B cell specificity and pattern in primary Sjögren’s syndrome - Studies in humans and a murine model

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterised by focal inflammation of exocrine glands, particularly salivary and lacrimal glands. Here, mononuclear cells, including B cells, infiltrate the glands, leading to dysfunction and later destruction of the glandular tissue. It thereby results in the common symptoms of dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia). Another distinctive feature of this disease is the systemic production of autoantibodies such as Ro/SSA and La/SSB. This autoantibody production results from the activation of B cells into antibody secreting short- and long-lived plasma cells. Hence, although the etiology of SS remains unclear, B cells do play an important part in the pathogenesis of this disease. In this doctoral work we address the concept of B cell specificity and pattern in primary SS (pSS), where we consider both the general and the autoantigenspecific B cell pattern in the peripheral blood and the salivary glands of patients with pSS. Additionally, we also account for the expression pattern of the Ro52 autoantigen in the salivary glands of pSS patients with regard to level of inflammation. Furthermore, in order to compare the plasma cell pattern before the onset of disease in relation to advanced disease and characterise the plasma cell compartment in the parotid and submandibular salivary glands and in the bone marrow, we explore a congenic NOD mouse strain, namely NOD.B10.H2b. Our general findings disclose a low number of autoantigen-specific memory B cells that are observed alongside high levels of plasma cells both in the peripheral blood and the salivary glands of patients with pSS. Moreover, we also demonstrate a correlation between the ductal epithelial expression of Ro52 and the level of inflammation in the salivary glands of pSS patients. By the application of the NOD.B10.H2b model, we observe an accumulation of longlived plasma cells in the parotid and submandibular salivary glands of mouse that coincides with our observations in lower labial salivary glands of the pSS patients

The expression of B & T cell activation markers in children’s tonsils following live attenuated influenza vaccine

Live attenuated influenza vaccines (LAIV) can prevent influenza illness and death in children. The absence of known correlates of protection induced by LAIV requires human studies of underlying mechanisms of vaccine-induced immunity, to further elucidate the immunological processes occurring. In this study, children scheduled for elective tonsillectomy were enrolled in a clinical trial to evaluate the immune response to LAIV, in order to compare T and B cell gene expression profiles. Twenty-three children (aged 3–17 years) were divided into 4 groups; unvaccinated controls, or vaccinated intranasally with LAIV at days 3–4, 6–7, and 12–15 before tonsillectomy. Total RNA extraction was performed on tonsillar tissue and high RNA quality was assured. The samples were then analyzed using a validated RT2 Profiler PCR Array containing 84 gene-specific primers involved in B and T cell activation, proliferation, differentiation, regulation and polarization. The gene expression after LAIV vaccination was subsequently compared to the controls. We observed that at d 3–4 post vaccination, 6 genes were down-regulated, namely APC, CD3G, FASLG, IL7, CD8A and TLR1. Meanwhile at 6–7 days post vaccination, 9 genes were significantly up-regulated, including RIPK2, TGFB1, MICB, SOCS1, IL2RA, MS4A1, PTPRC, IL2 and IL8. By days 12–15 the genes RIPK2, IL4, IL12B and TLR2 were overexpressed. RIPK2 was upregulated at all 3 time points. Our data suggests an overall proliferation, differentiation and regulation of B and T cells in the tonsils following LAIV, where the majority of genes were up-regulated at days 6–7 and normalized by days 12–15. These findings may provide a first step into defining future biomarkers or correlates of protection after LAIV immunization

Expression of NGAL-specific cells and mRNA levels correlate with inflammation in the salivary gland, and its overexpression in the saliva, of patients with primary Sjögren’s syndrome

