and Delta variants of SARS-CoV-2 has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters and whether existing mRNA vaccines for the wildtype strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1 specific salivary antibodies to the Omicron and Delta variants using a cohort of mRNA vaccinated (n=109) and convalescent (n=19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5%, 26.7%, and 14.7% in measurable signal to the Omicron strain and 13.4%, 2.4%, and âˆ'6.4% percent mean reduction to the Delta Variant for convalescent, Pfizer, and Moderna vaccinated groups respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves. Key Points: AMPERIAL assay developed to quantify salivary SARS-CoV-2 S1 IgG antibodies to Omicron and Delta variantsThere was a reduction in affinity to both Delta and Omicron VariantsThe reduction in affinity was more pronounced to Omicron than for Delta VariantsThere was a significant difference between IgG affinities in Individuals vaccinated with Pfizer versus Moderna Vaccines.

The Kinetics of COVID-19 Vaccine Response in a Community-Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG Abs to SARS-CoV-2 S1 Ag in saliva to investigate the kinetics of Ab response in a community-based population that had received either the Pfizer or Moderna mRNA-based vaccine. Samples were received from a total of 97 individuals, including a subset of 42 individuals who collected samples twice weekly for 3 mo or longer. In all, >840 samples were collected and analyzed. In all individuals, salivary SARS-CoV-2 S1 IgG Ab levels rose sharply in the 2-wk period after their second vaccination, with peak Ab levels seen at 10-20 d after vaccination. We observed that 20%, 10%, and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop, respectively, from peak levels during the duration of monitoring, and in two patients, Abs fell to prevaccination levels (5%). The use of noninvasive quantitative salivary Ab measurement can allow widespread, cost-effective monitoring of vaccine response.

The Kinetics of COVID-19 Vaccine Response in a Community Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG antibodies to SARS-CoV-2 S1 in saliva to investigate the kinetics of antibody response in a community-based population who had received either the Pfizer or Moderna mRNA-based vaccines. Samples were received from a total of 97 individuals including a subset of 42 individuals who collected samples twice-weekly for 3 months or longer. In all, 840 samples were collected and analyzed. In all individuals, salivary antibody levels rose sharply in the 2-week period following their second vaccination, with peak antibody levels being at 10-20 days post-vaccination. We observed that 20%, 10% and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop respectively from peak levels during the duration of monitoring and two patients fell to pre-vaccination levels (5%). The use of non-invasive quantitative salivary antibody measurement can allow widespread, cost-effective monitoring of vaccine response. ARTICLE SUMMARY LINE: COVID-19 antibodies were measured in saliva and 20% of vaccinated subjects experienced a 90% drop in peak antibody levels over the course of monitoring.

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.

Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva.

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Development and validation of a highly sensitive and specific electrochemical assay to quantify anti-SARS-CoV-2 IgG antibodies to facilitate pandemic surveillance and monitoring of vaccine response.

Amperial™ is a novel assay platform that uses immobilized antigen in a conductive polymer gel followed by an electrochemical detection. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARSCoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

RNAPro•SAL: a device for rapid and standardized collection of saliva RNA and proteins.

The stabilization and processing of salivary transcriptome and proteome biomarkers is a critical challenge due to the ubiquitous nature of nucleases and proteases as well as the inherent instability of these biomarkers. Furthermore, extension of salivary transcriptome and proteome analysis to point-of-care and remote sites requires the availability of self-administered ambient temperature collection and storage tools. To address these challenges, a self-contained whole saliva collection and extraction system, RNAPro•SAL, has been developed that provides rapid ambient temperature collection along with concurrent processing and stabilization of extracellular RNA (exRNA) and proteins. The system was compared to the University of California, Los Angeles (UCLA) standard clinical collection process (standard operating procedure, SOP). Both systems measured total RNA and protein, and exRNA IL-8, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ß-actin and ribosomal protein S9 (RPS9) by qPCR. Proteome analysis was measured by EIA analysis of interleukin-8 (IL-8), and ß-actin, as well as total protein. Over 97% of viable cells were removed by both methods. The system compared favorably to the labor-intensive clinical SOP, which requires low-temperature collection and isolation, yielding samples with similar protein and exRNA recovery and stability.

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.