Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Chronic Fatigue and Dysautonomia following COVID-19 Vaccination Is Distinguished from Normal Vaccination Response by Altered Blood Markers.

SARS-CoV-2 mRNA vaccination can entail chronic fatigue/dysautonomia tentatively termed post-acute COVID-19 vaccination syndrome (PACVS). We explored receptor autoantibodies and interleukin-6 (IL-6) as somatic correlates of PACVS. Blood markers determined before and six months after first-time SARS-CoV-2 vaccination of healthy controls (N = 89; 71 females; mean/median age: 39/49 years) were compared with corresponding values of PACVS-affected persons (N = 191; 159 females; mean/median age: 40/39 years) exhibiting chronic fatigue/dysautonomia (≥three symptoms for ≥five months after the last SARS-CoV-2 mRNA vaccination) not due to SARS-CoV-2 infection and/or confounding diseases/medications. Normal vaccination response encompassed decreases in 11 receptor antibodies (by 25-50%, p < 0.0001), increases in two receptor antibodies (by 15-25%, p < 0.0001) and normal IL-6. In PACVS, serological vaccination-response appeared significantly (p < 0.0001) altered, allowing discrimination from normal post-vaccination state (sensitivity = 90%, p < 0.0001) by increased Angiotensin II type 1 receptor antibodies (cut-off ≤ 10.7 U/mL, ROC-AUC = 0.824 ± 0.027), decreased alpha-2B adrenergic receptor antibodies (cut-off ≥ 25.2 U/mL, ROC-AUC = 0.828 ± 0.025) and increased IL-6 (cut-off ≤ 2.3 pg/mL, ROC-AUC = 0.850 ± 0.022). PACVS is thus indicated as a somatic syndrome delineated/detectable by diagnostic blood markers.

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Autoantibodies targeting G protein-coupled receptors: An evolving history in autoimmunity. Report of the 4th international symposium.

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions.

SARS-CoV-2 Virus-like Particles (VLPs) Specifically Detect Humoral Immune Reactions in an ELISA-Based Platform.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

Increased protease-activated receptor 1 autoantibodies are associated with severe COVID-19.

In patients with severe #COVID19, increased levels of autoantibodies against PAR1 were found. These might serve as allosteric agonists of PAR1 on endothelial cells and platelets, and thus might contribute to the pathogenesis of microthrombosis in COVID-19. https://bit.ly/3pqM9Vv.

Dysregulated autoantibodies targeting vaso- and immunoregulatory receptors in Post COVID Syndrome correlate with symptom severity.

Most patients with Post COVID Syndrome (PCS) present with a plethora of symptoms without clear evidence of organ dysfunction. A subset of them fulfills diagnostic criteria of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Symptom severity of ME/CFS correlates with natural regulatory autoantibody (AAB) levels targeting several G-protein coupled receptors (GPCR). In this exploratory study, we analyzed serum AAB levels against vaso- and immunoregulatory receptors, mostly GPCRs, in 80 PCS patients following mild-to-moderate COVID-19, with 40 of them fulfilling diagnostic criteria of ME/CFS. Healthy seronegative (n=38) and asymptomatic post COVID-19 controls (n=40) were also included in the study as control groups. We found lower levels for various AABs in PCS compared to at least one control group, accompanied by alterations in the correlations among AABs. Classification using random forest indicated AABs targeting ADRB2, STAB1, and ADRA2A as the strongest classifiers (AABs stratifying patients according to disease outcomes) of post COVID-19 outcomes. Several AABs correlated with symptom severity in PCS groups. Remarkably, severity of fatigue and vasomotor symptoms were associated with ADRB2 AAB levels in PCS/ME/CFS patients. Our study identified dysregulation of AAB against various receptors involved in the autonomous nervous system (ANS), vaso-, and immunoregulation and their correlation with symptom severity, pointing to their role in the pathogenesis of PCS.

Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching.

