Table_1_Low Prevalence of Anti-DFS70 Antibodies in Children With ANA-Associated Autoimmune Disease.pdf

IntroductionAnti-DFS70 antibodies occur in healthy individuals with various medical conditions. Unlike other anti-nuclear autoantibodies (ANA), they are not associated with systemic autoimmune disease in adult patients. To date, only a few studies have addressed the prevalence and/or clinical relevance of anti-DFS70 autoantibodies in children with and without autoimmune disease.MethodsIncluded in this retrospective cross-sectional mono-centric study were 308 pediatric patients with suspected or known autoimmune conditions who had a positive ANA in indirect immune fluorescence (IIF) screening and who were screened for anti-DFS70 antibodies by extractable nuclear antigen antibodies (ENA) immunoblot. Patients were assigned to four different diagnostic categories according to their diagnosis in the corresponding medical record: (a) absence of autoimmune or rheumatic disease (noARD, n = 116); (b) suspected autoimmunity without definitive diagnosis (sAI, n = 48); (c) other rheumatic disease (ORD) (n = 115); and (d) ANA-associated autoimmune disease (AARD, n = 29).ResultsThe prevalence of anti-DFS70 antibodies in the overall cohort was 33.8%. Among children without ARD (46.6%, 54/116), prevalence was significantly higher than among children with ORD (23.7%, 27/115, p = 0.0003) or AARD (17.2%, 5/29, p = 0.0054). Among all of the anti-DFS70 positive patients with AARD, other autoantibodies were found in the ENA immunoblot. In contrast, among anti-DFS70 positive patients with ORD (11.5%, 4/27), sAI (33.3%, 6/18) and noARD (16.7%, 9/54), other autoantibodies infrequently were detected (p = 0.0005). Patients with uveitis rarely were positive for anti-DFS70 antibodies (7.7%, 1/13). No association was found between anti-DFS70 antibodies and a history of allergic conditions (p = 0.51). The concordance between a typical DFS pattern in IIF and the detection of anti-DFS70 antibodies by immunoblot was 59.3%.ConclusionAs with adults, the higher prevalence of anti-DFS70 among children without autoimmune disease confirms the mutual exclusion for this autoantibody in the pathogenesis of ARD. Among ANA-positive children, monospecific anti-DFS70 antibodies may help to discriminate between AARD and not-AARD-related conditions.</p

Binding of [³H]photocholesterol to wildtype and mutant stomatin.

<p>COS-7 cells were transiently transfected with WT or mutant stomatin constructs. Subsequently they were incubated with a photoactivatable, radioactive cholesterol derivative ([³H]photocholesterol) and irradiated with UV light to crosslink [³H]photocholesterol to respective binding proteins. The cells were solubilized and stomatin was immunoprecipitated by monoclonal anti-stomatin antibody GARP-50. (<b>A</b>) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant stomatin. (<b>B</b>) The expression level of the constructs was determined by immunoblotting with monoclonal anti-stomatin antibody GARP-50.</p

Table2_Storage of packed red blood cells impairs an inherent coagulation property of erythrocytes.docx

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.</p

Table1_Storage of packed red blood cells impairs an inherent coagulation property of erythrocytes.docx

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.</p

Effect of CYC treatment and CYC treatment followed by RTX on serum Ig concentrations in AAV patients.

<p>Vertical dashed lines indicate the time of treatment with CYC (<b>A</b>) and CYC followed by RTX (<b>B</b>). Black circles stand for IgG concentrations, open circles for IgA concentrations, and black triangles for IgM concentrations. Vertical bars on the right represent the normal ranges of IgG, IgA and IgM serum concentrations. Median ± SE are reported.</p

Patients’ characteristics of the AAV cohort.

<p>Characteristics of patients with ANCA-associated vasculitis analysed for effects of cyclophosphamide (CYC) and rituximab (RTX).</p><p>GPA, granulomatosis with polyangiitis (Wegener’s granulomatosis); MPA, microscopic polyangiitis; CSS, Churg-Strauss syndrome; ANCA, antineutrophil cytoplasmic antibody; PR3, proteinase3; MPO, myeloperoxidase; IQR, interquartile range; CYC, cyclophosphamide; RTX, rituximab.</p><p>If not indicated otherwise, median and interquartile range are reported;</p>#<p>14 patients of CYC analysis group were later treated with RTX and subsequently also enrolled into the RTX analysis group.</p

Treatment characteristics of the AAV cohort.

<p>Treatment characteristics of all AAV patients, patients treated with CYC and patients treated with RTX after previous CYC therapy.</p><p>CYC, cyclophosphamide; RTX, rituximab; MTX, methotrexate; AZA, azathioprine; MMF, mycophenolate mofetil; LEF, leflunomide; CSA, cyclosporine A; IQR, interquartile range.</p>*<p>other than prednisone,</p>‡<p>before CYC or RTX treatment,</p>#<p>14 patients of the CYC group were later treated with RTX and subsequently also enrolled in the RTX analysis group.</p

List of patients and status of acanthocytosis.

<p>ID denotes the internal identification code; AC percent is the percentage of acanthocytes within the blood sample; age and age at onset is given in years.</p><p>List of patients and status of acanthocytosis.</p

Mapping of the PKAN-linked Pank2 Y227C mutation onto the Pank3 structure und partial alignment of human Pank proteins.

<p>A) Pank3 dimer structure (PDB ID 2i7P) is shown in blue (chain A) and yellow (chain B), respectively. B) The magnification reveals a polar interaction (dashed line) in the WT Pank3 between Y27 (magenta, corresponding to Y227 in Pank2) and R49 (green, corresponding to R249 in Pank2). Side chains of F14, L25, L45 and I85 (cyan) are in close proximity to Y27 (less than 5 Ångström) constituting a hydrophobic environment around it. C) A partial multiple alignment of human Pank proteins, Pank2 (GI: 85838513), Pank1 (GI:23510400) and Pank3 (GI:119581908) is shown. Elements of secondary structure, helices (α) and β-strands (-) are indicated above and numbered accordingly. Known sites of PKAN missense mutations are boxed in yellow, the Pank2 Y227 site described here is boxed in magenta, the Pank2 R249 site which is in polar contact with Pank2 Y227 and is itself a known PKAN-linked Pank2 mutation is boxed in green. D) The magnification shows the effect of a Pank3 Y27C mutation (magenta) corresponding to the PKAN-linked Pank2 Y227C mutation disrupting the tyrosine-specific polar contact and local hydrophobic packing. PANK3 structure views and mutation Y27C were edited and modeled by PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). The contact map of Y27 was calculated using the Protein Interactions Calculator (PIC) server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125861#pone.0125861.ref024" target="_blank">24</a>].</p

Distribution of acanthocytosis in the patient and control samples.

<p>The distribution of the amount of acanthocytes (in % of total cells) in the samples of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (n = 22) with respect to the c.680 A>G mutation in the PANK2 gene is shown in a Box-Whiskers blot. The circles are <i>outliers</i>, and the asterisk is a <i>far outlier</i>.</p