(Circulation. 1999;99:1348-1354.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Detection of Adenoviral Genome in the Myocardium of Adult Patients With Idiopathic Left Ventricular Dysfunction
From the Medical Clinic II, University Hospital Benjamin Franklin, Freie University Berlin, Germany (M.P., U.K., P.L.S., H.-P.S.), and the Departments of Pediatrics (N.E.B., F.J.F.-G., V.P., J.A.T.) and Molecular and Human Genetics (J.A.T.), Baylor College of Medicine, Houston, Tex.
Correspondence to Jeffrey A. Towbin, MD, Department of Pediatrics (Cardiology), Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030. E-mail jtowbin{at}bcm.tmc.edu
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Abstract |
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Background—The use of molecular biological techniques has demonstrated the importance of enteroviral infection of the myocardium in the pathogenesis of myocarditis and dilated cardiomyopathy in adults and adenovirus and enterovirus infection in children. The aim of this study was to determine the frequency of adenoviral infection of the myocardium of adults with impaired left ventricular function of unknown origin.
Methods and Results—Nested polymerase chain reaction (nPCR) was used to determine the frequency of detection of adenoviral DNA and enteroviral RNA in myocardial tissue samples from 94 adult patients with idiopathic left ventricular dysfunction and 14 control patients. Histological and immunohistological analyses were performed to detect myocardial inflammation. Adenoviral genomic DNA was detected by nPCR in 12 of the 94 patients with left ventricular dysfunction (in each case, adenovirus type 2), whereas enteroviral RNA was detected in another 12 patients. All control samples were negative for both viruses. In all patients, active myocarditis was excluded according to the Dallas criteria. However, there was significantly decreased CD2, CD3, and CD45RO T lymphocyte counts in the adenovirus-positive group compared with the adenovirus-negative group (P<0.05), whereas no differences were associated with enterovirus infection.
Conclusions—Although enteroviruses are an important causative agent in the pathogenesis of myocarditis and dilated cardiomyopathy, this study shows that adenovirus infection is also important in the pathogenesis of left ventricular failure in adults. However, the pathogenetic basis of disease associated with adenovirus infection may be different than that after infection with other agents, particularly with respect to activation of the host immune response.
Key Words: cardiomyopathy • myocarditis • viruses
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Myocarditis and its sequela, dilated cardiomyopathy (DCM), are believed to be initiated by a number of different agents, including viruses, parasitic organisms, and drugs; viral infection is believed to be most common. Since the development of molecular biological techniques, several studies have established a role for persistent enterovirus infection of the myocardium in the pathogenesis of myocarditis and DCM1 2 3 4 5 6 with molecular hybridization or polymerase chain reaction (PCR) methods. We have previously reported the prevalence of viral sequences, detected by PCR and reverse-transcription PCR (RT-PCR), within myocardial samples from pediatric patients with myocarditis or DCM.7 8 In these studies, adenoviral and enteroviral sequences were commonly identified in the myocardium of children with these diseases, with adenovirus detected in 28% and enterovirus detected in 16%.8 Since then, we have studied >300 samples from such patients and detected adenovirus in 19%, enterovirus in 13%, herpes simplex virus or cytomegalovirus (CMV) in
1%, and parvovirus or Epstein-Barr virus
in <1%.9 10 These findings contrast somewhat with the
studies reported in adult cases of myocarditis or DCM with which
enterovirus1 2 3 4 5 6 or CMV11 infections have been
most commonly associated. However, detection of adenoviral DNA in
myocardial samples from adult patients with myocarditis has been
reported in a very small number of patients. Both adenoviral DNA and
enteroviral RNA were detected in 4 of 7 patients with myocarditis
(57%) and in 0 of 6 control subjects.12 The aim of this study was to determine whether adenoviral genomic DNA can be detected in endomyocardial biopsies of patients with idiopathic left ventricular dysfunction by use of nested PCR (nPCR) and compare the frequency of detection with that for enteroviral RNA in the same patient population. Previously, Martin et al7 reported that in an number of adenovirus-associated pediatric cases of myocarditis, the level of inflammation was less than in enterovirus-positive cases. Therefore, we studied each of the endomyocardial biopsy samples histologically and immunohistologically for the presence of active or chronic inflammatory processes to determine whether a similar association exists in adult patient samples.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Study Design
In this study, 95 patients with idiopathic left ventricular dysfunction were enrolled. Fifty-six patients were randomly and blindly selected from a pool of patients who had undergone cardiac catheterization in 1995 and 1996. The remaining 39 samples were from consecutive patients enrolled prospectively during 1997. All patients underwent cardiac catheterization for further evaluation of impaired global or regional left ventricular function documented by echocardiography, which was performed on the basis of clinical presentation or history of palpitations, reduced exercise tolerance, atypical chest pain, left or right bundle-brunch block, or cardiac arrhythmias. In addition, right ventricular endomyocardial biopsies were obtained from all patients.
