Modulation of the acute defence reaction by eplerenone prevents cardiac disease progression in viral myocarditis.

AIMS: Left ventricular (LV) dysfunction in viral myocarditis is attributed to myocardial inflammation and fibrosis, inducing acute and long-time cardiac damage. Interventions are not established. On the basis of the link between inflammation, fibrosis, aldosterone, and extracellular matrix regulation, we aimed to investigate the effect of an early intervention with the mineralocorticoid receptor antagonist (MRA) eplerenone on cardiac remodelling in a murine model of persistent coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: SWR/J mice were infected with 5 × 104 plaque-forming units of CVB3 (Nancy strain) and daily treated either with eplerenone (200 mg/kg body weight) or with placebo starting from Day 1. At Day 8 or 28 post infection, mice were haemodynamically characterized and subsequently sacrificed for immunohistological and molecular biology analyses. Eplerenone did not influence CVB3 load. Already at Day 8, 1.8-fold (P < 0.05), 1.4-fold (P < 0.05), 3.2-fold (P < 0.01), and 2.1-fold (P < 0.001) reduction in LV intercellular adhesion molecule 1 expression, presence of monocytes/macrophages, oxidative stress, and apoptosis, respectively, was observed in eplerenone-treated vs. untreated CVB3-infected mice. In vitro, eplerenone led to 1.4-fold (P < 0.01) and 1.2-fold (P < 0.01) less CVB3-induced cardiomyocyte oxidative stress and apoptosis. Furthermore, collagen production was 1.1-fold (P < 0.05) decreased in cardiac fibroblasts cultured with medium of eplerenone-treated vs. untreated CVB3-infected HL-1 cardiomyocytes. These ameliorations were in vivo translated into prevention of cardiac fibrosis, as shown by 1.4-fold (P < 0.01) and 2.1-fold (P < 0.001) lower collagen content in the LV of eplerenone-treated vs. untreated CVB3-infected mice at Days 8 and 28, respectively. This resulted in an early and long-lasting improvement of LV dimension and function, as indicated by reduced LV end-systolic volume and end-diastolic volume, and an increase in LV contractility (dP/dtmax ) and LV relaxation (dP/dtmin ), respectively (P < 0.05). CONCLUSIONS: Early intervention with the MRA eplerenone modulates the acute host and defence reaction and prevents cardiac disease progression in experimental CVB3-induced myocarditis without aggravation of viral load. The findings advocate for an initiation of therapy of viral myocarditis as early as possible, even before the onset of inflammation-induced myocardial dysfunction. This may also have implications for coronavirus disease-19 therapy.

Serum alarmin S100A8/S100A9 levels and its potential role as biomarker in myocarditis.