Salivary gland involvement is a characteristic feature of primary Sjögren’s syndrome (pSS), where tissue destruction is mediated by infiltrating immune cells, and may be accompanied by the presence of adipose tissue. Optimally diagnosing this multifactorial disease requires the incorporation of additional routines. Screening for disease-specific biomarkers in biological fluid could be a promising approach to increase diagnostic accuracy. We have previously investigated disease biomarkers in saliva and tear fluid of pSS patients, identifying Neutrophil gelatinase-associated lipocalin (NGAL) as the most upregulated protein in pSS. In the current study, we aimed to explore for the first time NGAL expression at the site of inflammation in the pSS disease target organ. Immunohistochemical staining was conducted on minor salivary gland biopsies from 11 pSS patients and 11 non-SS sicca subjects, targeting NGAL-specific cells. Additional NGAL/PNAd double staining was performed to study NGAL expression in high endothelial venules, known as specialised vascular structures. Moreover, NGAL mRNA expression was measured utilising quantitative real-time polymerase chain reaction (qRT-PCR) on minor salivary gland biopsies from 15 pSS patients and 7 non-SS sicca individuals that served as tissue controls. Our results demonstrated NGAL expression in acinar and ductal epithelium within the salivary gland of pSS patients, where significantly greater levels of acinar NGAL were observed in pSS patients (p < .0018) when compared to non-SS subjects. Also, acinar expression positively correlated with focus score values (r 2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r 2 = 0.74, p < .003). Some PNAD+ endothelial venules also expressed NGAL. An increase in NGAL staining with increased fatty replacement was also observed in pSS patients. Concurringly, a 27% increase in NGAL mRNA levels were also detected in the minor salivary glands of pSS patients when compared to non-SS tissue control subjects. In conclusion, there is a positive association between increase in NGAL expression and inflammation in the pSS disease target organ, which also coincides with its previously demonstrated upregulation in the saliva of pSS patients. Additional functional analyses are needed to better understand the immunological implications of this potential biomarker

Expression of NGAL-specific cells and mRNA levels correlate with inflammation in the salivary gland, and its overexpression in the saliva, of patients with primary Sjögren’s syndrome

Salivary gland involvement is a characteristic feature of primary Sjögren’s syndrome (pSS), where tissue destruction is mediated by infiltrating immune cells, and may be accompanied by the presence of adipose tissue. Optimally diagnosing this multifactorial disease requires the incorporation of additional routines. Screening for disease-specific biomarkers in biological fluid could be a promising approach to increase diagnostic accuracy. We have previously investigated disease biomarkers in saliva and tear fluid of pSS patients, identifying Neutrophil gelatinase-associated lipocalin (NGAL) as the most upregulated protein in pSS. In the current study, we aimed to explore for the first time NGAL expression at the site of inflammation in the pSS disease target organ. Immunohistochemical staining was conducted on minor salivary gland biopsies from 11 pSS patients and 11 non-SS sicca subjects, targeting NGAL-specific cells. Additional NGAL/PNAd double staining was performed to study NGAL expression in high endothelial venules, known as specialised vascular structures. Moreover, NGAL mRNA expression was measured utilising quantitative real-time polymerase chain reaction (qRT-PCR) on minor salivary gland biopsies from 15 pSS patients and 7 non-SS sicca individuals that served as tissue controls. Our results demonstrated NGAL expression in acinar and ductal epithelium within the salivary gland of pSS patients, where significantly greater levels of acinar NGAL were observed in pSS patients (p < .0018) when compared to non-SS subjects. Also, acinar expression positively correlated with focus score values (r 2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r 2 = 0.74, p < .003). Some PNAD+ endothelial venules also expressed NGAL. An increase in NGAL staining with increased fatty replacement was also observed in pSS patients. Concurringly, a 27% increase in NGAL mRNA levels were also detected in the minor salivary glands of pSS patients when compared to non-SS tissue control subjects. In conclusion, there is a positive association between increase in NGAL expression and inflammation in the pSS disease target organ, which also coincides with its previously demonstrated upregulation in the saliva of pSS patients. Additional functional analyses are needed to better understand the immunological implications of this potential biomarker

Pulmonary changes in Norwegian fatal cases of pandemic influenza H1N1 (2009) infection: a morphologic and molecular genetic study

