Atrial-Radiofrequency Catheter Ablation Mediated Targeting of Mesenchymal Stroma - 《stemcells》干细胞学杂志干细胞之家



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作者：Patrick A. Schweizera,c, Ulf Krauseb,d, Ruediger Beckera, Anja Seckingerb, Alexander Bauera, Cornelia Hardte, Volker Ecksteinb, Anthony D. Hob, Michael Koenenc, Hugo A. Katusa, Joerg Zeheleina,c作者单位：aInnere Medizin III, Universittsklinikum Heidelberg, Heidelberg, Germany;bInnere Medizin V, Universittsklinikum Heidelberg, Heidelberg, Germany;cMax-Planck-Institut fr medizinische Forschung, Abteilung Zellphysiologie, Heidelberg, Germany;dNationales Centrum fr Tumorerkrankungen, Heidelberg, Germany 7 Q: f9 f5 n5 D/ w' z1 z

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      【摘要】
   Sinus node dysfunction and high-degree heart block are the major causes for electronic pacemaker implantation. Recently, genetically modified mesenchymal stromal cells (MSCs, also known as "mesenchymal stem cells") were demonstrated to generate pacemaker function in vivo. However, experimental approaches typically use open thoracotomy for direct cell injection into the myocardium. Future clinical implementation, however, essentially requires development of more gentle methods to precisely and efficiently apply specified stem cells at specific cardiac locations. In a "proof of concept" study, we performed selective power-controlled radiofrequency catheter ablation (RFCA) with eight ablation pulses (30 W, 60 seconds each) to induce heat-mediated lesions at the auricles of the cardiac right atrium of four healthy foxhounds. The next day, allogeneic MSCs (4.3 x 105 cells per kilogram of body weight) labeled with superparamagnetic iron oxide particles (SPIOs) were infused intravenously. Hearts were explanted 8 days later. High numbers of SPIO-labeled cells were identified in areas surrounding the RFCA-induced lesions by Prussian blue staining. Antibody staining revealed SPIO-labeled cells being positive for the typical MSC surface antigen CD44. In contrast, low levels of calprotectin, an antigen found on monocytes and macrophages, indicated negligible infiltration of monocytes in MSC-positive areas. Thus, RFCA allows targeting of MSCs to the cardiac right atrium, adjacent to the sinoatrial node, providing an opportunity to rescue or generate pacemaker function without open thoracotomy and direct injection of MSCs. This method presents a new strategy for cardiac stem cell application leading to an efficient guidance of MSCs into the myocardium.$ @4 C1 n9 s$ F4 P, G

Disclosure of potential conflicts of interest is found at the end of this article. $ e+ ]: I8 x( \3 n& ]
      【关键词】 Mesenchymal stromal cells Catheter ablation Pacemaker Atrium5 |; e, G. O/ D- n3 n' f9 y
   INTRODUCTION
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Symptomatic bradycardia caused by high degree sinoatrial and/or atrioventricular (AV) nodal diseases is commonly treated with an electronic pacemaker device. However, technical limitations such as battery durability, placement of the stimulating electrode, electrode dysfunction, infection, and the lack of response to the autonomic modulatory system are major disadvantages of electronic pacemakers . With respect to these concerns, ongoing research is focused on the implementation of biological pacemaker systems, which, autonomically regulated, achieve individually specified heart rhythms that reliably adapt to physiological requirements of the patient.4 X$ B. q" J5 a. p/ c
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Among the strategies discussed, tissue engineering approaches using embryonic stem cells (ESCs) or mesenchymal stromal cells (MSCs, also known as "mesenchymal stem cells" or "marrow stromal cells") show the most promising results. They induce spontaneous excitability, integrate into myocardium .: p$ c1 q  H7 W# [
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In a novel approach, radiofrequency catheter ablation (RFCA) was used to target MSCs to the right auricle of the canine heart, demonstrating that intravenously applied MSCs can be directed to sites adjacent to the sinoatrial node without open thoracotomy and direct myocardial injection. MSCs that colonize the atrium may integrate and electrically couple with intact myocytes to establish or induce cardiac pacemaker function." I: s7 M/ u. g+ w

