Karin Klingel

B19V is a common pathogen in microvascular disease and cardiomyopathy with infection of endothelial cells. B19V replication, however, is almost restricted to erythroid progenitor cells (ErPC). Endothelial regeneration attributable to bone marrow-derived circulating angiogenic cells (CAC) is a prerequisite for organ function. Due to many similarities of ErPC and CAC, we hypothesized B19V as perpetrator for impaired endogenous endothelial regeneration. B19V DNA and mRNA were quantified from endomyocardial biopsies (EMB), and bone marrow and circulating progenitor cells by PCR. Highest B19V DNA concentrations were found in CD34(+)KDR(+)-cells from 17 patients with chronic B19V cardiomyopathy. B19V replication intermediates could be detected in nearly half of the patients. Furthermore, chronic B19V infection was associated with impaired endothelial regenerative capacity. B19V infection of CAC in vitro resulted in expression of transcripts encoding B19V proteins. The capsid protein VP1 was identified as novel inducer of apoptosis in addition to non-structural proteins. Inhibition studies identified "death receptor" signalling with activation of caspases-8 and -10 to be responsible for apoptosis induction. B19V causally impaired endothelial regeneration with spreading of B19V in CAC in an animal model in vivo. We thus conclude that B19V infection and damage of CAC result in dysfunctional endogenous vascular repair supporting a primary bone marrow disease with secondary end-organ damage.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

B19V is a common pathogen in microvascular disease and cardiomyopathy with infection of endothelial cells. B19V replication, however, is almost restricted to erythroid progenitor cells (ErPC). Endothelial regeneration attributable to bone marrow-derived circulating angiogenic cells (CAC) is a prerequisite for organ function. Due to many similarities of ErPC and CAC, we hypothesized B19V as perpetrator for impaired endogenous endothelial regeneration. B19V DNA and mRNA were quantified from endomyocardial biopsies (EMB), and bone marrow and circulating progenitor cells by PCR. Highest B19V DNA concentrations were found in CD34(+)KDR(+)-cells from 17 patients with chronic B19V cardiomyopathy. B19V replication intermediates could be detected in nearly half of the patients. Furthermore, chronic B19V infection was associated with impaired endothelial regenerative capacity. B19V infection of CAC in vitro resulted in expression of transcripts encoding B19V proteins. The capsid protein VP1 was identified as novel inducer of apoptosis in addition to non-structural proteins. Inhibition studies identified "death receptor" signalling with activation of caspases-8 and -10 to be responsible for apoptosis induction. B19V causally impaired endothelial regeneration with spreading of B19V in CAC in an animal model in vivo. We thus conclude that B19V infection and damage of CAC result in dysfunctional endogenous vascular repair supporting a primary bone marrow disease with secondary end-organ damage.

The presence of pericardial effusion (PE) is considered to be suggestive of inflammation in suspected myocarditis. However, the incremental value of assessing for PE in addition to comprehensive cardiac magnetic resonance (CMR) imaging remains unclear. In total, 132 patients with suspected acute (AMC) or chronic myocarditis (CMC) were included. All patients underwent endomyocardial biopsy (EMB) and CMR. Imaging protocols included T2 imaging for the assessment of myocardial oedema (oedema ratio [ER]), T1 imaging before and after contrast agent administration for global relative enhancement (gRE) calculation, and late enhancement (LE). Furthermore, the presence and extent of PE were determined. The potential incremental diagnostic value of PE was determined by applying a two, three, or four out of four criteria approach including ER, gRE, LE, and PE. PE was present in 84 of the 132 patients (63.6%) and was more common in suspected AMC vs. CMC (56 of 70 vs. 28 of 62; P < 0.001). According to EMB results, PE was found in 86% of patients with AMC, 67% with chronic myocarditis, and 56% without evidence of myocardial inflammation. Implementing PE into a three out of four approach did not result in improved accuracy compared with the established two out of three approach using ER, gRE, and LE (59 vs. 68% for the total population, 69 vs. 79% for suspected AMC, and 48 vs. 52% for suspected CMC). The finding of PE in unselected patients with suspected AMC or CMC is not specific to myocarditis. Therefore, with the currently applied criteria and methods, assessment of PE does not improve the diagnostic performance of CMR in this patient cohort.

