Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's disease.

OBJECTIVES: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated. METHODS: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. RESULTS: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-ß, which were normalised by JAKi without cytotoxicity. CONCLUSIONS: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated.

Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's Disease.

Objectives: Inflammatory cytokines that signal through the JAK- STAT pathway, especially interferons (IFNs), are implicated in Sjögren's Disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signaling and effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been reported. Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single cell (sc) RNA sequencing (RNAseq), immunofluorescence microscopy (IF), and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (e.g., focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell-type specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFNß, which were normalized by JAKi without cytotoxicity. Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalizes this aberrant signaling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a Phase Ib/IIa randomized controlled trial to treat SjD with tofacitinib was initiated.

Disruption of Genes Encoding Putative Zwitterionic Capsular Polysaccharides of Diverse Intestinal Bacteroides Reduces the Induction of Host Anti-Inflammatory Factors.

Bacterial zwitterionic capsular polysaccharides (ZPS), such as polysaccharide A (PSA) of the intestinal commensal Bacteroides fragilis, have been shown to modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). We previously used a genomic screen to identify diverse host-associated bacteria with the predicted genetic capacity to produce ZPSs related to PSA of B. fragilis and hypothesized that genetic disruption (KO) of a key functional gene within these operons would reduce the anti-inflammatory activity of these bacteria. We found that ZPS-KO bacteria in two common gut commensals, Bacteroides uniformis and Bacteroides cellulosilyticus, had a reduced ability to induce Tregs and IL-10 in stimulations of human peripheral blood mononuclear cells (PBMCs). Additionally, we found that macrophage stimulated with either wildtype B. fragilis or B. uniformis produced significantly more IL-10 than KOs, indicating a potentially novel function of ZPS of shifting the cytokine response in macrophages to a more anti-inflammatory state. These findings support the hypothesis that these related ZPS may represent a shared strategy to modulate host immune responses.

GZMK+CD8+ T cells Target a Specific Acinar Cell Type in Sjögren's Disease.

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2- seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2.

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV1), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.

Intestinal microbial communities and Holdemanella isolated from HIV+/- men who have sex with men increase frequencies of lamina propria CCR5+ CD4+ T cells.

Men who have sex with men (MSM), regardless of HIV infection status, have an intestinal microbiome that is compositionally distinct from men who have sex with women (MSW) and women. We recently showed HIV-negative MSM have elevated levels of intestinal CD4+ T cells expressing CCR5, a critical co-receptor for HIV. Whether elevated expression of CCR5 is driven by the altered gut microbiome composition in MSM has not been explored. Here we used in vitro stimulation of gut Lamina Propria Mononuclear Cells (LPMCs) with whole intact microbial cells isolated from stool to demonstrate that fecal bacterial communities (FBCs) from HIV-positive/negative MSM induced higher frequencies of CCR5+ CD4+ T cells compared to FBCs from HIV-negative MSW and women. To identify potential microbial drivers, we related the frequency of CCR5+ CD4+ T cells to the abundance of individual microbial taxa in rectal biopsy of HIV-positive/negative MSM and controls, and Holdemanella biformis was strongly associated with increased frequency of CCR5+ CD4+ T cells. We used in vitro stimulation of gut LPMCs with the type strain of H. biformis, a second strain of H.biformis and an isolate of the closely related Holdemanella porci , cultured from either a HIV-positive or a HIV-negative MSM stool. H. porci elevated the frequency of both CCR5+ CD4+ T cells and the ratio of TNF-α/IL-10 Genomic comparisons of the 3 Holdemanella isolates revealed unique cell wall and capsular components, which may be responsible for their differences in immunogenicity. These findings describe a novel mechanism potentially linking intestinal dysbiosis in MSM to HIV transmission and mucosal pathogenesis.

Saliva as a potential matrix for evaluating pharmacologically active dolutegravir concentration in plasma.

Therapeutic drug monitoring (TDM) is used in certain clinically selected cases and in research settings to optimize the response to antiretroviral therapy. Plasma of blood is commonly used for TDM, but blood sampling is invasive and at risk for transmission of infectious agents. On the other hand, saliva sampling is noninvasive, safe, cheap, and easily performed compared to blood. Dolutegravir (DTG) is now widely prescribed as a key component of antiretroviral therapy for HIV infection. In this study, we examined the relationship between DTG concentrations in plasma and saliva of treated patients to explore the possibility of using saliva as an alternative body fluid of TDM. A total of 17 pairs of blood and saliva samples were obtained from 15 consented HIV-1-infected subjects treated with DTG containing regimens for more than one month. Both blood and saliva samples were collected within 1 h of each other. Drug concentrations were determined by liquid chromatography-tandem mass spectrometry using DTG-d5 as an internal standard. The LLOQ was 0.5 ng/mL. The calibration curves were prepared with pooled plasma or saliva containing DTG in a range of 0.5-100 ng/mL with precision of <14.4% and accuracy within ±14.7%. The DTG concentrations in the plasma and saliva were significantly correlated (Pearson's correlation coefficient r = 0.76, p < 0.001). The median ratio of the drug concentration in saliva to those in plasma was 0.0056, which is close to the rate of non-protein-bound DTG in plasma (0.70%), suggesting that only free DTG in plasma is transported to the salivary glands and secreted into saliva. The present study demonstrates that DTG concentration in saliva reflects the pharmacologically active drug concentration in plasma and may provide an easily accessible alternative for monitoring effective antiretroviral treatment.