Endomyocardial biopsies from 14 patients with coronary heart disease (n=1), toxic cardiomyopathy (n=3), primary arrhythmia (n=1), or ejection fraction >60% and without histological and immunohistological evidence of myocardial inflammation (n=9) were used as a control group. None of these 14 patients had a history of recent viral illness.
For evaluation of myocardial inflammation, histological examinations according to the Dallas classification13 and immunohistological staining for T lymphocytes, activated macrophages, and major histocompatibility complex antigens (MHC I and MHC II) were performed. All samples were coded before nucleic acid extractions and nPCR analysis, and detection of adenoviral DNA and enteroviral RNA was performed blinded.
Cardiac Catheterization
In all patients, catheterization of the left and
right sides of the heart, hemodynamic measurements,
coronary angiography, and left ventricular
angiogram were carried out before endomyocardial
biopsies were obtained. Endomyocardial biopsy
samples were obtained from the right ventricle by standard
percutaneous transvenous right femoral approach with a
Cordis bioptome modified by Olsen.14 Biopsy samples for
PCR analysis were immediately snap-frozen in liquid nitrogen
and stored below –80°C. Biopsy samples for
histological analysis were formalin fixed and
paraffin embedded; tissue for immunohistochemical analysis was
frozen in OCT media.
Other causes of left ventricular dysfunction, including coronary, hypertensive, valvular, restrictive, or constrictive heart diseases, were excluded in all patients.
Hemodynamic Evaluation
Left ventricular end-diastolic volume
index ( milliliters per square meter of body surface area [BSA]) and
ejection fraction were determined according to the methods of Dodge and
Sheehan15 with commercial software (Cardio 500, Kontron
GmbH). The left ventricular end-diastolic
pressure was determined with a left ventricular pigtail
catheter, whereas the cardiac index and stroke volume index were
determined by use of a flow-directed catheter.
Echocardiographic analysis was performed in all
patients to evaluate left ventricular
end-diastolic diameter and ejection fraction.
Primer Design and Synthesis
Primer pairs were designed and synthesized (GIBCO-BRL) to
amplify the genomic sequence of adenoviruses encoding the hexon protein
and the 5' nontranslated region of the enteroviruses (Table 1
). The adenovirus-specific
primers were designed to amplify all adenovirus serotypes for which
sequence data are available in GenBank; the enterovirus-specific
primers should amplify most enterovirus types. Primers corresponding to
sequences in the ß-actin gene were used as a positive control for the
isolation of intact DNA and RNA (Table 1).16
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RNA and DNA Template Preparation
Tissue samples were first homogenized in RNAzol by
use of disposable RNase-free pestles (PGC
Scientific). Total RNA and genomic/viral DNA were isolated
simultaneously from patient specimens with Tris-saturated
phenol (pH 6.6) RNAzol in a modification of the RNAzol
method17 as previously described4 5 and
resuspended in 25 µL of diethyl pyrocarbonate (DEPC)-treated
water.17
Adenovirus type 5 DNA and coxsackievirus B3 RNA, isolated from infected cultured cells, were used as positive viral controls for PCR analysis after nucleic acid extraction.
RT and PCR
For detection of enteroviral genomic nucleic acid of the
RNA viruses, RT-PCR was used.18 For synthesis of cDNA, 3
µL extracted total nucleic acid was mixed with 6 µg (2 µL of 3
mg/mL) random primers (GIBCO-BRL) and 6.2 µL DEPC-treated water in
the presence of 20 U (0.5 µL) of the RNase inhibitor
RNasin (Promega). This mixture was heated to 95°C for 5 minutes and
then snap-cooled on ice. To this, 4 µL of 5x RT buffer (GIBCO-BRL),
2 µL of 100 mmol/L dithiothreitol, 0.8 µL of 25 mmol/L
dNTPs, another 0.5 µL RNasin, and 200 U (1 µL) Moloney murine
leukemia virus RT (GIBCO-BRL) were added. The samples were incubated at
37°C for 1 hour, followed by 5 minutes at 95°C to
inactivate the enzyme. Then, 2 µL of this first-strand
cDNA was subjected to PCR amplification to detect enteroviral RNA
(nPCR) or ß-actin RNA (single PCR).