Background: During the pandemic outbreak of the 2009 swine influenza (A(H1N1) pdm09), 32 fatal cases occurred in Norway and 19 of these were included in this study. Objectives: We characterised pulmonary changes in these fatal Norwegian cases. Patients and Methods: Upon hospitalisation, detailed clinical information and specimens from the upper and lower respiratory pathways were collected. At post-mortem, lung tissue was collected, formalin-fixed and paraffin-embedded. Immunohistochemical and light microscopic examination was performed to visualise the local expression of the A(H1N1) pdm09 virus. Reverse transcription-polymerase chain reaction (RT-PCR) and pyrosequencing of the non-fixed specimens allowed the identification of mutations in the influenza virus surface glycoprotein (haemagglutinin gene) particularly at position 222. Results and Conclusions: The overall course of illness lasted from 2 to 40 days (median 9 days). Diffused alveolar damage (DAD) was evident in 11 cases, 4 of which had no apparent underlying illness. Obesity was prominent in 12 cases, where three individuals were classified as otherwise healthy. The HA D222G mutation was detected in six cases, 3 of which had no underlying illness. Immunohistochemistry showed the A(H1N1)pdm09 virus to be prominent at the site of inflammation both in close proximity to and inside alveolar structures in the lung tissue. In addition to a possible role for the HA D222G mutation, our findings indicate that host factors and underlying conditions in the infected individuals are fundamental for disease outcome in many cases. This study increases our understanding of determinants for the clinical outcome of pandemic influenza, which could guide future treatment

Severity of clinical dry eye manifestations influences protein expression in tear fluid of patients with primary Sjögren's syndrome

Ocular dryness is a characteristic feature of primary Sjögren’s syndrome (pSS). This may result in dry eye disease (DED), leading to damage of the ocular surface. Additional, non-invasive diagnostic techniques are needed when evaluating pSS patients. Hence, screening for disease-specific biomarkers in biological fluid could be promising. We have previously examined the proteome of tear fluid from pSS patients through Liquid chromatography-mass spectrometry (LC-MS), and conducted a thorough ocular evaluation of patients with pSS. In this study we further explored the association between dry eye manifestations and protein expression in tear fluid of pSS patients. Medical history of 27 patients and 32 healthy controls was gathered. Subjective complaints were registered through questionnaires. Objective findings including tear osmolarity, tear film break up time (TFBUT), Schirmer’s test, and ocular and corneal surface staining were also recorded. LC-MS was conducted formerly on tear fluid from all subjects in order to generate proteomic biomarker profiles. Scaffold was employed to analyse the LC-MS data for quantitative differences between patient and control groups, and the mean spectral counts were calculated for the five most upregulated proteins in relation to DED manifestations. Dysregulated cellular processes were identified in pSS patients using FunRichv3 enrichment analysis. The five most upregulated proteins previously identified in pSS patients were DNA (apurinic or apyrimidinic site) lyase (APEX1), thioredoxin-dependent peroxidase reductase (PRDX3), copine (CPNE1), aconitate hydratase (ACO2), and LIM domain only protein 7 (LMO7), in descending order. A significant increase in mean spectral counts for these proteins were observed in pSS patients with pathological DED manifestations compared to healthy controls (p<0.0001). Consequently, dysregulated cellular pathways involving innate and adaptive immunity were also detected. In conclusion, our observations suggest a relationship between presence of dry eye signs and upregulated proteins in tear fluid from patients with pSS. Further studies are needed in order to replicate the concepts explored and analyses performed in a greater cohort of pSS patients, where sensitivity and specificity of the methods conducted can also be verified further

Proteomic and histopathological characterisation of sicca subjects and primary Sjögren’s syndrome patients reveals promising tear, saliva and extracellular vesicle disease biomarkers

Background Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren’s syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals. Methods Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses. Results Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44). Conclusions Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression

Interdisciplinary, Comprehensive Oral and Ocular Evaluation of Patients with Primary Sjögren’s Syndrome

