Genome-wide association study identifies Sjögren's risk loci with functional implications in immune and glandular cells.

Sjögren's disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren's cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.

Proteomic Profiling of Saliva and Tears in Radiated Head and Neck Cancer Patients as Compared to Primary Sjögren's Syndrome Patients.

Patients with head and neck cancer (HNC) and patients with primary Sjögren's syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkers.

Inflammatory Stratification in Primary Sjögren's Syndrome Reveals Novel Immune Cell Alterations in Patients' Minor Salivary Glands.

There is a critical need to deconvolute the heterogeneity displayed by the minor salivary glands of primary Sjögren's syndrome (pSS) patients. This is challenging primarily because the disease etiology remains unknown. The hypothesis includes that initial events in the disease pathogenesis target the salivary glands, thereby triggering the development of focal infiltrates (≥50 mononuclear cells) and finally germinal center-like structures. However, the proportion of key mononuclear immune cells residing at these sites, in combination with the overall ratio of morphometric tissue atrophy and adipose infiltration within the minor salivary glands (MSG) parenchyma at distinct phases of inflammatory disease establishment and progression have not been quantified in detail. In this cross-sectional study, we intended to address this problem by stratifying 85 patients into mild (S1), moderate (S2), and severe (S3) stages using the Inflammatory severity index. We found that mild (<3%) and marked (≥3%) levels of atrophy were accompanied by the respective levels of adipose infiltration in the non-SS sicca controls (p <0.01), but not in pSS patients. The percentage of adipose infiltration significantly correlated with the age of patients (r = 0.458, p <0.0001) and controls (r = 0.515, p <0.0001). The CD4+ T helper cell incidence was reduced in the focal infiltrates of the MSG of S2 patients compared to S1 (p <0.01), and in S2 compared to S1 and S3 combined (p <0.05). CD20+ B cells increased from S1 to S3 (p <0.01) and S2 to S3 (p <0.01), meanwhile CD138+ plasma cells diminished in S3 patients compared to both S1 and S2 groups combined (p <0.01). The proportion of patients with anti-Ro/SSA+, anti-La/SSB+, and RF+ increased over the course of inflammatory disease progression and they were significantly more common in the S3 group relative to S1 (p <0.05). On the other hand, S2 patients measured a higher mean salivary flow relative to S1 and S3 patients combined (p <0.05). Our results demonstrate how the proposed Inflammatory severity index stratification revealed pathological cell and tissue-associated aberrations in the salivary component over the course of inflammatory progression, and their correlations to clinical outcomes. This could be directly transferred to the optimization of available diagnostic strategies applied for pSS patients.

Oral and ocular late effects in head and neck cancer patients treated with radiotherapy.

A broader understanding of oral and ocular late effects in head and neck cancer (HNC) patients who underwent intensity-modulated radiotherapy (IMRT) may provide valuable information in follow-up and improve quality of life. Twenty-nine HNC patients treated at least 6 months earlier and 30 age-matched controls were recruited. After completing several questionnaires: Oral Health Impact Profile-14 (OHIP-14), Shortened Xerostomia Inventory (SXI), Ocular Surface Disease Index (OSDI) and McMonnies Dry Eye questionnaire (MDEQ), participants underwent oral and ocular examinations. Oral examination included clinical oral dryness score (CODS) and secretion rates of unstimulated and stimulated saliva (UWS, SWS). Ocular examination included tear film break-up time, Schirmer test and ocular surface staining. The patients had more problems related to dry mouth than controls based on CODS and SXI, and more complaints of dry eye disease based on OSDI and MDEQ. UWS and SWS rates and oral health related quality of life were significantly lower in the patient group. Subjective oral dryness (SXI) correlated significantly with subjective ocular dryness (OSDI and MDEQ). Our study demonstrates that HNC patients treated with IMRT experience late effects in terms of xerostomia and ocular dryness underlining the importance of interdisciplinary approach in the evaluation and follow-up of HNC patients.

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome.

OBJECTIVES: Clinical presentation of primary Sjögren's syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. METHODS: We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. RESULTS: We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10-62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. CONCLUSION: Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.

Cytokines Explored in Saliva and Tears from Radiated Cancer Patients Correlate with Clinical Manifestations, Influencing Important Immunoregulatory Cellular Pathways.