AIMS: The alarmin S100A8/S100A9 (S100A8/A9) is released by activated monocytes/macrophages and neutrophils in the setting lymphocytic myocarditis (MC). We recently demonstrated its therapeutic potential in experimental acute MC. Now, we investigated the diagnostic relevance of S100A8/A9 serum levels in patients with suspected acute and chronic MC and in patients with heart failure without cardiac inflammation. METHODS AND RESULTS: Serum S100A8/A9 levels were analysed in patients with a recent onset of MC [≤ 30 days, n = 32; ejection fraction (EF): 45.4 ± 12.9%], dilated cardiomyopathy patients with inflammation (n = 112; EF: 29.0 ± 11.4%), or without inflammation (n = 58; EF: 26.6 ± 9.3%), and controls (n = 25; EF: 68.5 ± 4.6%), by using specific ELISAs. Blood samples were collected at Time Point 1 (T1), where also endomyocardial biopsies (EMBs) were withdrawn. Patients with a recent onset of MC showed a 4.6-fold increase in serum S100A8/A9 levels vs. controls (MC: 1948 ± 1670 ng/mL vs. controls: 426 ± 307 ng/mL; P < 0.0001). Serum S100A8/A9 correlated with the disease activity, represented by EMB-derived counts of inflammatory cells (CD3: r = 0.486, P = 0.0047, lymphocyte function-associated antigen-1: r = 0.558, P = 0.0009, macrophage-1 antigen: r = 0.434, P = 0.013), the EMB mRNA levels of S100A8, S100A9 (r = 0.541, P = 0.002), and left ventricular ejection fraction (LVEF: r = 0.498, P = 0.0043). EMB immunofluorescence co-stainings display macrophages/monocytes and neutrophils as the main source of S100A8 and S100A9 in recent onset MC. The diagnostic value of serum alarmin levels (cut-off 583 ng/mL) was characterized by a specificity of 92%, a sensitivity of 90.6%, positive predictive value of 93.5%, negative predictive value of 88.5%, and an accuracy of 0.949 (95% confidence interval [0.89-1]). In a subgroup of MC patients, S100A8/A9 serum levels and EMBs at T1 (n = 12) and a follow-up visit (T2, n = 12, mean follow-up 8.5 months) were available. A fall of serum S100A8/A9 (T1: 2208 ± 1843 ng/mL vs. T2: 888.8 ± 513.7 ng/mL; P = 0.00052) was associated with a reduced cardiac inflammation (CD3 T1: 70.02 ± 107.4 cells per square millimetre vs. T2: 59.18 ± 182.5 cells per square millimetre; P = 0.0342, lymphocyte function-associated antigen-1 T1: 133.5 ± 187.1 cells per square millimetre vs. T2: 74.12 ± 190.5 cells per square millimetre; P = 0.0186, and macrophage-1 antigen T1: 132.6 ± 129.5 cells per square millimetre vs. T2: 54.41 ± 65.16 cells per square millimetre; P = 0.0015). Serum S100A8/A9 levels were only slightly increased in patients within the chronic phase of MC and in heart failure patients without inflammation vs. controls. CONCLUSIONS: Serum S100A8/A9 might serve as an additional tool in the diagnostic workup of suspected acute MC patients.

Mesenchymal stromal cells inhibit NLRP3 inflammasome activation in a model of Coxsackievirus B3-induced inflammatory cardiomyopathy.

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1ß, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1ß secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1ß-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

Pathogenic Role of the Damage-Associated Molecular Patterns S100A8 and S100A9 in Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The alarmins S100A8 and S100A9 are damage-associated molecular patterns, which play a pivotal role in cardiovascular diseases, inflammation, and viral infections. We aimed to investigate their role in Coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: S100A8 and S100A9 mRNA expression was 13.0-fold (P=0.012) and 5.1-fold (P=0.038) higher in endomyocardial biopsies from patients with CVB3-positive myocarditis compared with controls, respectively. Elimination of CVB3 led to a downregulation of these alarmins. CVB3-infected mice developed an impaired left ventricular function and displayed an increased left ventricular S100A8 and S100A9 protein expression versus controls. In contrast, CVB3-infected S100A9 knockout mice, which are also a complete knockout for S100A8 on protein level, showed an improved left ventricular function, which was associated with a reduced cardiac inflammatory and oxidative response, and lower CVB3 copy number compared with wild-type CVB3 mice. Exogenous application of S100A8 to S100A9 knockout CVB3 mice induced a severe myocarditis similar to wild-type CVB3 mice. In CVB3-infected HL-1 cells, S100A8 and S100A9 enhanced oxidative stress and CVB3 copy number compared with unstimulated infected cells. In CVB3-infected RAW macrophages, both alarmins increased MIP-2 (macrophage inflammatory protein-2) chemokine expression, which was reduced in CVB3 S100A8 knockdown versus scrambled siRNA CVB3 cells. CONCLUSIONS: S100A8 and S100A9 aggravate CVB3-induced myocarditis and might serve as therapeutic targets in inflammatory cardiomyopathies.

Placenta-Derived Adherent Stromal Cells Improve Diabetes Mellitus-Associated Left Ventricular Diastolic Performance.