MATERIALS AND METHODS

All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH publication number 85¨C23, revised 1996) and the European Community guidelines for the use of experimental animals." s. R( i/ W9 [$ S8 E
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Radiofrequency Catheter Ablation+ d/ v# n/ h2 P8 u
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An i.v. cannula in the forelimb (brachial vein) was used for administration of fluids and drugs. Four foxhounds of 22¨C26 kg of body weight were anesthetized with propofol (1.5 mg/kg); anesthesia was maintained with periodic repeat boil of propofol as required. Buprenorphine 0.03 mg/kg i.v. was applied before starting any procedure. Lesions in the right auricle were induced by transvenous power-controlled radiofrequency catheter ablation (Cerablate easy catheter, bipolar, 4-mm tip and HAT 200 RF generator; Dr. Osypka GmbH, Rheinfelden, Germany, http://www.osypka.de). The ablation catheter was implemented via preparation of the right femoral vein and guided under x-ray monitoring until the tip was located at the wall of the auricle in the right atrium. In the fourth dog, precise placing of the RFCA catheter was technically difficult due to unstable catheter position. Each dog obtained eight ablation pulses (30 W/500 kHz, power-controlled mode, each pulse 60 seconds). Electrocardiographic leads I, II, III, aVR, aVL, and aVF were continuously monitored with a VR12 recorder (Electronics for Medicine, Pleasantville, NY).( u* y  T3 r! A, I! Q5 _
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Isolation of Canine MSCs/ F- C: N9 X7 E) z* U6 f5 O
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Mononuclear cells were obtained by Biocoll density gradient centrifugation (density = 1.077 g/cm3; Biochrom AG, Berlin, http://www.biochrom.de) of canine bone marrow (mean: 34 ml, range: 15¨C56 ml) and plated in fibronectin-coated tissue culture flasks (Nunc, Rochester, NY, http://www.nuncbrand.com). Expansion medium consists of 58% low-glucose Dulbecco's modified Eagle's medium (Cambrex, Walkersville, MD, http://www.cambrex.com), 40% MCDB201 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 2% fetal calf serum (FCS; HyClone, Logan, UT, http://www.hyclone.com) supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, insulin transferrin selenium, linoleic acid, 10 nM dexamethasone, 0.1 mM L-ascorbic-acid-2-phosphate (all from Sigma-Aldrich), platelet-derived growth factor-BB, and epidermal growth factor (10 ng/ml each; R&D Systems Inc., Minneapolis, http://www.rndsystems.com). At 80% confluency, cells were trypsinized (0.25% trypsin/1 mM EDTA; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), washed with phosphate-buffered saline (PBS, Cambrex), and reseeded at a density of 5,000¨C10,000 cells per cm2 . Antibodies were tested in advance for specificity in vitro. Appropriate controls were performed in all experiments.
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Dog Leukocyte Antigen Typing

Dog class I gene dog leukocyte antigen (DLA)-88 histocompatibility typing was performed by sequencing as described elsewhere .. {  z* I/ H2 p9 w" `
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Labeling of MSCs  e+ q7 o4 i" U- _' K( M: j
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To track MSCs after transplantation, cells were labeled with superparamagnetic iron oxides (SPIOs) as published elsewhere . In brief, cells were incubated for 18 hours in expansion medium containing 25 µg iron (Endorem; Guerbet, Villepinte, France, http://www.guerbet.com) and 0.75 µg poly(L-lysine) (PLL; Sigma-Aldrich) per ml. Afterward, cells were washed three times with PBS, trypsinized as above, and resuspended in PBS containing 1% FCS and 2 mM EDTA at a final concentration of 1 x 107 cells per milliliter.
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MSC Application and Terminal Experiment
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MSCs (4.3 x 105 per kilogram body weight) were applied 1 day after RFCA via an i.v. cannula in the forelimb followed by 10 ml 0.9% saline to wash the cannula. Final experiments were performed under deep propofol anesthesia 1 week after MSC application using 4 mol eq KCl i.v. to arrest the heart. Hearts were then exposed through a midsternotomy and washed in 0.9% NaCl. After optical inspection, right auricles were excised, cut into two pieces, and frozen on isopentane/dry ice or fixed in 3% formaldehyde at 4¡ãC for at least 72 hours.6 E( ^' a4 l. c: I& H4 S