OBJECTIVE: To determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc). METHODS: C57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4+ or CD8+ T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope. A monoclonal (m)AT1R Ab was generated by hybridoma technique and transferred into C57BL/6J and AT1Ra/b knockout mice. The induced phenotype was examined by histology, immunohistochemistry, immunofluorescence, apoptosis assay and ELISA. In vitro, Abs responses towards AT1R were measured in cells of different origins and species. RESULTS: AT1R-immunised mice developed perivascular skin and lung inflammation, lymphocytic alveolitis, weak lung endothelial apoptosis and skin fibrosis accompanied by Smad2/3 signalling, not present in controls or mice deficient for CD4+ T and B cells. The AT1R peptide 149-172 provoked lung inflammation. Application of the mAT1R Ab induced skin and lung inflammation, not observed in AT1Ra/b knockout mice. In vitro, AT1R Abs activated rat cardiomyocytes and human monocytes, enhanced angiotensin II-mediated AT1R activation in AT1R-transfected HEK293 cells via AT1R binding and mAT1R Ab-activated monocytes mediated the induction of profibrotic markers in dermal fibroblasts. CONCLUSION: Our immunisation strategy successfully induced AT1R Abs, contributing to inflammation and, possibly, to fibrosis via activation of AT1R. Therefore, AT1R Abs are valuable targets for future therapies of SSc and other AT1R Ab-related diseases.

Autoantibodies targeting GPCRs and RAS-related molecules associate with COVID-19 severity.

COVID-19 shares the feature of autoantibody production with systemic autoimmune diseases. In order to understand the role of these immune globulins in the pathogenesis of the disease, it is important to explore the autoantibody spectra. Here we show, by a cross-sectional study of 246 individuals, that autoantibodies targeting G protein-coupled receptors (GPCR) and RAS-related molecules associate with the clinical severity of COVID-19. Patients with moderate and severe disease are characterized by higher autoantibody levels than healthy controls and those with mild COVID-19 disease. Among the anti-GPCR autoantibodies, machine learning classification identifies the chemokine receptor CXCR3 and the RAS-related molecule AGTR1 as targets for antibodies with the strongest association to disease severity. Besides antibody levels, autoantibody network signatures are also changing in patients with intermediate or high disease severity. Although our current and previous studies identify anti-GPCR antibodies as natural components of human biology, their production is deregulated in COVID-19 and their level and pattern alterations might predict COVID-19 disease severity.

Non-HLA Antibodies in Hand Transplant Recipients Are Connected to Multiple Acute Rejection Episodes and Endothelial Activation.

The role of anti-HLA antibodies in transplant rejection is well-known but the injury associated with non-HLA antibodies is now widely discussed. The aim of our study was to investigate a role of non-HLA antibodies in hand allografts rejection. The study was performed on six patients after hand transplantation. The control group consisted of: 12 kidney transplant recipients and 12 healthy volunteers. The following non-HLA antibodies were tested: antibody against angiotensin II type 1 receptor (AT1R-Ab), antibody against endothelin-1 type-A-receptor (ETAR-Ab), antibody against protease-activated receptor 1 (PAR-1-Ab) and anti-VEGF-A antibody (VEGF-A-Ab). Chosen proinflammatory cytokines (Il-1, IL-6, IFNγ) were used to evaluate the post-transplant humoral response. Laboratory markers of endothelial activation (VEGF, sICAM, vWF) were used to assess potential vasculopathy. The patient with the highest number of acute rejections had both positive non-HLA antibodies: AT1R-Ab and ETAR-Ab. The same patient had the highest VEGF-A-Ab and very high PAR1-Ab. All patients after hand transplantation had high levels of laboratory markers of endothelial activation. The existence of non-HLA antibodies together with multiple acute rejections observed in patient after hand transplantation should stimulate to look for potential role of non-HLA antibodies in humoral injury in vascular composite allotransplantation.

Stem-cell mobilization of healthy sibling donors with pegfilgrastim-A prospective open-label phase II trial (EudraCT no: 2005-004971-39).