To verify the isolation of both RNA and DNA from the endomyocardial biopsy samples, 2 µL of cDNA or total nucleic acid was subjected to PCR. The template was amplified in a 20-µL reaction containing 1x PCR buffer (GIBCO-BRL), 2.5 mmol/L magnesium chloride, 0.25 mmol/L dNTPs, 0.5 µmol/L oligonucleotide primers, and 2.5U Taq DNA polymerase (GIBCO-BRL). After an initial 5-minute incubation at 94°C, 35 rounds of amplification were performed with a Stratagene Robocycler under the following conditions: 94°C for 45 seconds, 64°C for 45 seconds, and 72°C for 45 seconds. This was followed by a 72°C incubation for 5 minutes.
For detection of viral genomic sequences, 2 µL cDNA or total nucleic acid was subjected to nPCR. The primary reaction was performed under the same conditions as described for amplification of ß-actin. For the secondary amplification, 2 µL of the primary reaction was diluted in 98 µL TE (10 mmol/L Tris, pH 7.5, 1 mmol/L EDTA), and then 2 µL diluted product was subjected to 30 cycles of PCR amplification as described for the primary amplification.
Analysis of PCR Products
The products of each reaction were analyzed by
1.75% agarose gel electrophoresis containing 0.5 µg/mL ethidium
bromide (Sigma Chemical Co), and the DNA product was visualized by
UV transillumination. In all cases, positive (purified viral nucleic
acid) and multiple negative (water or nucleic acid from a tissue sample
known to be negative) control reactions were performed
simultaneously with the test samples. All samples were
analyzed without prior knowledge of clinical or
histological data for each patient, and all
PCR-positive samples were required to have duplicate results. Any
sample not giving a signal with the ß-actin primers was excluded.
DNA Sequencing
The adenovirus amplimers were reamplified and the PCR
products were purified with a PCR purification kit (Qiagen)
according to the manufacturer's instructions and resuspended in 30
µL TE. The DNA sequence was determined by cycle sequencing by use of
primer ADH-I3 according to the kit manufacturer's instructions
(Stratagene) with a 8% denaturing polyacrylamide gel.
Histology and Immunohistology
For evaluation of myocardial inflammation,
histological examinations of sections of
formalin-fixed, paraffin-embedded endomyocardial
biopsies were performed according to the Dallas
classification.13
For immunohistochemical staining, endomyocardial biopsies were directly embedded in OCT (Miles Laboratories, Inc) and frozen at -70°C. Sections (5 µm) were fixed in acetone for 10 minutes and then incubated with monoclonal antibodies directed against CD2, CD3, CD4, CD8, and CD45RO T lymphocytes. In addition, monoclonal antibodies against activated macrophages and MHC I and II antigens were used. Unbound antibodies were removed by washing twice with PBS. Peroxidase-conjugated rabbit-anti-mouse antibody (Dianova GmbH), diluted at 1:200 in PBS containing 10% FCS, was then added to each section. Quantification of T lymphocytes, activated macrophages, and cells expressing MHC I and MHC II antigens was performed by 2 independent observers, as described by Kühl et al.19
Statistical Analysis
The SPSS statistical software package was used for statistical
analysis. Statistical significance was determined by use of
Student's t test, with a confidence level of
P=0.05.
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Results |
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Clinical Presentation
One enrolled patient was determined to be a child (age, 14 years) with myocarditis and was excluded from data analysis. All enrolled adult patients (age
17 years; n=94) had global left
ventricular dysfunction with an ejection fraction <55% or
regional left ventricular dysfunction with wall motion
disturbances in 2 wall segments. Detailed
hemodynamic data of these and the control patients are
listed in Table 2. Coronary,
hypertensive, valvular, restrictive, and constrictive heart
diseases were excluded in all patients by cardiac
catheterization of both sides of the heart. With
respect to clinical data, the adenovirus-positive group was
significantly older than theadenovirus-negative group
(P<0.05) (Table 3). There
were no statistically significant differences in any other clinical
criteria between the enterovirus-positive and enterovirus-negative
patients (Table 4).