Histology and Immunohistochemistry
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Frozen right auricles were sliced from the basis toward the apex. Sections of 15 µm and 30 µm were prepared at ¨C15¡ãC to ¨C20¡ãC using a cryotome (microtome 5030; Bright Instrument Co. Ltd., Huntingdon, England, http://www.brightinstruments.com) and stored at ¨C20¡ãC. Prussian blue staining was performed with 15-µm, 30-µm, and 300-µm slices prepared from fixed auricles at 2-mm intervals. Slices were rinsed with PBS (3 x 2 minutes) and incubated for 15 minutes with 1% potassium ferrocyanide (Perl's reagent for Prussian blue staining) in 1% hydrochloric acid. Following several washes with PBS, sections were counterstained with hematoxylin and eosin (H&E) following standard protocols and analyzed using a Stemi SV6 loupe microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Immunofluorescence analysis was performed in a two-step staining protocol. Sections were rinsed with PBS, fixed in 1% formaldehyde for 30 minutes at room temperature, and incubated in blocking solution (0.1 M glycine, 2% bovine serum albumin, and 2% horse serum in PBS) for 2¨C3 hours. MSCs were detected by overnight staining at 4¡ãC using the primary anti-CD44 antibody (rat anti-CD44 IgG; EMD Biosciences, San Diego, CA, http://www.emdbiosciences.com) diluted 1:200 in blocking solution. After washes with PBS (3 x 2 minutes), sections were incubated for 3 hours at 4¡ãC with a secondary antibody AlexaFluor 568 goat anti-rat IgG antibody (Invitrogen), rinsed with PBS (3 x 2 minutes), and counterstained with 4,6-diamidino-2-phenylindole (DAPI). The presence of macrophages and granulocytes was analyzed using a mouse monoclonal antibody directed against calprotectin (dilution 1:1,000; Chemicon, Temecula, CA, http://www.chemicon.com) and goat anti-mouse IgG antibody, AlexaFluor 488, (Invitrogen) as secondary antibody. Sections were mounted in Citifluor Glycerol/PBS Solution (Agar Scientific, Stansted, England, http://www.agarscientific.com) and analyzed by light and fluorescence microscopy using a Zeiss Axioplan 2 fluorescence microscope with an Intas LC110C camera (Intas, Göttingen, Germany, http://www.intas.de). Combined laser detected transmission and confocal microscopy was accomplished with a Leica SP2 AOBS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany, http://www.leica.com).* q. {; Q  h! m2 T9 }0 x- g  a" y! H

RESULTS$ J& Y1 e& |$ e9 F% f

Radiofrequency Catheter Ablation8 `- G8 e" G! ?6 H$ l

We performed selective RFCA at the right auricles of four healthy foxhounds. Atrial fibrillation was observed in the first experiment, spontaneously converting into sinus rhythm after ablation. Otherwise, no ECG abnormalities were detected.6 I( m. x3 L6 s: `3 R/ y
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Isolation and Labeling of Canine MSCs

MSCs were successfully isolated and expanded from six consecutive dogs tested. Like their human counterparts, canine MSCs are characterized by typical cell surface markers such as CD44 and CD166, whereas hematopoietic markers such as CD14, CD34, and CD45 were absent (data not shown). For subsequent experiments, we used MSCs isolated from two unmatched donor dogs. To track MSCs after application, cells were labeled with SPIOs. Exposure of canine MSCs to the recommended concentration of SPIO/PLL (25 µg iron, 0.75 µg PLL) yielded completely labeled cell populations as characterized by Prussian blue staining in vitro (data not shown).  e# P% P' ]" e, \2 [' z
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Histocompatibility Typing of Donors and Recipients

Typing exon 2 and 3 of the DLA-88 gene of donor and recipient dogs revealed four alleles already described (DLA-88*00601, *050101, *50201, *50801). Three additional alleles were novel, of which one was similar to DLA-88*01501 and one similar to DLA-88*02301 . Pedigree analysis showed histoincompatibility between donors and recipients.! K# c9 V- }7 E* u
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Application of MSCs and Final Experiment+ [% N* M; v% V. C! R' c/ V