BACKGROUND: Pegfilgrastim is a covalently bound conjugate of filgrastim and mono-methoxypolyethylene glycol with a longer half-life. STUDY DESIGN AND METHODS: We report on phase II prospective monocentric trial examining the feasibility of stem cell mobilization with 12 mg single dose pegfilgrastim in related donors. The objectives were to determine the optimal collection day, defined as CD34+ concentration in peripheral blood (PB) >50 cells/µl, the number of donors collected with single leukapheresis, and the peak level of pegfilgrastim in donor-serum. Furthermore, the cell composition of grafts was assessed and compared to published data. RESULTS: The results included about 28 matched related donors. The median pegfilgrastim serum level remained >200 ng/mL for 48 hours before declining, with the maximal measured concentration of 259.49 ng/ml 24 h after application. The median white blood cell count and CD34 count in PB peaked on day four with 52.6 (range 22.8-85.0) Gpt/l and 66.25 (range 22.9-136.6) cells/µl, respectively. A CD34+ count >50 cells/µl on day four was detected in 75% of donors. 79% of the donors underwent a single collection. Conventional filgrastim was administered additionally in two donors, due to insufficient CD 34+ concentration in PB. 89% of donors showed CD34+ yields ≥4 (median 6.5, range 4.6-14.5) × 10/kg body weight of the recipient. All grafts were administered without rejections. DISCUSSION: The results of this trial showed that stem cell mobilization with pegfilgrastim is a feasible, and attractive option. This is the first trial presenting the kinetics of pegfilgrastim serum levels in healthy donors.

Autoantibody Release in Children after Corona Virus mRNA Vaccination: A Risk Factor of Multisystem Inflammatory Syndrome?

Multisystem inflammatory syndrome (MIS) is a new systemic inflammatory acute onset disease that mainly affects children (MIS-C) and, at a lesser frequency, adults (MIS-A); it typically occurs 3-6 weeks after acute SARS-CoV infection. It has been postulated and shown in adults that MIS may occur after SARS-CoV-2 vaccination (MIS-V). Our current case is one of the first published cases with a multisystem inflammatory syndrome in an 18-year-old adolescent after the SARS-CoV-2 vaccine from Pfizer/BionTech (BNT162b2), who fulfills the published level 1 criteria for a definitive disease: age < 21 years, fever > 3 consecutive days, pericardial effusion, elevated CRP/NT-BNP/Troponin T/D-dimeres, cardiac involvement, and positive SARS-CoV-2 antibodies. The disease starts 10 weeks after the second vaccination, with a fever (up to 40 °C) and was treated with amoxicillin for suspected pneumonia. The SARS CoV-2-PCR and several antigen tests were negative. With an ongoing fever, he was hospitalized 14 days later. A pericardial effusion (10 mm) was diagnosed by echocardiography. The C-reactive protein (174 mg/L), NT-BNP (280 pg/mL), and Troponin T (28 pg/mL) values were elevated. Due to highly elevated D-dimeres (>35,000 µg/L), a pulmonary embolism was excluded by thoracal computer tomography. If the boy did not improve with intravenous antibiotics, he was treated with intravenous immunoglobulins; however, the therapy was discontinued after 230 mg/kg if he developed high fever and hypotension. A further specialized clinic treated him with colchicine and ibuprofen. The MIS-V was discovered late, 4 months after the onset of the disease. As recently shown in four children with MIS-C after SARS-CoV-2 infection and a girl with Hashimoto thyroiditis after BNT162b2 vaccination, we found elevated functional autoantibodies against G-protein-coupled receptors that may be important for pathophysiology but are not conclusive for the diagnosis of MIS-C. Conclusion: We are aware that a misattribution of MIS-V as a severe complication of coronavirus vaccination can lead to increased vaccine hesitancy and blunt the global COVID-19 vaccination drive. However, the pediatric population is at a higher risk for MIS-C and a very low risk for COVID-19 mortality. The publication of such cases is very important to make doctors aware of this complication of the vaccination, so that therapy with intravenous immunoglobulins can be initiated at an early stage.

Ambrisentan, an endothelin receptor type A-selective antagonist, inhibits cancer cell migration, invasion, and metastasis.

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan's inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Soluble (pro)renin receptor in elderly chronic heart failure patients.