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PCR Analysis
The sensitivity of nPCR compared with single PCR had previously
been determined to be 10-fold greater in our hands. In the case of
adenovirus-specific PCR, nPCR achieved an estimated sensitivity of 5 to
50 genomes or 1 adenovirus genome per 1000 cells. A similar sensitivity
was achieved for the enterovirus-specific PCR, representing
a detection limit of 1 to 10 genomes or 10-2
cfu.
All samples were positive for the presence of ß-actin sequences by
PCR and RT-PCR, indicating the successful isolation of both RNA and
DNA, respectively. Of the 94 enrolled adult patients, 12 were
adenovirus positive by nPCR (Figure 1),
and another 12 were enterovirus positive (Figure 2). None of the 14 control samples was
positive for either virus. For 18 of the 94 enrolled patients, 2
biopsies were analyzed; for the other 76, single biopsy samples
were studied. Of these 18 patients, 3 had 1 sample positive for
adenoviral DNA (in 1 patient, both were positive). Of these 18
patients, 5 had at least 1 sample positive for enteroviral RNA,
including 2 patients for whom both were positive. BLAST search
analysis of the DNA sequences revealed that in all 12
adenovirus-positive samples from adults, type 2 adenovirus was detected
(Figure 3). The single pediatric sample
was also positive for adenovirus, type 5 in this case (Figure 3, sample 5).
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Histopathology and Immunohistochemistry
The histopathological analysis of the enrolled adult
patients (n=94) and the control group (n=14) excluded active
myocarditis in each patient according to the Dallas
classification.13
There were significantly fewer CD2 (Figure 4), CD3, and CD45RO T lymphocytes in
adenovirus-positive compared with adenovirus-negative patients (Table 3
), but no significant differences existed in the number of CD4
and CD8 T lymphocytes or activated macrophages. In
addition, the number of MHC I and MHC II antigen-expressing cells was
statistically indistinguishable between both subgroups (Table 3). There was no difference in the degree of myocardial
inflammation or types of cells present associated with the presence
of enterovirus (Table 4).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Application of molecular biological methods (slot-blot technique, in situ hybridization, PCR) has facilitated detection of viral nucleic acid, especially enteroviral RNA, in endomyocardial biopsies of adult patients with myocarditis or DCM. Adenoviral infection of the myocardium in myocarditis or DCM patients has primarily been reported in studies of myocardial samples from children.7 8 20 21 22 23 These have revealed that adenoviral infection is at least as common as enterovirus infection. Adenoviral infection of the myocardium has also been reported in a study of a small number of samples from adult myocarditis patients.12 The aim of this study was to determine whether adenoviral genomic DNA can be detected in endomyocardial biopsies of adult patients with idiopathic left ventricular dysfunction by use of nPCR.
The data presented here demonstrate that in adult patients with
idiopathic left ventricular dysfunction (after exclusion of
coronary, hypertensive, valvular, restrictive, and
constrictive heart diseases), adenoviral DNA can be detected in a
significant proportion (12 of 94, 13%). Enteroviral RNA could be
detected with a similar incidence (12 of 94). These are similar to the
data from pediatric patients with DCM in whom adenoviral DNA and
enteroviral RNA have been detected in 16 of 132 patients (12%) and 10
of 132 patients (8%), respectively.7 10 No patients were
infected with both adenovirus or enterovirus. In 18 patients, 2
biopsies were studied: In 8 patients, virus was detected in 1 sample,
but virus was detected in both samples in only 3 patients. These data
suggest that viral infection of the myocardium is focal and
that multiple samples should be studied to accurately determine the
frequency of virus infection. These data are consistent with
the pathological data that have shown that histological
evidence of myocarditis is not always evident in each
sample.23
DNA sequence analysis of the PCR products showed that in all 12 adult samples positive for adenovirus, the serotype detected was type 2 adenovirus. This is similar to the data from neonatal and pediatric patients with myocarditis or DCM in whom the major serotype of adenovirus detected was type 2 (a group C adenovirus, like type 5), with the remainder being type 5.7 10 20
Despite the considerable evidence that adenoviruses and enteroviruses are the major causative agents of myocarditis and DCM identified to date, it has been something of a conundrum why 2 such distinctly different virus families infect myocytes and cause a similar pathology. The description of the common coxsackievirus B and adenovirus (subgroup C) receptor offers at least a partial explanation,24 25 because the expression of this receptor by cardiomyocytes would facilitate the uptake of either of these viruses.
