Towards stratification of patients with Sjögren’s syndrome : Single cell analyses and immune profiling

Primary Sjögren’s syndrome (pSS) is a systemic, chronic autoimmune disorder that is characterized by progressive lymphocytic infiltration in the exocrine glands i.e., the salivary and lacrimal glands, leading to immune-mediated glandular destruction. It mostly affects middle-aged women, making diagnosis of the disease challenging, as the symptoms (primarily dryness of the mouth and eyes and fatigue) are often confused with the side effects of drugs, other comorbidities or aging. Currently there is no cure, as the exact mechanism of the disease pathogenesis is not known, and treatment strategies mainly aim at alleviating the symptoms. Like most autoimmune diseases, pSS progression and phenotype are complex and multi-faceted, with a wide spectrum of clinical manifestations, ranging from local to systemic, including fatal conditions like B cell lymphoma. Patient heterogeneity is a major obstacle to disease management. Therefore, it is imperative to identify potential disease markers that may help in diagnosis, prediction, stratification of the patients and/or identification of new therapeutic targets. The overall aim of this thesis was to study the peripheral blood immune system in pSS, to identify disease-specific immune profiles and potential biomarkers that may help in patient stratification. In paper I, phosphoflow cytometry was used to compare basal and TLR7 and -9 stimulated phosphorylation states in immune cells of pSS patients and healthy donors. Both basal and stimulation-induced phosphorylation differed significantly between pSS patients and healthy individuals, and between patient subgroups. Plasma cytokine levels, measured by Luminex assay, also differed significantly between the patients and controls as well as between patient subgroups, and correlated with autoantibody status and other clinical parameters. In paper II, single cell analysis of peripheral blood immune cells, with special emphasis on intracellular signaling, was done using mass cytometry. We compared the frequencies of different immune cell subsets among the patient subgroups and healthy individuals and analyzed their signaling profiles, upon stimulation with IFNα2b and IFNγ separately. Significant differences in cell frequencies were observed among the SSA- and SSA+ pSS patients and controls, along with increased activation status in many cell types, particularly in the SSA+ subgroup. Upon IFNα2b and IFNγ stimulation, aberrations in phospho-signaling were detected in the various immune cell subsets of the patient subgroups, which were most prominent in the SSA+ pSS patients. In paper III, we used flow cytometry and qPCR to analyze the expression of TAM receptors in various immune cells of pSS patients and healthy controls. Significant differences in the mRNA levels of some of the TAM receptors as well as in cell frequencies were observed between the patients and healthy donors. Differential TAM receptor expressions in the immune cells were detected between the pSS patients and healthy controls, with most of them being expressed at slightly lower levels in the patients. In conclusion, aberrations in cellular compositions, cytokine and TAM receptor levels as well as discrepancies in intracellular signaling pathways were detected in the pSS patients and their subgroups compared to healthy individuals. Further research can shed light on new biomarkers for stratification of patients for personalized treatment. Optimized therapeutic strategies can greatly alter the disease outcome and quality of life.Doktorgradsavhandlin

Co-seismic spring flow changes attributed to the March 29, 1999 Chamoli earthquake of Garhwal Himalaya

The moderate magnitude Chamoli earthquake that occurred on March 29, 1999, in the Garhwal Higher Himalaya produced, among many other observable effects, changes in flow of several artisan springs. Qualitative observations of significant changes in the flow of ten springs located in regions of higher intensity show a strong spatial correlation with our preliminary estimates of perturbing pore pressure field induced in the water saturated shallow rocks of the region by the earthquake its coseismic phase. The results are significant for it is the first successful attempt in the Himalayan region to investigate the response pattern of the local groundwater flow system to perturbations induced to the ambient tectonic stress regime by a major earthquake

A simulation of earthquake induced undrained pore pressure changes with bearing on some soil liquefaction observations following the 2001 Bhuj earthquake

The Bhuj earthquake of January 26th, 2001, induced wide spread liquefaction within the Kachch peninsula. It has been pointed out that inundation due to soil liquefaction was short lived in some parts than in others in the affected region. Several geological, seismological and hydrological factors would have cumulatively contributed to these observed changes. We simulate in this article, undrained or short-term change in pore pressure in a poroelastic half space, in response to a simplified model of the Bhuj earthquake source. We find that the regions of relatively shorter lived inundation due to soil liquefaction may fall in the region where pore pressure responsible for soil liquefaction attributable to strong ground shaking was counteracted by pore pressure changes due to undrained poroelastic effect and vice versa