Overactivation of renin-angiotensin system (RAS) is one of the main pathophysiological features in the evolution of chronic heart failure (CHF). The (pro)renin receptor ((P)RR) represents an important player in a tissue renin-angiotensin system (tissue RAS), which mediates tissue injury through fibrosis and hypertrophy of the affected organs in CHF patients. In our study we used plasma samples from 556 elderly subjects with CHF and 198 healthy participants in order to evaluate prognostic and diagnostic potential of s(P)RR in setting of CHF. The patients with CHF showed significantly higher plasma levels of s(P)RR than the healthy volunteers (p=0.0005). We observed association between higher s(P)RR plasma concentrations and lower left ventricular ejection fraction and higher degree of left ventricular dilatation on baseline echocardiography examination of the CHF patients. Elderly CHF patients with higher baseline s(P)RR plasma concentration were at same risk for death, stroke and hospitalization due to heart failure worsening at mean follow-up from forty-eight months in comparison to low s(P)RR counterparts.

GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis.

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.

Antibodies to Multiple Receptors are Associated with Neuropsychiatric Symptoms and Mortality in Alzheimer's Disease: A Longitudinal Study.

BACKGROUND: Endogenous antibodies to signaling molecules and receptors (Abs) are associated with Alzheimer's disease (AD). OBJECTIVES: To investigate the association of 33 Abs to dopaminergic, serotoninergic, muscarinic, adrenergic, vascular, and immune receptors with cognitive, neuropsychiatric, and mortality outcomes. METHODS: Ninety-one patients with mild AD were followed annually for 5 years with the Mini-Mental State Examination (MMSE) and the Neuropsychiatric Inventory (NPI; composite outcomes: "psychosis" (item 1 + 2), "mood" (item 4 + 5 + 7), and "agitation" (item 3 + 8 + 9)). Abs were quantified in sera obtained at baseline by ELISA and reduced to principal components (PCs). Associations between Abs and outcomes were assessed by a mixed model (MMSE decline), zero-inflated fixed effects count models (composite NPI scores), and Cox regression (mortality). The resulting p-values were adjusted for multiple testing according to a false discovery rate of 0.05 (Benjamini-Hochberg). RESULTS: The measured levels of the 33 Abs formed four PCs. PC1 (dopaminergic and serotonergic Abs) was associated with increased mortality (Hazard ratio 2.57, p < 0.001), PC2 (serotonergic, immune, and vascular Abs) with decreased agitation symptoms (ß - 0.19, p < 0.001), and PC3 (cholinergic receptor Abs) with increased mood symptoms (ß 0.04, p = 0.002), over time. There were no associations between Abs and MMSE decline. CONCLUSION: The associations between Abs, mortality, and neuropsychiatric symptoms reported in this cohort are intriguing. They cannot, however, be generalized. Validation in independent sample sets is required.

Antibodies to Signaling Molecules and Receptors in Alzheimer's Disease are Associated with Psychomotor Slowing, Depression, and Poor Visuospatial Function.

BACKGROUND: Alzheimer's disease (AD) is associated with several antibodies as well as signaling molecules and receptors. These may be detrimental in the presence of a disrupted blood-brain barrier (BBB). OBJECTIVE: To investigate whether the levels of antibodies toward 33 signaling molecules involved in neurotransmitter, vascular, and immune functions were associated with AD and, within the AD group; cognitive function and mood. METHODS: Antibodies in sera from patients with mild AD [(n = 91) defined as a Mini-Mental State Examination ≥ 20 or a Clinical Dementia Rating Scale≤1] and healthy controls (n = 102) were measured with enzyme-linked immunosorbent assays. Levels in AD and controls were compared by Mann-Whitney test. In the AD group, associations between antibodies and psychometric test scores were analyzed by robust regression. The false discovery threshold was set to 0.05. RESULTS: Antibodies to serotonin receptors [5-HT2AR (effect size (r) = 0.21, p = 0.004), 5-HT2CR (r = 0.25, p = 0.0005) and 5-HT7R (r = 0.21, p = 0.003)], vascular endothelial growth factor receptor 1 [VEGFR1 (r = 0.29, p < 0.001)] and immune-receptors (Stabilin-1 (r = 0.23, p = 0.001) and C5aR1 (r = 0.21, p = 0.004) were higher in AD. Psychomotor speed was associated with D1R-abs (ß 0.49, p < 0.001), depression with ETAR-abs (ß 0.31, p < 0.001), and visuospatial function with 5-HT1AR-abs (ß 0.27, p = 0.004) despite similar antibody levels compared to controls. CONCLUSIONS: Antibody levels to VEGFR1, serotonergic receptors, and receptors in the immune system were increased in AD. Antibodies at similar levels as in controls were associated cognitive dysfunction and depression in AD.