One day after RFCA, SPIO-labeled allogeneic MSCs (4.33 x 105 cells per kilogram of body weight) were applied intravenously. Cell viability was always >95% as determined by trypan blue exclusion test. Final experiments were carried out 7 days later. Hearts were explanted through midsternotomy and washed in ice-cold 0.9% NaCl.6 ~* x6 ?, J9 L% k/ z
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Morphology of RFCA-Induced Lesions- f7 {( X5 i9 [% x# t4 D& \: w7 m
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At the macroscopic level, lesions of the right auricle could be discovered easily from the epicardial side as well defined areas coated with a white fibrin layer and partially covered with coagula (Fig. 1). Subsequently, right auricles were excised and turned inside out for endocardial macroscopic evaluation. Endocardial lesions appeared as reddish coagula-covered spots with a destroyed trabecula structure. Right auricles of the first two animals had two and three circular transmural lesions, each with an effective diameter of about 1 cm. The right auricle of the third animal showed a single transmural lesion of about 1.5 cm in diameter, with a solid central thrombus. In the fourth animal, RFCA pulses yielded small, nontransmural lesions of about 0.2¨C0.5 cm in diameter visible only by endocardial evaluation.* J8 P. H1 u4 {3 Q

Figure 1. Macroscopic view of the heart of foxhound 1. (A): Lesion at the right auricle 8 days after radiofrequency catheter ablation. (B): Isolated right auricle with a transmural lesion. The sectioning plane used for histological analysis is indicated. Scale bars = 1 cm./ d# g, p  c. u- |* y% A* k5 o$ U

Distribution of Prussian Blue-Stained Cells- h6 }( n, n# y8 z4 J8 D
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To examine whether lesion-induced signaling has attracted SPIO-labeled MSCs, right auricles were sliced every 300 µm from the basis to the apex using a vibratome (VT1000S, Leica). Slices stained for Prussian blue to examine macroscopic structures displayed a demarcated brownish necrosis zone of burned tissue surrounded by a wide-stretched bluish border zone stretched from the margin of the necrosis to regions of intact myocardium (Fig. 2A¨C2D), indicating the presence of SPIO-labeled MSCs. Corresponding to the ablation protocol, transmural lesions appeared in the first three animals, whereas the auricle of the fourth animal displayed smaller nontransmural lesions (Fig. 2E, 2F). Distribution and intensity of the Prussian blue staining suggested that even small lesions sufficiently attract iron-positive cells to migrate into the affected region. Moreover, staining levels indicated a high density of immigrated cells, organized in a reticular structure with injured and healthy myofibers. Even at tiny, punctiform damages (Fig. 2F), the lesion is surrounded by Prussian blue-positive structures, easily visible at low magnification. Thus, even small lesions sufficiently stimulate iron-positive MSCs to immigrate.
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Figure 2. Loupe-microscopic view of vibratome sections. (A): Complete section (300 µm) of a transmural lesion from the right auricle of foxhound 1. Reflected light analysis of Prussian blue-stained tissue reveals pronounced signals at the border zone. Scale bar = 5 mm. (B¨CD): Transmitted light analysis of the segments framed in (A) at higher magnification. Note the more lucent resolution showing the reticular structured distribution of the blue stained border zone. (E): Reflected light examination of a Prussian blue-stained section of a right auricle with a nontransmural lesion (dark brown). Interestingly, iron-positive cells also appear at the epicardial layer opposite the endocardial lesion. (F): Section of the right auricle of foxhound 4 showing smaller, nontransmural lesions using reflected light. Even tiny, punctiform damages induce the formation of a Prussian blue-stained border zone. Scale bars (B¨CF) = 1 mm.
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Histological Characterization of the Lesion" Q/ A# `) I8 l" P