Although radiotherapy is a common form of treatment for head and neck cancer, it may lead to tissue damage in the salivary and lacrimal glands, possibly affecting cytokine expression in the gland fluid of treated individuals. Cytokine profiles in saliva and tear fluid of 29 radiated head and neck cancer patients and 20 controls were screened using a multiplex assay. Correlations between cytokine expression and clinical oral and ocular manifestations were examined, and cellular pathways influenced by these cytokines were assessed using the Functional Enrichment Analysis Tool. Significantly elevated cytokines identified in patient saliva were CCL21, IL-4, CX3CL1, CCL2, CXCL1 and CCL15. Many of these cytokines correlated positively with objective signs of oral dryness, and reduced saliva production in the patients. Although CCL21 and IL-4 levels were significantly lower in patient tear fluid, they correlated with subjective ocular symptoms. These increased salivary cytokines affected pro-inflammatory and apoptotic cellular pathways, including T cell signalling, several interleukin signalling pathways, TNF and TGF-ß receptor signalling, and the apoptotic p53 pathway. In conclusion, the upregulated salivary cytokines identified suggest an interplay between innate and adaptive immunity, affecting immunoregulatory cellular pathways. Whether this is due to late effects of radiotherapy or tissue repair remains to be investigated.

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 (r2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r2 = 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.

Autoantigen-specific B cells and plasma cells are prominent in areas of fatty infiltration in salivary glands of patients with primary Sjögren's syndrome.

Salivary and lacrimal gland involvement is a characteristic feature of primary Sjögren's syndrome (pSS), where tissue destruction is mediated by mononuclear cell infiltration, resulting in lacrimal and salivary gland impairment. We have previously shown distinct prevalence of adipose tissue replacement in the minor salivary gland tissue from pSS patients. The salivary gland microenvironment was further examined through microarray analysis, identifying signalling pathways that promoted adipose tissue development, inflammation, and lymphoma. As B cells may also contribute to disease progression, we now aimed to study the B cell pattern with regard to adipocyte development in pSS. Double immunohistochemical staining of paraffin-embedded salivary gland tissue from 22 pSS patients and 11 non-SS tissue controls was employed, using the characteristic pSS autoantigens Ro52 or Ro60, alongside CD27. Additional CD138/CD20 double staining was also performed to identify the plasma- and general B- cell pattern. Our results demonstrated CD27-positive Ro52 and Ro60 specific cells observed within and in close proximity to the adipose tissue. CD138-positive plasma cells were also seen in areas of adipose tissue replacement, while the CD20+ cells were located within focal infiltrates, forming distinct B cell zones. The quantification of CD138+ and CD20+ cells revealed elevated numbers of CD138+ cells in areas of fatty infiltration, and also interstitially, in the salivary glands of pSS patients when compared to non-SS controls. A significant increase (p < .01) in CD138+ cells close to areas of fatty infiltration, and interstitially, with increasing fatty infiltration and focus score was further observed in pSS patients. A correlation between the number of CD20+ B cell zones/mm2 of salivary gland tissue and focus score values was also witnessed in the patients (r2 = 0.6047, p < .001). In conclusion, autoantigen-specific B cells and plasma cells appear prominent in areas of fatty infiltration in salivary glands of pSS patients, where an increase in CD138+ plasma cells and CD20+ B cells, in relation to both fatty and focal infiltration, suggests their active involvement in promoting inflammation. Further studies are needed to assess whether these adipocytes are also a result of tissue repair.

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.

Elevated cytokine levels in tears and saliva of patients with primary Sjögren's syndrome correlate with clinical ocular and oral manifestations.

Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity in pSS.

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.

Signalling pathways identified in salivary glands from primary Sjögren's syndrome patients reveal enhanced adipose tissue development.

A characteristic feature of primary Sjögren's syndrome (pSS) is the destruction of salivary and lacrimal glands mediated by mononuclear cell infiltration. Adipocytes can also occupy a large portion of the salivary gland (SG) tissue area, although little is known about their significance in pSS. We have previously investigated adipose tissue infiltration in SG biopsies from pSS patients and non-SS sicca controls. Our findings indicated the distinct incidence of adipose tissue replacement in pSS patients, where adipocytes were detected in interleukin (IL) 6 rich regions. We now aimed to examine the development of adipocytes in the SG microenvironment, and delineate their possible involvement in immune reactions. A microarray analysis was performed on SG from 6 pSS patients and 6 non-SS controls, where the expression levels of genes involved in adipose tissue development, inflammatory responses, and lymphoma development were assessed. Real-time PCR was carried out on SG from 14 pSS patients and 15 non-SS controls to account for IL6, IL10, and IL17 mRNA levels. Immunohistochemical staining of frozen SG tissue using IL17 was also conducted. Our results indicate signalling pathways identified in SG of pSS patients displayed genes leading to prominent adipose tissue development and reduced mitochondrial fatty acid beta-oxidation (ARID5B, OXCT1, BDH1, SOX8, HMGCS2, FTO, ECHS1, PCCA, ACADL and ACADVL), inflammatory responses (IL1R1, IL7R, IL10RA, IL15, IL18RAP, CCL2, CCL5, CCL22, CXCR6, CD14, and CD48), and lymphoma development via JAK-STAT signalling (STAT2, TYK2, EBI3, FAS, TNFRSF1B, MAP3K8, HMOX1, LTB, TNF, STAT1, and BAK1). Genes involved in interferon production and signalling were also detected (IRF1, IRF9, and IRF7), in addition to IL6, IL10, and IL17. Higher mRNA levels of IL6, IL17 and IL10 were observed in the SG of pSS patients compared to controls. Moreover, IL17 positive cells were detected mostly interstitially in the SG and around adipocytes, also within the focal infiltrates. In conclusion, adipocyte development seems to be more prominent in the SG of pSS patients, where adipose tissue replacement is also evident. Whether this is due to disease progression, or the repair process, remains to be investigated. Detection of IL17 positive adipocytes in the target organ suggests their involvement in immune reactions.