Comparative assessment of urinary prostate cancer antigen 3 and TMPRSS2:ERG gene fusion with the serum [-2]proprostate-specific antigen-based prostate health index for detection of prostate cancer.

BACKGROUND: We compared urinary prostate cancer antigen 3 (PCA3), transmembrane protease, serine 2 (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) gene fusion (T2:ERG), and the serum [-2]proprostate-specific antigen ([-2]proPSA)-based prostate health index (Phi) for predicting biopsy outcome. METHODS: Serum samples and first-catch urine samples were collected after digital rectal examination (DRE) from consented outpatients with PSA 0.5-20 µg/L who were scheduled for prostate biopsy. The PCA3 score (PROGENSA PCA3, Hologic Gen-Probe) and T2:ERG score (Hologic Gen-Probe) were determined. Measurements of serum PSA, free PSA, and [-2]proPSA (Beckman Coulter) were performed, and the percentages of free PSA (%fPSA) and Phi ([-2]proPSA/fPSA × âˆšPSA) were determined. RESULTS: Of 246 enrolled men, prostate cancer (PCa) was diagnosed in 110 (45%) and there was no evidence of malignancy (NEM) in 136 (55%). A first set of biopsies was performed in 136 (55%) of all men, and 110 (45%) had ≥1 repeat biopsies. PCA3, Phi, and T2:ERG differed significantly between men with PCa and NEM, and these markers showed the largest areas under the ROC curve (AUCs) (0.74, 0.68, and 0.63, respectively). PCA3 had the largest AUC of all parameters, albeit not statistically different from Phi. Phi showed somewhat lower specificities than PCA3 at 90% sensitivity. Combination of both markers enhanced diagnostic power with modest AUC gains of 0.01-0.04. Although PCA3 had the highest AUC in the repeat-biopsy cohort, the highest AUC for Phi was observed in DRE-negative patients with PSA in the 2-10 µg/L range. CONCLUSIONS: PCA3 and Phi were superior to the other evaluated parameters but their combination gave only moderate enhancements in diagnostic accuracy for PCa at first or repeat prostate biopsy.

Cells on a chip--the use of electric properties for highly sensitive monitoring of blood-derived factors involved in angiotensin II type 1 receptor signalling.

BACKGROUND: We developed a highly sensitive cardiomyocyte based screening system for the non-destructive electronic detection of chronotropic drugs and tissue-secreted factors involved in AT1 receptor-mediated cardiovascular diseases. METHODS: For this purpose we cultured spontaneously beating neonatal rat cardiomyocytes on microelectrode arrays (MEAs), and tested the optimised, stable culture parameters for a reproducible real-time recording of alterations in contraction frequency. After the evaluation of culture parameters, computer-based electronic measurement systems were used for counting of contractions by recording of the field potential of cardiomyocytes. RESULTS: Using the biosensor, angiotensin II, the predominant ligand of the AT1 receptor, was detected at very low concentrations of 10(-11) M via altered contractions of cardiomyocytes. Moreover, we demonstrated that cardiomyocyte coupled microarrays allow the detection of blood-derived low concentrated anti-AT1 receptor autoimmune antibodies of pregnant women suffering from preeclampsia. CONCLUSION: This study demonstrates the first well-suited electrophysiological recording of cardiomyocytes on multielectrode arrays as a benefit for functional biomonitoring for the detection of AT1 receptor/ligand interactions and other marker proteins in sera directed to cardiovascular diseases.