Evaluation of H&E counterstained slides of 30 µm (Fig. 3B¨C3E) revealed the absence of living cells from the center of the lesion. Instead and consistent with a necrosis, we observed a brownish mass of detritus (Fig. 3C). Around the necrosis, cardiomyocytes were destroyed and interstitially rearranged. More distally toward the periphery of the affected zone, cardiomyocytes showed vacuoles and a swollen cytoplasm, indicating a less affected state. Intercellular spaces were filled with small cells heavily loaded with Prussian blue-positive particles thought to cause the bluish appearance of the border zone (Fig. 3D, 3E). Typically, the border zone ranged from the margin of the central necrosis to the surrounding intact cardiac tissue with Prussian blue-stained cells arranged in a reticular structure around damaged cardiomyocytes. Interestingly, myocardial vessels of this region were surrounded by accumulated Prussian blue-stained cells (Fig. 3D). Thus, based on cellular morphology and staining results, we reasoned that Prussian blue-stained cells were immigrated SPIO-labeled MSCs (Fig. 3E).( R0 ~3 ]2 Z, s# j  l1 J

Figure 3. Microscopic analysis of a Prussian blue-stained lesion at the right auricle. (A): Loupe-microscopic view of a vibratome section of 300 µm examined with reflected light showing necrotic tissue at the center of the lesion, the slightly blue stained border zone, and unaffected intact myocardium. Scale bar = 1 cm. (B): Cryosection (30 µm) of unaffected intact myocardium framed in (A) and counterstained with H&E lacks any Prussian blue signals even at higher magnification. Scale bar = 100 µm. (C): H&E counterstained cryosection (30 µm) of Prussian blue-stained necrotic tissue framed in (A). The section lacks any Prussian blue staining signal. Scale bar = 100 µm. (D): At the border zone adjoining the radiofrequency catheter ablation induced lesion, framed in (A), Prussian blue-stained cells appear distributed around small vessels (arrows) and along intercellular spaces, indicating migration of mesenchymal stromal cells toward the affected myocardium. Cryosection is 30 µm. Scale bar = 200 µm. (E): Higher magnification of the region framed in (D). Small Prussian blue-positive cells colonize spaces around the injured cardiomyocytes specified by swollen cytoplasm and vacuoles. Scale bar = 100 µm.# c. H1 ~* a7 f1 ]+ r# a
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Histological Characterization of Peripheral Organs! H# ?) o: ]6 k2 v
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Lung, liver, spleen, and kidney samples of experimental and control animals were sliced into sections of 30 µm and 300 µm. Prussian blue staining of lung, spleen, and kidney revealed no iron-labeled MSCs. Due to abundant iron-positive Kupffer cells, liver samples of experimental as well as control animals displayed an almost identical staining, which impaired detection of Prussian blue-stained MSCs.
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Immunohistochemical Analysis) S* o! w" Q: L# f- a

To assess whether the iron-positive areas arise from engrafted SPIO-labeled MSCs and not from extracellular debris, we performed anti-CD44 immunostaining of Prussian blue-stained slices of 15 µm. Dual stain, as illustrated in Figure 4, displayed a close morphological correlation of Prussian blue-stained cells and CD44-positive mesenchymal structures, indicating the presence of SPIO-labeled MSCs. However, not every CD44-positive cell was Prussian blue-positive, a fact that might be explained by an unequal distribution of SPIO during cell division yielding MSCs lacking iron. Moreover, intracytoplasmically localized iron might not be visible in each section of the particular cell. In CD44-positive regions counterstained with nuclear stain, the density of nuclei was strikingly higher than in CD44-negative myocardial tissue, indicating immigrated MSCs. In order to rule out the possibility that iron particles might be phagocytosed by macrophages after having "attacked" allogeneic MSCs in the injured region, we performed immunostaining with an antimacrophage/-monocyte antibody, which specifically labels calprotectin. As a result, regions attributed by intense Prussian blue staining and high numbers of small cells almost lack macrophage evidence (Fig. 4C, 4D), which pointed to a low immunogenicity, a well known characteristic of MSCs. In contrast, many macrophages were detected at the necrotic center of the lesion (Fig. 4E, 4F). Nonetheless, using confocal microscopy we observed, at a much higher magnification, that Prussian blue-stained cells always express CD44 antigen at their surface (Fig. 4G, 4H).