Left ventricular (LV) diastolic dysfunction is among others attributed to cardiomyocyte stiffness. Mesenchymal stromal cells (MSC) have cardiac-protective properties. We explored whether intravenous (i.v.) application of PLacenta-eXpanded (PLX) MSC-like cells (PLX) improves LV diastolic relaxation in streptozotocin (STZ)-induced diabetic mice and investigated underlying mechanisms. Diabetes mellitus was induced by STZ application (50 mg/kg body weight) during five subsequent days. One week after the first STZ injection, PLX or saline were i.v. applied. Two weeks later, mice were hemodynamically characterized and sacrificed. At this early stage of diabetic cardiomyopathy with low-grade inflammation and no cardiac fibrosis, PLX reduced LV vascular cell adhesion molecule-1, transforming growth factor-ß1, and interferon-γ mRNA expression, induced the percentage of circulating regulatory T cells, and decreased the splenic pro-fibrotic potential in STZ mice. STZ + PLX mice exhibited higher LV vascular endothelial growth factor mRNA expression and arteriole density versus STZ mice. In vitro, hyperglycemic PLX conditioned medium restored the hyperglycemia-impaired tube formation and adhesion capacity of human umbelical vein endothelial cells (HUVEC) via increasing nitric oxide (NO) bioavailability. PLX further induced the diabetes-downregulated activity of the NO downstream protein kinase G, as well as of protein kinase A, in STZ mice, which was associated with a raise in phosphorylation of the titin isoforms N2BA and N2B. Concomitantly, the passive force was lower in single isolated cardiomyocytes from STZ + PLX versus from STZ mice, which led to an improvement of LV diastolic relaxation. We conclude that i.v. PLX injection improves diabetes mellitus-associated diastolic performance via decreasing cardiomyocyte stiffness. Stem Cells Translational Medicine 2017;6:2135-2145.

NOD2 (Nucleotide-Binding Oligomerization Domain 2) Is a Major Pathogenic Mediator of Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. METHODS AND RESULTS: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(-/-) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2-/- CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2-/- decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2-/- was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1ß (interleukin-1ß) protein expression under in vivo and in vitro CVB3 conditions. CONCLUSIONS: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling.

: Cardiac-derived adherent proliferating cells (CardAPs) are cells derived from human endomyocardial biopsy specimens; they share several properties with mesenchymal stromal cells. The aims of this study were to evaluate whether intramyocardial injection of CardAPs modulates cardiac fibrosis and hypertrophy in a mouse model of angiotensin II (Ang II)-induced systolic heart failure and to analyze underlying mechanisms. Intramyocardial application of 200,000 CardAPs improved left ventricular function. This was paralleled by a decline in left ventricular remodeling, as indicated by a reduction in cardiac fibrosis and hypertrophy. CardAPs reduced the ratio of the left ventricle to body weight and cardiac myosin expression (heavy chain), and decreased the Ang II-induced phosphorylation state of the cardiomyocyte hypertrophy mediators Akt, extracellular-signal regulated kinase (ERK) 1, and ERK2. In accordance with the antifibrotic and antihypertrophic effects of CardAPs shown in vivo, CardAP supplementation with cardiac fibroblasts decreased the Ang II-induced reactive oxygen species production, α-SMA expression, fibroblast proliferation, and collagen production. Coculture of CardAPs with HL-1 cardiomyocytes downregulated the Ang II-induced expression of myosin in HL-1. All antifibrotic and antihypertrophic features of CardAPs were mediated in a nitric oxide- and interleukin (IL)-10-dependent manner. Moreover, CardAPs induced a systemic immunomodulation, as indicated by a decrease in the activity of splenic mononuclear cells and an increase in splenic CD4CD25FoxP3, CD4-IL-10, and CD8-IL-10 T-regulatory cells in Ang II mice. Concomitantly, splenocytes from Ang II CardAPs mice induced less collagen in fibroblasts compared with splenocytes from Ang II mice. We conclude that CardAPs improve Ang II-induced cardiac remodeling involving antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. SIGNIFICANCE: Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.

The use of massage therapy for reducing pain, anxiety, and depression in oncological palliative care patients: a narrative review of the literature.

A considerable number of cancer patients use complementary medicine therapies in order to alleviate different symptoms such as pain, anxiety, and depression, occurring in connection with cancer. This paper explores the question to what extent massage therapies are able to reduce the amount of pain, anxiety, and depression. For this purpose, a systematic literature analysis was carried out in the electronic databases and specialist journals. There is already evidence that massage therapies can influence the symptoms of pain, anxiety, and depression in a positive way.

