Utilizing CD44v6 and V600EBRAF-mutation for in vitro targeted combination therapy of thyroid carcinomas.

Aim: The aim of this study was to assess the feasibility of targeted therapy of thyroid carcinoma, first exploring potential targets BRAF, EGFR and CD44v6 in patient material through immunohistochemistry and mutation analysis. Materials and methods: A patient cohort (n = 22) consisting of seven papillary (PTC), eight anaplastic (ATC) and seven follicular (FTC) thyroid carcinomas were evaluated. Additionally, eight thyroid carcinoma cells lines were analyzed for CD44v6-expression and sensitivity to the multi-kinase inhibitor sorafenib (Nexavar®), which targets numerous serine/threonine and tyrosine kinases, including the Raf family kinases. Targeted therapy using 131I-AbN44v6, a novel anti-CD44v6 antibody, and/or sorafenib was evaluated in 3D multicellular tumor spheroids. Results: Of the two cell surface proteins, EGFR and CD44v6, the latter was overexpressed in >80 % of samples, while EGFR-expression levels were moderate at best in only a few samples. BRAF mutations were more common in PTC patient samples than in ATC samples, while FTC samples did not harbor BRAF mutations. CD44v6-expression levels in the thyroid carcinoma cell lines were more heterogenous compared to patient samples, while BRAF mutational status was in line with the original tumor type. Monotherapy in 3D multicellular ATC tumor spheroids with either 131I-AbN44v6 or sorafenib resulted in delayed spheroid growth. The combination of 131I-AbN44v6 and sorafenib was the most potent and resulted in significantly impaired spheroid growth. Conclusion: This "proof of concept" targeted therapy study in the in vitro ATC 3D multicellular tumor spheroids indicated applicability of utilizing CD44v6 for molecular radiotherapy both as a monotherapy and in combination with sorafenib.

Transcription profiling of peripheral B cells in antibody-positive primary Sjögren's syndrome reveals upregulated expression of CX3CR1 and a type I and type II interferon signature.

B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS). The aim of this study was to analyse the transcriptome of CD19+ B cells from patients with pSS and healthy controls to decipher the B cell-specific contribution to pSS. RNA from purified CD19+ B cells from 12 anti-SSA antibody-positive untreated female patients with pSS and 20 healthy blood donors was subjected to whole transcriptome sequencing. A false discovery rate corrected significance threshold of α < 0.05 was applied to define differential gene expression. As validation, gene expression in B cells from 17 patients with pSS and 16 healthy controls was analysed using a targeted gene panel. RNA-sequencing identified 4047 differentially expressed autosomal genes in pSS B cells. Upregulated expression of type I and type II interferon (IFN)-induced genes was observed, establishing an IFN signature in pSS B cells. Among the top upregulated and validated genes were CX3CR1, encoding the fractalkine receptor involved in regulation of B-cell malignancies, CCL5/RANTES and CCR1. Increased expression of several members of the TNF superfamily was also identified; TNFSF4/Ox40L, TNFSF10/TRAIL, TNFSF13B/BAFF, TNFRSF17/BCMA as well as S100A8 and -A9/calprotectin, TLR7, STAT1 and STAT2. Among genes with downregulated expression in pSS B cells were SOCS1 and SOCS3, CD70 and TNFAIP3/A20. We conclude that B cells from patients with anti-SSA antibody-positive pSS display immune activation with upregulated expression of chemokines, chemokine receptors and a prominent type I and type II IFN signature, while suppressors of cytokine signalling are downregulated. This adds insight into the autoimmune process and suggests potential targets for future functional studies.

Diminished CXCR5 expression in peripheral blood of patients with Sjögren's syndrome may relate to both genotype and salivary gland homing.

Genetic investigations of Sjögren's syndrome (SS) have identified a susceptibility locus at p23.3 of chromosome 11, which contains the CXCR5 gene. C-X-C motif chemokine receptor 5 (CXCR5) is a chemokine receptor expressed on B and T cell subsets, and binds the chemotactic ligand C-X-C motif chemokine ligand 13 (CXCL13). In this study we aimed to link the genetic association with functional effects and explore the CXCR5/CXCL13 axis in SS. Expression quantitative trait loci analysis of the 11q23.3 locus was performed using B cell mRNA expression data from genotyped individuals. Lymphocyte surface markers were assessed by flow cytometry, and CXCL13 levels by a proximity extension assay. CXCR5+ and CXCL13+ cells in minor salivary glands were detected using immunohistochemistry. Our results demonstrated that SS-associated genetic polymorphisms affected the expression of CXCR5 (P < 0·01). Notably, a decreased percentage of CXCR5+ cells, with lower CXCR5 expression, was observed for most circulating B and T cell subsets in SS patients, reaching statistical significance in CD19+ CD27+ immunoglobulin (Ig)D+ marginal zone (P < 0·001), CD19+ CD27+ IgD- memory (P < 0·05) and CD27-IgD double-negative (P < 0·01) B cells and CD4+ CXCR3- CCR6+ Th17 cells (P < 0·05). CXCL13 levels were increased in patient plasma (P < 0·001), and immunohistochemical staining revealed expression of CXCL13 and higher numbers of CXCR5+ cells (P < 0·0001) within focal infiltrates and interstitially in salivary glands of SS patients. In conclusion, we link a genetic susceptibility allele for SS to a functional phenotype in terms of decreased CXCR5 expression. The decrease of CXCR5+ cells in circulation was also related to homing of B and T cells to the autoimmune target organ. Therapeutic drugs targeting the CXCR5/CXCL13 axis may be useful in SS.

