Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

Introduction: Systemic sclerosis (SSc) is a heterogenous disorder that appears to result from interplay between vascular pathologies, tissue fibrosis and immune processes, with evidence for deregulation of chemokines, which normally control immune trafficking. We recently identified altered levels of chemokine CCL21 in SSc associated pulmonary arterial hypertension (PAH). Here, we aimed to define target organ expression and biomarker characteristics of CCL21. Materials and methods: To investigate target organ expression of CCL21, we performed immunohistochemistry (IHC) on explanted lung tissues from SSc-PAH patients. We assessed serum levels of CCL21 by ELISA and Luminex in two well-characterized SSc cohorts from Oslo (OUH, n=552) and Zurich (n=93) University hospitals and in 168 healthy controls. For detection of anti-CCl21 antibodies, we performed protein array analysis applying serum samples from SSc patients (n=300) and healthy controls. To characterize circulating CCL21 in SSc, we applied immunoprecipitation (IP) with antibodies detecting both full length and tailless and a custom-made antibody detecting only the C-terminal of CCL21. IP products were analyzed by SDS-PAGE/western blot and Mass spectrometry (MS). Results: By IHC, we found that CCL21 was mainly expressed in the airway epithelial cells of SSc patients with PAH. In the analysis of serum levels of CCL21 we found weak correlation between Luminex and ELISA (r=0.515, p<0.001). Serum levels of anti-CCL21 antibodies were higher in SSc patients than in healthy controls (p<0.001), but only 5% of the SSc population were positive for anti-CCL21 antibodies in SSc, and we found no correlation between anti-CCl21 and serum levels of CCL21. By MS, we only identified peptides located within amino acid (aa) 23-102 of CCL21, indicating that CCL21 in SSc circulate as a truncated protein without the C-terminal tail. Conclusion: This study demonstrates expression of CCL21 in epithelial lung tissue from SSc patients with PAH, and indicate that CCL21 in SSc circulates as a truncated protein. We extend previous observations indicating biomarker potential of CCL21, but find that Luminex is not suitable as platform for biomarker analyses. Finally, in vivo generated anti-CCL21 antibodies exist in SSc, but do not appear to modify serum CCL21 levels in patients with SSc-PAH

The Danish 22q11 research initiative

Background: Neurodevelopmental brain disorders such as schizophrenia, autism and attention deficit hyperactivity disorder are complex disorders with heterogeneous etiologies. Schizophrenia and autism are difficult to treat and often cause major individual suffering largely owing to our limited understanding of the disease biology. Thus our understanding of the biological pathogenesis needs to be substantiated to enable development of more targeted treatment options with improved efficacy. Insights into the pre-morbid disease dynamics, the morbid condition and the underlying biological disease mechanisms may come from studies of subjects with homogenous etiologies. Breakthroughs in psychiatric genetics have shown that several genetic anomalies predispose for neurodevelopmental brain disorders. We have established a Danish research initiative to study the common microdeletion at chromosome 22q11.2, which is one of the genetic anomalies that confer high risk of schizophrenia, autism and attention deficit hyperactivity disorder. Methods/design: The study applies a "cause-to-outcome" strategy to identify pre-morbid pathogenesis and underlying biological disease mechanisms of psychosis and secondarily the morbid condition of autism and attention deficit hyperactivity disorder. We use a population based epidemiological design to inform on disease prevalence, environmental risk factors and familial disposition for mental health disorders and a case control study design to map the functional effects across behavioral and neurophysiological traits of the 22q11 deletion in a recruited sample of Danish individuals. Discussion: Identification of predictive pre-morbid clinical, cognitive, functional and structural brain alterations in 22q11 deletion carriers may alter current clinical practice from symptomatic therapy of manifest mental illness into early intervention strategies, which may also be applicable to at risk subjects without known etiology. Hopefully new insights into the biological disease mechanisms, which are mandatory for novel drug developments, can improve the outcome of the pharmacological interventions in psychiatry

Deficit of homozygosity among 1.52 million individuals and genetic causes of recessive lethality

Genotypes causing pregnancy loss and perinatal mortality are depleted among living individuals and are therefore difficult to find. To explore genetic causes of recessive lethality, we searched for sequence variants with deficit of homozygosity among 1.52 million individuals from six European populations. In this study, we identified 25 genes harboring protein-altering sequence variants with a strong deficit of homozygosity (10% or less of predicted homozygotes). Sequence variants in 12 of the genes cause Mendelian disease under a recessive mode of inheritance, two under a dominant mode, but variants in the remaining 11 have not been reported to cause disease. Sequence variants with a strong deficit of homozygosity are over-represented among genes essential for growth of human cell lines and genes orthologous to mouse genes known to affect viability. The function of these genes gives insight into the genetics of intrauterine lethality. We also identified 1077 genes with homozygous predicted loss-of-function genotypes not previously described, bringing the total set of genes completely knocked out in humans to 4785.publishedVersio