Disaster Nursing Knowledge and Competencies Among Nursing University Students Participated in Relief Activities Following the 2016 Kumamoto Earthquakes.

OBJECTIVE: This study examined disaster nursing knowledge and competencies among university nursing students who participated in relief activities following the 2016 Kumamoto earthquakes. METHODS: Participants were university nursing students involved in disaster relief activities. Of the 260 individuals to whom a self-report questionnaire was distributed at nursing universities and hospitals located in Kyushu, southwestern Japan, 201 participants returned the questionnaires by mail (response rate 77.3%), of which 108 questionnaires were complete (valid response rate 41.5%). Questions involved experiences related to their volunteer activities, their motive for volunteering, type of relief activities performed, and use of disaster nursing knowledge and competencies. RESULTS: We identified four categories of disaster nursing competencies: "understanding and implementation of assistance to victims in collaboration with other members of the disaster response team," "understanding the natural disaster's influence on victims," "ethical practice in a disaster recovery area," and "understanding of their role within the disaster relief organization." Those who had completed a disaster nursing program had superior measures of performance across all four competencies compared with those who had not yet completed the program. CONCLUSIONS: Four domains of disaster nursing knowledge and competencies used by university nursing students during the 2016 Kumamoto earthquake relief activities were identified. Those who had completed a disaster nursing program had higher competencies than were those who were either currently or not yet enrolled in it.

Factors contributing to salivary human immunodeficiency virus type-1 levels measured by a Poisson distribution-based PCR method.

Objective To elucidate the mechanism underlying secretion of human immunodeficiency virus type 1 (HIV-1) into the oral cavity, by examining the relationships between various oral and systemic factors and the viral load in saliva. Methods Plasma and saliva samples from HIV-1 infected patients were assayed using the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, version 1.0 and a Poisson distribution-based polymerase chain reaction (PCR) method for quantifying HIV-1 RNA and DNA. Results Forty-four pairs of samples were obtained from 18 patients. Salivary viral load was approximately 10% of the plasma viral load, but higher than the plasma load in two patients. The salivary viral DNA load was < 1% of the total HIV-1 nucleic acid load except in one patient who had more viral DNA than RNA. Multiple regression analysis showed that salivary viral load was significantly correlated with plasma viral load (partial correlation coefficient, 0.90) and the community periodontal index (-0.63). Conclusions The present results suggest that excretion through salivary glands, but not occult bleeding, may be a major pathway of HIV-1 into the oral cavity.

Plasma and saliva concentrations of abacavir, tenofovir, darunavir, and raltegravir in HIV-1-infected patients
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OBJECTIVE: We studied the relationships between plasma and saliva concentrations of antiretroviral drugs to explore whether saliva can be used for therapeutic drug monitoring (TDM). METHODS: Abacavir (ABC), tenofovir (TFV), darunavir (DRV), and raltegravir (RAL) in plasma and saliva from 30 HIV-1-infected patients were quantified using liquid chromatography-tandem mass spectrometry. RESULTS: Mean saliva-to-plasma concentration ratios were 0.623 (ABC), 0.024 (TFV), 0.065 (DRV), and 0.0135 (RAL), which agree with the plasma protein binding rates except TFV. Significant correlations were evident between saliva and plasma concentrations of ABC, DRV, and RAL. CONCLUSIONS: This study suggests that plasma concentrations of ABC, DRV, and RAL can be estimated from their saliva concentrations and that the saliva concentration of some antiretroviral drugs reflects the unbound drug concentration in plasma.
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Determination of abacavir, tenofovir, darunavir, and raltegravir in human plasma and saliva using liquid chromatography coupled with tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry assay for the determination of abacavir (ABC), tenofovir (TFV), darunavir (DRV), and raltegravir (RAL) in human plasma and saliva was developed and validated to investigate the applicability of saliva as an appropriate specimen for therapeutic drug monitoring. As internal standards, TFV was chosen for ABC, ABC was chosen for TFV, RAL for DRV, and DRV for RAL. Sample preparation involved protein precipitation with acetonitrile, evaporation of solvent using a centrifugal evaporator, and reconstitution by dissolving the residue in mobile phase. Liquid chromatography was performed on a C18 reverse phase column (1.5 × 50 mm, 5 µm) isocratically at a flow rate of 0.2 mL/min using 5mM formic acid-3% (v/v) acetonitrile as the mobile phase for ABC and TFV and 5mM formic acid-35% (v/v) acetonitrile as the mobile phase for DRV and RAL. The run time was 6 min, and the retention time was approximately 2.0 min for TFV, 2.5 min for RAL, and 4-4.5 min for ABC and DRV. Analytes were detected using tandem mass spectrometry in positive electrospray ionization mode. The precursor/product ion transitions (m/z) were 287.3/191.2 for ABC, 288.5/176.2 for TFV, 548.3/392.3 for DRV, and 445.3/109.5 for RAL, and were monitored on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. The linearity of the assay was assessed in the range 1-10,000 ng/mL for all four drugs. Within-run and between-run mean accuracy, precision, and the extraction recovery for all drugs were -14.5-18.1%, 1.2-13.1%, and 86.0-111.1%, respectively. The proposed assay is sufficiently sensitive and accurate to quantify these drugs in plasma and saliva, and is suitable for investigating the relationship between drug concentrations in plasma and saliva.