Gum Arabic (GA), a nutrient from dried exudate of Acacia senegal, is widely used as emulsifier and stabilizer. It stimulates sodium and water absorption in diarrhea. This study explored the effects of GA in colonic tissue. Mice were treated with GA (10% wt/vol) in drinking water and gene array was performed. As GA modified several tumor-relevant genes, chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by 3 cycles of 3% dextrane sodium sulphate in drinking water) was induced with or without GA treatment. Within 4 days, GA treatment decreased the colonic transcript levels of the angiogenetic factors angiogenin 1, angiogenin 3, and angiogenin 4 by 78 +/- 18%, 88 +/- 15%, and 92 +/- 13%, respectively (n = 5 each), and of further genes including CD38 antigen, aquaporin4, interleukin18, vav-3-oncogene, gamma(+)-amino acid transporter, sulfatase1, ubiquitinD, and chemokine ligand5. According to Western blotting, GA treatment similarly decreased angiogenin protein expression, and according to immunohistochemistry, it decreased ss-catenin expression. Chemical cancerogenesis resulted in multiple colonic tumors within 12 wk. GA treatment (10% wt/wt) in drinking water significantly decreased the number of tumors by 70%. The observations disclose a powerful anticarcinogenic effect of GA. The nutrient could thus be used for the prophylaxis against colon carcinoma particularly in individuals at enhanced risk.

In situ hybridization studies have proved that myocardial enterovirus infections are detectable in all stages of acute and chronic enterovirus-induced myocarditis as well as in some patients with end-stage dilated cardiomyopathy, suggesting the possibility of myocardial enterovirus persistence. Possible enterovirus persistence in the human heart is supported by the discovery of enterovirus persistence in different murine models of chronic myocarditis, demonstrating that coxsackievirus B3, typically a cytolytic enterovirus, is capable of evading immunological surveillance in a host-dependent fashion. Progress is currently being made in unraveling the molecular mechanisms of enterovirus persistence, the diversity of host and virus genetics and their impact on the nature and severity of the disease. Apart from providing an etiologic diagnosis, there are therapeutic implications from the in situ demonstration of myocardial enterovirus infection. Evaluation of specific antiviral agents, ...

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a con...

Common causative agents in the development of inflammatory cardiomyopathy include cardiotropic viruses such as coxsackievirus B3 (CVB3). Here, we investigated the role of the ubiquitin-like modifier interferon-stimulated gene of 15 kDa (ISG15) in the pathogenesis of viral cardiomyopathy. In CVB3-infected mice, the absence of protein modification with ISG15 was accompanied by a profound exacerbation of myocarditis and by a significant increase in mortality and heart failure. We found that ISG15 in cardiomyocytes contributed significantly to the suppression of viral replication. In the absence of an intact ISG15 system, virus titers were markedly elevated by postinfection day 8, and viral RNA persisted in ISG15(-/-) mice at postinfection day 28. Ablation of the ISG15 protein modification system in CVB3 infection predisposed mice to long-term disease with deposition of collagen fibers, all leading to inflammatory cardiomyopathy. We found that ISG15 acts as part of the intrinsic immunit...

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.

Proteasomes are known to be the main suppliers of MHC class I (MHC-I) ligands. In an attempt to identify coxsackievirus B3 (CVB3)-MHC-I epitopes, a combined approach of in silico MHC-I/transporters associated with antigen processing (TAP)-binding and proteasomal cleavage prediction was applied. Accordingly, 13 potential epitopes originating from the structural and non-structural protein region of CVB3 were selected for further in vitro processing analysis by proteasomes. Mass spectrometry demonstrated the generation of seven of the 13 predicted MHC-I ligands or respective ligand precursors by proteasomes. Detailed processing analysis of three adjacent MHC-I ligands with partially overlapping sequences, i.e. VP2(273-281), VP2(284-292) and VP2(285-293), revealed the preferential generation predominantly of the VP2(285-293) epitope by immunoproteasomes due to altered cleavage site preferences. The VP2(285-293) peptide was identified to be a high affinity binder, rendering VP2(285-293) a likely candidate for CD8 T cell immunity in CVB3 infection. In conclusion, the concerted usage of different in silico prediction methods and in vitro epitope processing/presentation studies was supportive in the identification of CVB3 MHC-I epitopes.