Two cases of lysergamide intoxication by ingestion of seeds from Hawaiian Baby Woodrose.

We describe two cases of human consumption of seeds from Argyreia nervosa (Hawaiian Baby Woodrose), which resulted in one fatality due to falling from a building and one surviving witness. The principal psychoactive constituent of the seeds, lysergamide (LSA), was recovered from blood and urine samples by mixed-mode cation exchange solid-phase extraction and quantified by ultra performance liquid chromatography-time of flight mass spectrometry (UPLC-ToF/MS). The LSA concentrations were determined by UPLC-ToF/MS to be 4.9 microg/L in blood and 1.0mg/L in urine in the dead person and 1.8 microg/L in blood and 0.50mg/L in urine in the living person. These analytical findings were found to be in accordance with the case story, which indicated that seeds had been ingested and also noted psychological reactions, i.e. the will to jump out of the window. Other findings in the dead person were 22 microg/L THC in blood, 0.71 g/L ethanol in blood and 1.0 g/L ethanol in vitreous humor. Constituents originating from the seeds of A. nervosa, i.e. LSA, ergonovine, lysergic acid alpha-hydroxyethylamide were also identified in the biological samples. The 2-hydroxy-3-oxo metabolites of LSA and ergonovine were identified in the urine sample of the deceased.

[Massage and music therapy for relief of anxiety of cancer patients in palliative care]. / Massagen und Musiktherapie zur Reduktion der Angst von onkologischen Patienten bei der Palliativversorgung.

In palliative care between 13.9 and 25 percent of all patients suffering from cancer show signs of anxiety disorders. Up to 75 percent of these patients suffer from non-pathologic anxiety, which has a negative impact on the patients' quality of life. Therefore it is important to provide interventions that are able to reduce anxiety of cancer patients. Massage and music therapy are effective interventions for minimizing anxiety of cancer patients in palliative care. An empathic attitude of the nurse increases the effect of the mentioned interventions. While there is evidence of the interventions mentioned it is yet necessary to further explore these in additional clinical trials to consolidate the already existing results.

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of NBS lymphoblasts.

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced gamma-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 (-/-) and NBS1 (+/-) cells are not due to an altered p53 stabilization or phosphorylation in NBS1 (-/-) cells. gamma-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 (-/-) cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after gamma-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.

Time-course of radiation-induced chromosomal aberrations in tumor patients after radiotherapy.

PURPOSE: Radiation-induced chromosome aberrations are routinely used in biologic dosimetry to monitor radiation exposure. Translocations are considered stable aberrations with time after exposure. This study was performed to determine the temporal persistence of radiation-induced translocations during a 36-month period in therapeutically irradiated testicular seminoma patients who underwent partial body exposure (>10% of bone marrow). METHODS AND MATERIALS: Chromosome analyses were carried out in peripheral lymphocytes of 11 patients with testicular seminoma (n = 9), germinoma (n = 1), or follicular non-Hodgkin's lymphoma (n = 1). All patients received radiotherapy with photons from a linear accelerator; in 1 case, additional electron beams were used. Doses ranged from 26 Gy (seminoma) to 45 Gy (non-Hodgkin's lymphoma). None of the patients received chemotherapy. From each patient, blood samples were taken during the 36 months after irradiation at defined points. Chromosomal aberrations were scored after fluorescence in situ hybridization painting of chromosomes 1, 4, and 12 in combination with a pancentromeric probe. RESULTS: For 9 patients (7 with testicular seminoma, 1 with germinoma, and 1 with non-Hodgkin's lymphoma), a significant temporal decline of translocations, with a mean decline rate of 4.4% +/- 0.4% monthly, could be detected. Two testicular seminoma patients showed no temporal decline of aberration frequencies. CONCLUSION: Most partial body irradiated patients (9 of 11) showed a significant temporal decline of translocation frequencies during a 36-month period. Thus, reciprocal translocations after partial body irradiation cannot be regarded as stable over time. The temporal decline of aberration frequencies has to be taken into account for retrospective dose estimations.