Therapeutic optimization of atrioventricular delay in cardiosurgical ICU patients by noninvasive cardiac output measurements versus pulse contour analysis.

BACKGROUND: Optimizing atrioventricular (AV) delay improves cardiac output and postoperative outcome. Impedance cardiography (ICG) is a non-invasive method for CO measurement. This study evaluates the ability of two ICG methods to determine the optimal AV delay (OAVD) and to compare ICG with invasive PICCO measurements. METHODS: In 14 cardiosurgical ICU patients (age 70.4 +/- 12.0 yrs) with temporary pacing wires, OAVD was determined by pulse contour analysis (PICCO) and ICG (conventional ICG [CI] and electrical velocimetry [EV] ICG monitors). Cardiac output (CO) and stroke volume (SV) were measured during DDD pacing with AVD varying from 70 to 270 ms in 20-ms increments. RESULTS: Measured OAV showed a linear correlation between PICCO and ICG: CI (r = 0.82, P < 0.0002) and EV (r = 0.84, P < 0.0002). The mean OAVD deviation between PICCO and ICG was 15.7 +/- 21.0 ms (CI) and 17.1 +/- 20.5 ms (EV). Hemodynamic parameters (SV increase OAVD against worst case) improved significantly (+ 11.7 +/- 7.2 %, P < 0.0001). CONCLUSION: Inappropriate selection of AVD can compromise the hemodynamic situation of cardiosurgical patients. As it is totally noninvasive, ICG is a reliable and effective tool for tailoring AVD. Both systems (CI and EV) offer valid OAV determination.

Modern noninvasive risk stratification in primary prevention of sudden cardiac death.

OBJECTIVE: Since the publication of MADIT II and SCD-HeFT, an implantable cardioverter defibrillator (ICD) for primary prevention represents an established, guideline-implemented therapeutic strategy. Facing such an enormous amount of potential ICD recipients, the identification of an effective risk stratification remains crucial. METHODS: This article reviews the tools of noninvasive risk stratification which are currently used and defines an optimal test configuration. This analysis focuses on the capacity of the tests regarding to the negative predictive value to reduce unneeded devices. RESULTS: Presently, no marker exists in terms of risk stratification which qualifies itself as gold standard. However, encouraging results can be stated for microvolt T-wave alternans (mTWA) providing a high negative predictive value. An increased QT variability (QTv) and an impaired deceleration capacity are associated with an excellent positive predictive value. Currently, only mTWA and QTv seem to be suitable in ischemic and non-ischemic disease, but available data, especially in non-ischemic patients, are too small to provide clear recommendations. CONCLUSION: The most hopeful tools at hand in modern noninvasive risk evaluation of sudden cardiac death in primary prevention seem to be mTWA and QTv. These noninvasive methods provide the best negative predictive or positive predictive value of all known parameters, while a higher rate of complete coronary revascularizations in acute coronary syndromes might also reduce the number of fatal arrhythmic events and therefore complicate the invention of an ideal risk marker.

Significance of Wedensky Modulation testing in the evaluation of non-invasive risk stratification for ventricular tachyarrhythmia in patients with coronary artery disease and implantable cardioverter-defibrillator.

OBJECTIVE: Evaluation of the significance of the Wedensky Modulation (WM) examination for ventricular tachyarrhythmias (VT) in patients with coronary artery disease and implantable cardioverter-defibrillator therapy (ICD). DESIGN: Prospective, single-centre study conducted from 2004 to 2006. SETTING: University of Bonn, Department of Medicine - Cardiology, Bonn, Germany. PATIENTS: 37 consecutive patients with coronary artery disease receiving an ICD for primary or secondary prevention. MAIN OUTCOME MEASURES: Correlation of a positive WM-Index (WMI) with established non-invasive Holter parameter, the occurrence of VT after ICD implantation with regard to primary or secondary prevention, and inducibility of VT during electrophysiological (EP) studies. RESULTS: The WMI was positive in 15 patients (67 (SD 8) years, 31% (SD 12%) EF) and showed significant correlation with heart rate variability (standard deviation of normal to normal intervals (SDNN): 143 (SD 80) ms vs 102 (SD 29) ms, p = 0.04, r = 0.45; total power (TP). 11 885 (SD 19 674) ms(2) vs 2229 (SD 1779) ms(2), p = 0.03, r = 0.384; very low frequency component (VLF): 2777 (SD 3039) ms(2) vs 1184 (SD 565) ms(2), p = 0.03; low frequency component (LF): 2955 (SD 5734) ms(2) vs 468 (SD 725) ms(2), p = 0.05, r = 0.375; high frequency component (HF): 4885 (SD 9939) ms(2) vs 382 (SD 609) ms(2), p = 0.05, r = 0.315) and turbulence (turbulence onset (TO): -0.002 (SD 0.008) vs +0.005 (SD 0.01), p = 0.05, r = 0.301; turbulence slope (TS): 3.4 (SD 3.1) vs 1.7 (SD 1.5), p = 0.04, r = 0.419). The positive predictive value of the WMI considering the inducibility of VT during EP testing was 100%. Those patients who received an ICD for primary prevention showed a higher WMI (p = 0.049) than the secondary prevention group. With respect to the occurrence of adequate VT episodes, a negative WM test result demonstrated a negative predictive value of 95%. CONCLUSION: The data presented show that the WM-Index predicts VT inducibility during EP testing and indicates a high negative predictive value regarding the occurrence of VT.