Abstract A comprehensive evaluation of oral and ocular symptoms and findings in primary Sjögren’s syndrome (pSS) patients may provide valuable information for management. Medical history was obtained from female pSS patients, and sex- and age-matched non-SS patients with sicca symptoms (non-SS sicca controls) as well as healthy subjects without sicca complaints (healthy controls). Oral (Summated Xerostomia Inventory, SXI) and ocular (McMonnies Dry Eye questionnaire, MDEIS, and Ocular Surface Disease Index, OSDI) subjective complaints were recorded. Objective findings including clinical oral dryness scores (CODS), unstimulated and stimulated saliva secretion rates (UWS/SWS), Schirmer I test, tear osmolarity, tear film break-up time (TFBUT), and ocular surface staining (OSS) were determined. The pSS and non-SS sicca controls were extensively troubled by subjective dryness, while the pSS group had higher CODS, significantly lower saliva and tear secretion, shorter TFBUT and higher OSS than both control groups. Furthermore, candida counts were significantly higher in the pSS patients. In the pSS group, subjective oral dryness significantly correlated with ocular dryness (MDEIS: r = 0.5, OSDI: r = 0.413) and SWS was significantly correlated with Schirmer I (r = 0.419). The findings imply that interdisciplinary subjective and objective evaluation of patients with xerostomia and xerophthalmia not only have implications for patient care, but also may guide clinicians in differentiating between pSS and non-SS sicca patients

Patients with non-Sjögren's sicca report poorer general and oral health-related quality of life than patients with Sjögren's syndrome: a cross-sectional study

Understanding the impact of the disease on quality of life is crucial in patient management. In this cross-sectional study, general and oral health-related quality of life questionnaires, and thorough examinations of oral and ocular dryness were performed in age- and sex-matched patients with primary Sjögren’s syndrome (pSS group), non-Sjögren’s syndrome sicca (non-SS group) and healthy controls. General and oral health-related quality of life were investigated with the 36-Item Short Form Health Survey and the 14-Item Oral Health Impact Profile questionnaires, respectively. Subjective symptoms of xerostomia and ocular dryness were recorded using the Summated Xerostomia Inventory and Ocular Surface Disease Index, respectively. Clinical examinations included evaluation of clinical oral dryness scores, candida counts, unstimulated and stimulated saliva secretory rates, tear osmolarity, tear film break-up time, Schirmer I test and ocular surface staining. Both patient groups had pronounced signs and symptoms of xerostomia and ocular dryness. Even though the non-SS patients had less severe clinical signs than the pSS patients, they demonstrated much poorer general and oral health-related quality of life. In conclusion, non-SS patients require more attention in order to improve their quality of life

Patients with non-Sjögren's sicca report poorer general and oral health-related quality of life than patients with Sjögren's syndrome: a cross-sectional study

Understanding the impact of the disease on quality of life is crucial in patient management. In this cross-sectional study, general and oral health-related quality of life questionnaires, and thorough examinations of oral and ocular dryness were performed in age- and sex-matched patients with primary Sjögren’s syndrome (pSS group), non-Sjögren’s syndrome sicca (non-SS group) and healthy controls. General and oral health-related quality of life were investigated with the 36-Item Short Form Health Survey and the 14-Item Oral Health Impact Profle questionnaires, respectively. Subjective symptoms of xerostomia and ocular dryness were recorded using the Summated Xerostomia Inventory and Ocular Surface Disease Index, respectively. Clinical examinations included evaluation of clinical oral dryness scores, candida counts, unstimulated and stimulated saliva secretory rates, tear osmolarity, tear flm break-up time, Schirmer I test and ocular surface staining. Both patient groups had pronounced signs and symptoms of xerostomia and ocular dryness. Even though the non-SS patients had less severe clinical signs than the pSS patients, they demonstrated much poorer general and oral health-related quality of life. In conclusion, non-SS patients require more attention in order to improve their quality of life