Molly S. Hein

Changes in Novel Biomarkers of Disease Activity in Juvenile and Adult Dermatomyositis are Sensitive Biomarkers of Disease Course

OBJECTIVE Muscle enzyme levels are insensitive markers of disease activity in juvenile and adult dermatomyositis (DM), especially during the active treatment phase. To improve our ability to monitor DM disease activity longitudinally, especially in the presence of immunomodulating agents, we prospectively evaluated whether interferon (IFN)-dependent peripheral blood gene and chemokine signatures could serve as sensitive and responsive biomarkers for change in disease activity in adult and juvenile DM. METHODS Peripheral blood and clinical data were collected from 51 patients with juvenile or adult DM prospectively over 2 study visits. We performed disease activity measurements and calculated whole-blood type I IFN gene and chemokine scores. We also measured serum levels of other proinflammatory cytokines, including interleukin-6 (IL-6). RESULTS Changes in juvenile and adult DM global disease activity correlated positively and significantly with changes in the type I IFN gene score before adjustment for medication use (r = 0.33, P = 0.023) and with changes in the IFN chemokine score before and after adjustment for medication use (r = 0.53, P < 0.001 and r = 0.50, P < 0.001, respectively). Changes in muscle and extramuscular visual analog scale (VAS) scores correlated positively with changes in IFN gene and chemokine scores (P = 0.002, P < 0.001, P = 0.095, P < 0.001). Serum levels of IL-6, IL-8, and tumor necrosis factor α (TNFα) correlated positively with changes in global, muscle, and extramuscular VAS scores (P < 0.05). CONCLUSION Our findings suggest that changes in type I IFN gene and chemokine scores as well as in levels of IL-6, IL-8, and TNFα may serve as sensitive and responsive longitudinal biomarkers of change in disease activity in juvenile and adult DM, even in the presence of immunomodulating agents.

BAFF expression correlates with idiopathic inflammatory myopathy disease activity measures and autoantibodies

Objective. To investigate B cell survival cytokine messenger RNA (mRNA) levels as biomarkers of idiopathic inflammatory myopathies (IIM). Methods. We measured and compared mRNA levels of B cell survival cytokines by quantitative real-time polymerase chain reaction in 98 patients with IIM, 38 patients with systemic lupus erythematosus, and 21 healthy controls. The cytokines were B cell-activating factor belonging to the tumor necrosis factor family (BAFF); ΔBAFF; and a proliferation-inducing ligand (APRIL); and their receptors BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor, and B cell maturation antigen (BCMA). We also identified autoantibodies, including anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-topoisomerase 1, anti-hystidyl-tRNA synthetase, anti-ribosomal P, and anti-chromatin. Clinical disease activity was assessed by the International Myositis Assessment and Clinical Studies core set tool. We examined correlation of mRNA with disease activity, medication use, and autoantibodies. Results. We found a positive correlation of BAFF and ΔBAFF expression with 3 disease activity measures, with ΔBAFF having a stronger correlation. Similarly, anti-SSA/Ro-52 and/or anti-SSA/Ro-60 had a strong positive correlation with mRNA levels of BAFF and ΔBAFF, and with relative ratios of BAFF/APRIL and BCMA/BAFF-R. Conclusion. These findings highlight the potential importance of BAFF, ΔBAFF, and BAFF-R in the pathogenesis of IIM, and suggest an important role in the assessment of disease activity.

Gene Expression Profiling in Blood and Affected Muscle Tissues Reveals Differential Activation Pathways in Patients with New-onset Juvenile and Adult Dermatomyositis

Objective. To identify shared and differential molecular pathways in blood and affected muscle between adult dermatomyositis (DM) and juvenile DM, and their association with clinical disease activity measures. Methods. Gene expression of transcription factors and cytokines involved in differentiation and effector function of T cell subsets, regulatory T cells and follicular Th cells, were analyzed in the blood from 21 newly diagnosed adult and 26 juvenile DM subjects and in 15 muscle specimens (7 adult and 8 juvenile DM) using a custom RT2 Profiler PCR Array. Disease activity was determined and measured by established disease activity tools. Results. The most prominent finding was the higher blood expression of Th17-related cytokines [retinoic acid-related orphan receptor-γ, interferon regulatory factor 4, interleukin (IL)-23A, IL-6, IL-17F, and IL-21] in juvenile DM at baseline. In contrast, adult patients with DM showed increased blood levels of STAT3 and BCL6 compared with juvenile DM. In muscle, GATA3, IL-13, and STAT5B were found at higher levels in juvenile patients with DM compared with adult DM. Among 25 patients (11 adult and 14 juvenile DM) who had blood samples at baseline and at 6 months, increased expression of IL-1β, STAT3, STAT6, STAT5B, and BCL6 was associated with an improvement in global extramuscular disease activity. Conclusion. We observed differences in gene expression profiling in blood and muscle between new-onset adult and juvenile DM. Cytokine expression in the blood of juvenile patients with new-onset DM was dominated by Th17-related cytokines compared with adult patients with DM. This may reflect the activation of different Th pathways between muscle and blood.

