Presentation_3_Determination of Autoantibody Isotypes Increases the Sensitivity of Serodiagnostics in Rheumatoid Arthritis.PDF

<p>Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.</p

Presentation_1_Determination of Autoantibody Isotypes Increases the Sensitivity of Serodiagnostics in Rheumatoid Arthritis.PDF

<p>Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.</p

Study of pheonotype of the H9c2 cells derived from rat cardiac myoblasts

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martina Kutichová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Supervisor - specialist: Doc. MUDr. Michaela Adamcová, Ph.D. Title of diploma thesis: Study of phenotype H9c2 cells derived from rat cardiac myoblasts The cell-based in vitro investigation represents an important and frequently employed platform in current biomedical research. In experimental cardiovascular research, enzymatically dispersed neonatal or adult cardiomyocytes from various animal species are considered as a "gold standard" for such experiments. However, besides isolated primary cultures, permanent cell lines are becoming extremely popular among cardiovascular scientists. H9c2(2-1) is the first permanent cardiomyocyte-derived cell line. Although these cells are known since the mid-seventies, there is not much information about their phenotype. This study followed the results of the shotgun proteomic analysis, which identified about 1500 proteins. The aim of this thesis was to continue with obtained proteomic results with focus on detection of specific proteins by Western blot method. H9c2 cell were studied for the presence of several muscle and/or cardiac specific proteins in both non-proliferating H9c2..

Detailed results for lymphocyte subsets in wild type lupus (PIL^+/+), miR155-deficient lupus (PIL^-/-) and controls (CO^+/+) as assessed by flow cytometry.

<p>Detailed results for lymphocyte subsets in wild type lupus (PIL^+/+), miR155-deficient lupus (PIL^-/-) and controls (CO^+/+) as assessed by flow cytometry.</p

Flow cytometry analysis of lymphocyte subsets at 8 months after disease induction.

<p>A, B, C, MiR155-deficient PIL^-/- mice had lower frequencies of CD19⁺ B-cells, total CD4⁺ and CD4⁺CD25⁺FoxP3- activated T_effector-cells compared to the WT lupus group PIL^+/+. D, Frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_reg) were reduced in PIL^+/+ compared to the respective control group (CO^+/+). T_reg were even lower in both miR155 knockout groups (PIL^-/- and CO^-/-) without intergroup differences. E, F, G, Upon <i>in vitro</i> restimulation, PIL^+/+ had significant higher frequencies of CD4⁺IL-4⁺ Th2 and CD4⁺IL-17⁺ Th17 cells compared to PIL^-/- and CO^+/+, a similar, albeit not significant trend was observed for CD4⁺IFNγ Th1 cells when comparing PIL^+/+ to PIL^-/-. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181015#pone.0181015.t001" target="_blank">Table 1</a> for exact numbers and p values. Results are representative of 2 independent experiments. PIL were induced with pristane, CO with PBS; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts; bars show mean with SD. * = <i>P</i> < 0.05, by Mann-Whitney test.</p

Anti-dsDNA, anti-chromatin and anti-histone auto-antibody levels at 8 months after disease induction.

<p>A, Anti-dsDNA IgG antibody levels were significantly lower in mir155-deficient PIL^-/- than in lupus wild type PIL^+/+. Both pristane-induced groups, PIL^+/+ and PIL^-/-, had significantly increased antibody levels compared to CO^+/+. B, C, In addition, PIL^-/- also had lower levels of anti-chromatin-antibodies and anti-histone-antibodies than PIL^+/+. Antibody levels in PIL^-/- were only slightly elevated compared to CO^+/+. Results are representative of 2 independent experiments. PIL were induced with pristane, CO with PBS; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts; bars show mean with SD. * = <i>P</i> < 0.05, by Mann-Whitney test.</p

Immunohistochemical stainings of affected lungs.

<p>A, E, The miR155-deficient group PIL^-/- showed less accumulation of CD3 positive T cells that the lupus wild type group PIL^+/+. The same effect was observed in other immunohistochemical stainings: B, F, CD45R positive B cells, C, G, F4/80 positive macrophages and D, H, Neu7/4 positive neutrophil granulocytes. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181015#pone.0181015.t001" target="_blank">Table 1</a> for exact numbers and p values. Results are representative of 2 independent experiments. PIL were induced with pristane; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts.</p

Renal and pulmonary involvement in fully established pristane-induced lupus (PIL).

<p>A, B, C, The summative kidney biopsy score and the glomerular activity score of wildtype lupus PIL^+/+ was significantly higher than that of mir155-deficient PIL^-/-, while there was no difference in the chronic lesion score. D, E, F, If affected, PIL^-/- showed only mild mesangial glomerulonephritis. PIL^+/+ had more severe renal manifestation with increased cellularity and hyaline deposits (black arrow). 40% of PIL^+/+, but no PIL^-/-, fulfilled the criteria for proliferative glomerulonephritis, while controls were not affected. G, H, I, Although all PIL mice showed at least some pulmonary pathology, the inflammatory perivascular area as well as the area of peribronchial and total pulmonary inflammation were greater in PIL^+/+ than in PIL^-/-, while controls were not affected. J, K, L, Typical examples show the marked pulmonary inflammation in PIL^+/+ compared to PIL^-/- or controls; PIL^+/+ also had considerable hemosiderin deposits as a late consequence of intrapulmonary bleeding (white arrow). Results are representative of 2 independent experiments upon analysis at 8 months after disease induction. PIL were injected with pristane, CO with PBS; +/+ stands for wild type, -/- for miR155-deficient knockouts; bars show mean with SD, * = p< 0.05.</p

Analysis of gene-expression patterns of INF-signature related genes and T-cell subset related cytokines.

<p>A, After induction of PIL in contrast to the mir155-deficient group PIL^-/- and controls (CO^+/+), INF-related genes were upregulated in the lupus wild type group PIL^+/+. B, Cytokines related to the T-cell subsets (Th1, Th2, Th17) were significantly upregulated in PIL^+/+, but not in PIL^-/- and CO^+/+. Results are representative of 2 independent experiments. PIL were induced with pristane, CO with PBS; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts; bars show mean with SD. * = <i>P</i> < 0.05, by Mann-Whitney test.</p

In-depth analysis of the organ involvement of miR155-deficient PIL^-/- compared to wild type lupus PIL^+/+ and wild type controls CO^+/+.

<p>In-depth analysis of the organ involvement of miR155-deficient PIL^-/- compared to wild type lupus PIL^+/+ and wild type controls CO^+/+.</p