Lymphangiogenic factors are associated with pulmonary arterial hypertension in systemic sclerosis

OBJECTIVES Pulmonary arterial hypertension (PAH) is a major complication in systemic sclerosis (SSc), a disease marked by vascular and lymphatic vessel abnormalities. In this study, we aimed to assess lymphangiogenic factors Vascular Endothelial Growth Factor-C (VEGFC) and Angiopoietin 2 (Ang2) and soluble forms of their cognate receptors; VEGFR3 and Tie-2 in SSc; and evaluate their predictive ability of PAH development. METHODS In this cohort study, we used multiplex bead assays to assess serum levels of lymphangiogenic factors in sera samples from two well-characterized SSc cohorts; an unselected identification cohort from Oslo and a PAH enriched validation cohort from Zurich and Oslo, all assessed by right heart catheterizations (RHC). We defined PAH according to ESC/ERS 2015 Guidelines. Statistics included logistic- and Cox-regression analyses. RESULTS We found in the identification cohort (n=371) lower mean serum levels of VEGF-C and higher Ang-2 levels in SSc patients compared to healthy controls, and the same trend in SSc cases with PAH compared to those without PH. We confirmed VEGF-C associations to SSc and PAH in the PAH enriched RHC validation cohort (n=149). For prediction analysis, we assembled all 251 cases assessed by RHC from the identification and validation cohorts. In multivariable Cox analysis we found, after adjusting for age and gender, that VEGF-C (HR 0.53 95% CI 0.29-0.97, p=0.040) and sVEGFR3 (HR 1.21 95% CI 1.01-1.45, p=0.042) predicted PAH development. CONCLUSION This study supports the notion of deregulated lymphangiogenesis during PAH development in SSc, and indicates VEGF-C as a promising marker for early PAH detection

Effects of faecal microbiota transplantation on small intestinal mucosa in systemic sclerosis

OBJECTIVES In systemic sclerosis (SSc), gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling. METHODS We analysed duodenal biopsies obtained pre- (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling. RESULTS In patients receiving FMT, the number of podoplanin and CD64-expressing cells in the mucosa were lower at week 2 compared to baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T-cell receptor complex, and chemokine receptor, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16. CONCLUSION Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement

Serum markers of cardiac complications in a systemic sclerosis cohort

Abstract Primary cardiac involvement is one of the leading causes of mortality in systemic sclerosis (SSc), but little is known regarding circulating biomarkers for cardiac SSc. Here, we aimed to investigate potential associations between cardiac SSc and candidate serum markers. Serum samples from patients of the Oslo University SSc cohort and 100 healthy controls were screened against two custom-made candidate marker panels containing molecules deemed relevant for cardiopulmonary and/or fibrotic diseases. Left (LV) and right ventricular (RV) dysfunction was assessed by protocol echocardiography, performed within three years from serum sampling. Patients suspected of pulmonary hypertension underwent right heart catheterization. Vital status at study end was available for all patients. Descriptive analyses, logistic and Cox regressions were conducted to assess associations between cardiac SSc and candidate serum markers. The 371 patients presented an average age of 57.2 (± 13.9) years. Female sex (84%) and limited cutaneous SSc (73%) were predominant. Association between LV diastolic dysfunction and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (OR 0.41, 95% CI 0.21–0.78, p = 0.007) was identified. LV systolic dysfunction defined by global longitudinal strain was associated with angiopoietin 2 (ANGPT2) (OR 3.42, 95% CI 1.52–7.71, p = 0.003) and osteopontin (OPN) (OR 1.95, 95% CI 1.08–3.52, p = 0.026). RV systolic dysfunction, measured by tricuspid annular plane systolic excursion, was associated to markers of LV dysfunction (ANGPT2, OPN, and TRAIL) (OR 1.67, 95% CI 1.11–2.50, p = 0.014, OR 1.86, 95% CI 1.25–2.77, p = 0.002, OR 0.32, 95% CI 0.15–0.66, p = 0.002, respectively) and endostatin (OR 1.86, 95% CI 1.22–2.84, p = 0.004). In conclusion, ANGPT2, OPN and TRAIL seem to be circulating biomarkers associated with both LV and RV dysfunction in SSc