Longitudinal Peripheral Blood Lymphocyte Subsets Correlate with Decreased Disease Activity in Juvenile Dermatomyositis

Objective: To determine the clinical characteristics and subsets of peripheral blood lymphocytes (PBL), which correlate with decreased disease activity in patients with juvenile dermatomyositis (JDM). Methods. Peripheral blood mononuclear cells from 24 patients with JDM were collected at Mayo Clinic Rochester between 2007 and 2011. These were analyzed using fluorescence-activated cell sorting and flow cytometry. Clinical disease activity was determined by visual analog scales (VAS) collected in 2 consecutive visits and correlated with PBL subsets. Results. The change in CD3+CD69+ T cells correlated with the change in global VAS scores. The change in HLA-DR- CD11c+ myeloid dendritic cells also correlated with the change in extramuscular VAS scores. There were trends toward decreased levels of HLA-DR- CD11c+ cells with decreased muscle and global VAS scores, but these did not reach significance. The change in HLA-DR- CD123+ plasmacytoid dendritic cells negatively correlated with the change in muscle VAS scores. Although not statistically significant, decreased levels of CD3-CD16- CD56+ natural killer (NK) cells and HLA-DR- CD86+ myeloid dendritic cells, and increased levels of CD16+CD56- NK cells, correlated with decreased VAS scores. Conclusion. Changes in CD3+CD69+ T cells, HLA-DR- CD11c+ myeloid dendritic cells, and HLA-DR- CD123+ plasmacytoid dendritic cells are associated with improved clinical course in JDM and could be used as markers for disease activity, but findings need to be verified in a larger, independent cohort. Lack of significant differences among most of our PBL subsets suggests that lymphocyte phenotyping may be difficult to definitively correlate with disease activity in JDM.

Maternal microchimerism in Hirschsprung's disease.

Hirschsprungs disease (HD) presents with severe constipation due to absent ganglion cells in the distal rectum. We sought to determine whether maternal chimeric cells are present in aganglionic bowel. We hypothesize that chimeric cells are part of the unfavorable microenvironment that leads to the destruction of enteric neurons in HD. Intestinal biopsies and resections from seven male patients with HD were compared with four male patients with chronic constipation and six with bowel atresia. Fluorescence in situ hybridization was used to identify chimeric cells based on male/female (XX/XY) differences. The location and immunophenotype of chimeric cells were also studied. Chimeric cells were present more often in the small intestine and rectum, compared with the appendix and colon. Patients with HD had a greater number of chimeric cells per 10× magnification field than patients with chronic constipation or congenital atresia. Chimeric cells were predominantly in the submucosa and outer longitudinal muscle layer in HD. Immunophenotyping identified over 40% of chimeric cells as inflammatory. Chimeric cells are present in greater numbers in aganglionic bowel than in other disorders. Clustering of chimeric cells in areas of absent ganglia lends support to the proposed role of maternal microchimerism in allo-autoimmune responses.

Genotype-phenotype correlation of hereditary erythrocytosis mutations, a single center experience

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3‐bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen‐sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL, BPGM, EPOR, EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing, we have identified 762 patients with 81 distinct HOA Hb variants (61 β, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms.

Increased expression of ADAMTS13 mRNA correlates with ischemic cerebrovascular disease in systemic lupus erythematosus patients

Objective: We investigated ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) messenger RNA levels as a biomarker of disease features in systemic lupus erythematosus. Methods: We measured and compared messenger RNA (mRNA) levels of ADAMTS13 in peripheral blood cells in patients with systemic lupus erythematosus and healthy control subjects by whole-genome microarray. We retrospectively analyzed the correlations of ADAMTS13 mRNA expression with clinical features, laboratory parameters, therapeutic features, and disease activity (according to the Systemic Lupus Erythematosus Disease Activity Index). We also examined the association of three single nucleotide polymorphisms (rs4962145, rs2285467, and rs685523) of the ADAMTS13 gene with patient characteristics. Results: In 309 patients, the median ADAMTS13 mRNA expression levels were significantly higher in blood cells of systemic lupus erythematosus patients than in 23 healthy controls (p = .03). Notably, ADAMTS13 mRNA expression levels were significantly higher in systemic lupus erythematosus patients with a history of stroke (p = .02) or transient ischemic attack (p = .02). Among the three single nucleotide polymorphisms analyzed, rs2285467 was significantly associated with stroke (p = .03) and anticardiolipin antibodies (p = .04). Conclusions: Increased expression of ADAMTS13 mRNA in blood cells is associated with the presence of ischemic cerebrovascular disease in systemic lupus erythematosus patients and suggests a potential role for ADAMTS13 in the pathogenesis of ischemic cerebrovascular disease in these patients.