Figure 4. Characterization of Prussian blue-positive cells. (A): Prussian blue-stained cryosection (15 µm) of the border zone of the right auricle. (B): The same section labeled with antibodies directed against the mesenchymal cell surface marker CD44 (red) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue) reveals small cells at spaces that correlate well with the iron staining. (C, D): Section (15 µm) of the right auricle stained with Prussian blue (C). Dual stain with anticalprotectin antibodies (green) and DAPI (blue) reveals the absence of macrophages at the border zone, indicating a low immunogenicity at this region (D). (E, F): The necrotic center of the lesion lacks iron-positive cells (E). Anticalprotectin staining counterstained with DAPI (blue) shows that macrophages (green) infiltrated the necrosis (F). Scale bar (A¨CF) = 100 µm. (G, H): At much higher magnification, confocal microscopy shows cells simultaneously stained with Prussian blue (black, arrows), anti-CD44 antibodies (red), and the nuclear stain DAPI (blue) accumulated at spaces around cardiomyocytes (G). Colocalization of Prussian blue staining and anti-CD44 staining on the level of single cells strongly suggests that iron-labeled mesenchymal stromal cells colonize the border zone of radiofrequency catheter ablation induced lesions (H). Scale bar (G) = 20 µm and (H) = 5 µm.7 d4 }1 G: d+ N& n1 u2 Q0 ]  Y

DISCUSSION7 Z0 {- g6 v5 X; i4 M+ [
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Mesenchymal stromal cells are multipotent cells with the property of extensive self-renewal and the ability to regenerate tissue of mesenchymal lineages . Thus, an unmatched allogeneic transplantation of MSCs without additional immunosuppression is feasible.4 y  u3 S' d0 S6 U$ Q

Experimental evaluation of MSCs revealed the expression of ion channels  were able to create a biological pacemaker system in a canine model, representing a new strategy to treat cardiac bradyarrhythmias. However, using open thoracotomy, cells were injected directly into the free wall of the left ventricular myocardium located very distal from the sinoatrial node. There, cells might be unable to initiate physiological transmission of excitation and ordered myocardial contraction.

In a "proof of concept" study, we show that targeted RFCA induced migration of intravenously applied MSCs to the desired region. By this, open thoracotomy is bypassed and the flexibility in targeting selected myocardial regions is clearly improved. In four foxhounds examined, iron-labeled MSCs, targeted by RFCA, migrated to the right atrium, closely related to the sinoatrial node. MSCs that integrate and electrically couple with the surrounding intact myocardium close to the sinoatrial node might facilitate repair of pacemaker dysfunction, or establishment of a new pacemaker might become feasible.

Focusing on principle aspects, we used identical RFCA conditions to induce lesions at the right auricles of four dogs. RFCA-induced cell damage strictly depends on the exposure of myocardium to heat. Since heat transfer in tissue is a predictable biophysical phenomenon, RFCA can be used as a precise instrument for the induction of defined tissue damages .

We induced larger transmural lesions with multiple pulses at a single site in three dogs and, probably due to unstable RFCA catheter positioning in the right auricle, smaller punctiform lesions at different sites in one dog. Our results suggest that RFCA-guided MSC homing is independent of the size of the RFCA-induced necrosis, which favors the idea that homing is induced by injured but viable cells at the border of the lesion.
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Recent studies about stem cell behavior after myocardial infarction (MI) showed that i.v. administered MSCs migrate to infarcted regions most efficiently when transplanted early post-MI (8 O# ~4 H! q* f- N- J

In rat and porcine models of myocardial infarction, i.v.-infused MSCs not only migrate to and colonize the ischemic heart tissue but also spread out to nontargeted organs , we observed no iron-loaded MSCs at nontargeted organs. This is a discrepancy that could be explained by the different number of applied cells, by timing of cell application, or by the period up to organ explantation.! M/ e7 {- e* |+ f) i% o0 {
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CONCLUSION- c% Y0 M5 p0 y

We describe a promising strategy to target MSCs to specific cardiac regions. RFCA guidance of MSCs close to the sinoatrial node may allow future implementation of pacemaker function adjacent to the native conduction system.
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DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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The excellent technical assistance of Patricia Kraft is gratefully acknowledged. The work was supported by the Young Investigator Award of the Faculty of Medicine, University of Heidelberg (P.A.S.), the ADUMED Stiftung, Germany (U.K.), and the Tandemprojekt of the Max-Planck-Gesellschaft in cooperation with the Universitätsklinikum Heidelberg, Innere Medizin III (J.Z.). P.A.S. and U.K. contributed equally to this work.! ?) E5 H$ ]( l! G
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