Oral disorders, saliva secretion, and oral health-related quality of life in patients with primary Sjögren's syndrome.

Chemosensory function, burning sensations in the tongue (BST), halitosis, saliva secretion, and oral health-related quality of life (OHRQoL) were investigated in patients with primary Sjögren's syndrome (pSS). In 31 patients with pSS and 33 controls, olfactory and gustatory functions were evaluated. Self-reported complaints of dysgeusia, BST, and halitosis were recorded. Saliva secretion rates were measured and OHRQoL was assessed using the short-form Oral Health Impact Profile (OHIP-14). Patients had significantly lower olfactory (8.8 ± 3.5 vs. 10.7 ± 1.2) and gustatory (18.9 ± 7.1 vs. 25.4 ± 4.3) scores than controls, and significantly more patients complained of dysgeusia (58.1% vs. 0%), BST (54.8% vs. 6.1%), and halitosis (41.9% vs. 0%). A significantly greater proportion of patients with pSS had ageusia (19% vs. 0%), hypogeusia (32% vs. 12%), anosmia (13% vs. 0%), or hyposmia (29% vs. 9%). Significantly lower saliva secretion rates (ml min-1 ) were observed in patients with pSS for stimulated (0.62 ± 0.40 vs. 1.57 ± 0.71) and unstimulated (0.08 ± 0.07 vs. 0.29 ± 0.17) saliva. The mean OHIP-14 score was significantly higher in patients with pSS (16.2 ± 10.8 vs. 2.7 ± 3.1) and was positively correlated with dysgeusia, BST, and halitosis. In conclusion, patients with pSS reported higher occurrence of dysgeusia, BST, and halitosis, and demonstrated relatively impaired chemosensory and salivary functions. The patients' poorer OHRQoL was associated with dysgeusia, BST, and halitosis.

Identification of potential saliva and tear biomarkers in primary Sjögren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis.

BACKGROUND: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren's syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. METHODS: Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis. RESULTS: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients. CONCLUSIONS: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.

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.

Adipose tissue is prominent in salivary glands of Sjögren's syndrome patients and appears to influence the microenvironment in these organs.

A minor salivary gland (SG) biopsy with focal lymphocytic sialadenitis and a focus score of ≥1 is today's widely accepted pathological finding confirming the SG component of Sjögren's syndrome (SS). Adipocytes can occupy a large percentage of the SG area although little is known about their significance in SS lesions. This study aimed to characterise adipose tissue infiltration in labial SG biopsies from 27 SS patients and 28 non-SS sicca controls. Biopsies were evaluated by one oral pathologist and assessed for focus score, acinar atrophy, fatty replacement and non-specific chronic inflammation. Moreover, to explore the SG microenvironment, immunohistochemical staining of paraffin-embedded SG tissue was performed using interleukin-6 (IL-6). The fatty replacement was evident in all SS patients possessing autoantibodies (Ro/SSA and/or La/SSB) as well as a positive SG biopsy (focus score ≥1). Additionally, 62% of SS patients having autoantibodies but a negative biopsy showed fatty infiltration (FI) while non-SS controls demonstrated fatty replacement in only 32% of the cases. Overall, the SS group (mean age 53.0 years) had a significantly higher incidence (p value 0.005) of FI than the non-SS controls (mean age 54.8 years). Interestingly, adipocytes were located in IL-6 rich areas, and IL-6 positive adipocytes were detected. As fat deposition seems to be more recurrent in SGs affected by SS, we propose the assessment of adipose tissue replacement as a helpful tool for diagnostic evaluation in SS. Detection of IL-6 positive adipocytes suggests their involvement in immune reactions. Still, functional studies are needed to investigate the SG microenvironment further.

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice, a model for primary Sjögren's syndrome.

Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren's syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU(+) cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138(+) PCs were however BrdU(-) and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138(+) cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138(+) cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.