On the aftershock sequence of a 4.6 mb earthquake of the Garhwal Himalaya

Locally recorded data for eighteen aftershocks of a magnitude(mb) 4.6 earthquake occurring near Ukhimath in the Garhwal Himalaya were analysed. A master event technique was adopted to locate seventeen individual aftershock hypocentres relative to the hypocentre of the eighteenth aftershock chosen as the master event. The aftershock epicentres define an approximately 30 km2 rupture zone commensurate with the magnitude of the earthquake. The distribution of epicentres within this zone and the limited amount of first motion data support the view that a group of parallel, sub-vertical, sinistral strike-slip faults oriented N46°, transverse to the regional NW-SE trend of the Garhwal Himalaya, was involved in this seismic episode. Since the estimated focal depth range for aftershocks of this sequence is 3-14 km, we infer that this transverse fault zone extends through the upper crustal layer to a depth of 14 km at least

Estimation of hypocentral parameters of local earthquakes when crustal layers have constant P-velocities and dipping interfaces

The paper describes an algorithm for estimating the hypocentral coordinates and origin time of local earthquakes when the wave speed model to be employed is a layered one with dipping interfaces. A constrained least-squared error problem has been solved using the penalty function approach, in conjunction with the sequential unconstrained optimization technique of Fiacco and McCormick. Joint confidence intervals for the computed parameters are estimated using the approach of Bard for nonlinear problems. These results show that when a hypocentre lies outside the array of recording stations and head waves from a dipping interface are involved, then its inclination must be taken into account for dip angles exceeding 5°

Single cell based phosphorylation profiling identifies alterations in toll-like receptor 7 and 9 signaling in patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and −9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and −9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways.publishedVersio

Aberrant signaling of immune cells in Sjögren's syndrome patient subgroups upon interferon stimulation

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, characterized by mononuclear cell infiltrates in the salivary and lacrimal glands, leading to glandular atrophy and dryness. Patient heterogeneity and lack of knowledge regarding its pathogenesis makes pSS a difficult disease to manage. Methods: An exploratory analysis using mass cytometry was conducted of MAPK/ERK and JAK/STAT signaling pathways in peripheral blood mononuclear cells (PBMC) from 16 female medication free pSS patients (8 anti-Sjögren’s syndrome-related antigen A negative/SSA- and 8 SSA+) and 8 female age-matched healthy donors after stimulation with interferons (IFNs). Results: We found significant differences in the frequencies of memory B cells, CD8+ T central and effector memory cells and terminally differentiated CD4+ T cells among the healthy donors and patient subgroups. In addition, we observed an upregulation of HLA-DR and CD38 in many cell subsets in the patients. Upon IFNα2b stimulation, slightly increased signaling through pSTAT1 Y701 was observed in most cell types in pSS patients compared to controls, while phosphorylation of STAT3 Y705 and STAT5 Y694 were slightly reduced. IFNγ stimulation resulted in significantly increased pSTAT1 Y701 induction in conventional dendritic cells (cDCs) and classical and non-classical monocytes in the patients. Most of the observed differences were more prominent in the SSA+ subgroup, indicating greater disease severity in them. Conclusions: Augmented activation status of certain cell types along with potentiated pSTAT1 Y701 signaling and reduced pSTAT3 Y705 and pSTAT5 Y694 induction may predispose pSS patients, especially the SSA+ subgroup, to upregulated expression of IFN-induced genes and production of autoantibodies. These patients may benefit from therapies targeting these pathways.publishedVersio

International Teaching Programme

Nicolaides-Baraitser syndrome (NBS) is an infrequently described condition, thus far reported in five cases. In order to delineate the phenotype and its natural history in more detail, we gathered data on 18 hitherto unreported patients through a multi-center collaborative study, and follow-up data of the earlier reported patients. A detailed comparison of the 23 patients is provided. NBS is a distinct and recognizable entity, and probably has been underdiagnosed until now. Main clinical features are severe mental retardation with absent or limited speech, seizures, short stature, sparse hair, typical facial characteristics, brachydactyly, prominent finger joints and broad distal phalanges. Some of the features are progressive with time. The main differential diagnosis is Coffin-Siris syndrome. There is no important gender difference in occurrence and frequency of the syndrome, and all cases have been sporadic thus far. Microarray analysis performed in 14 of the patients gave normal results. Except for the progressive nature there are no clues to the cause. (C) 2009 Wiley-Liss, Inc

Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and −9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and −9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways

A generalized invariant imbedding equation II

Research into fetal development and medicin