None of the enrolled patients showed active myocarditis according to the Dallas criteria. Thus, left ventricular function cannot be explained by active myocarditis. Therefore, the clinical data (impaired regional or global left ventricular dysfunction), combined with the histological results, indicate either a diagnosis of DCM or DCM as the precipitating event. The observation of a significant difference between the number of CD2, CD3, and CD45RO T lymphocytes in the adenovirus-positive group compared with the adenovirus-negative group is interesting. We have previously shown that in pediatric patients the inflammatory infiltrate is often less in patients positive for adenovirus than those positive for enterovirus.7 The reduction in the number of activated lymphocytes in the adenovirus-infected myocardium compared with myocardial samples infected with other viruses or of unknown origin may have importance in the pathogenesis of adenovirus-induced myocardial disease. The adenoviruses have a number of strategies for modulating the immune response. Several adenovirus-encoded proteins are capable of interacting with host immune components (for a review, see Reference 2626 ). These include proteins encoded by the E3 region, which can protect cells from tumor necrosis factor–mediated lysis,27 as well as downregulation of MHC class I antigen expression.28 The E1A proteins are capable of promoting the induction of apoptosis29 and inhibiting IL-6 expression,30 as well as interfering with IL-6 signal transduction pathways.31 These functions of E1A may be particularly pertinent in explanations of the myocardial pathology observed in DCM patients. First, IL-6 promotes lymphocyte activation, which was reduced in the adenovirus-infected patient samples in this study. Second, it has been reported that in a small number of cases, apoptotic cells were detected in myocardial tissue samples from patients with idiopathic DCM by an in situ labeling protocol, including adenovirus-infected samples.32 33
In this study, we have shown that in adult patients with idiopathic left ventricular dysfunction, including patients with DCM, both adenoviral DNA and enteroviral RNA can be detected with similar frequencies. Furthermore, adenovirus types 2 and 5 (group C adenoviruses) appear to be cardiovirulent serotypes of adenovirus in adults and children. However, infection of the myocardium with adenovirus may result in less immune cell activation than with other agents, suggesting the possibility of a different mechanism of pathogenesis of adenovirus-induced chronic myocardial disease.
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Acknowledgments |
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This work was supported by the Texas Children's Hospital Foundation Chair in Pediatric Cardiac Research (Dr Towbin) and an Abercrombie Cardiac Fund Grant (Drs Bowles and Towbin). This work was performed in the Phoebe Willingham Muzzy Pediatric Molecular Cardiology Laboratory at Baylor College of Medicine, Houston, Tex.
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Footnotes |
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Guest Editor for this article was Michael J. Sole, MD, Toronto General Hospital, Toronto, Ontario, Canada.
Received June 22, 1998; revision received November 11, 1998; accepted November 30, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bowles NE, Richardson PJ, Olsen EGJ, Archard LC. Detection of coxsackie-B-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet. 1986;17:1120–1123.
2.
Kandolf R, Ameis D, Kirschner P, Canu A, Hofschnieder
PH. In situ detection of enteroviral genomes in myocardial cells by
nucleic acid hybridization: an approach to the diagnosis of viral heart
disease. Proc Natl Acad Sci U S A. 1987;84:6272–6276.
3.
Bowles NE, Rose ML, Taylor P, Banner NR, Morgan-Capner
P, Cunningham L, Archard LC, Yacoub MH. End-stage dilated
cardiomyopathy: persistence of enterovirus RNA in
myocardium at cardiac transplantation and lack of immune
response. Circulation. 1989;80:1128–1136.
4.
Jin O, Sole MJ, Butany JW, Chia W-K, McLaughlin PR,
Liu P, Liew CC. Detection of enterovirus RNA in myocardial biopsies
from patients with myocarditis and cardiomyopathy
using gene amplification by polymerase chain reaction.
Circulation. 1990;82:8–16.
5. Grasso M, Arbustini E, Silini E, Diegoli M, Percivalle E, Ratti G, Bramerio M, Gavazzi A, Vigano M, Milanesi G. Search for coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction. Am J Cardiol. 1992;69:658–664.[Medline] [Order article via Infotrieve]
6. Tracy S, Chapmann NM, McManus BM, Pallansch MA, Beck MA, Carstens J. A molecular and serologic evaluation of enteroviral involvement in human myocarditis. J Mol Cell Cardiol. 1990;22:403–414.[Medline] [Order article via Infotrieve]
7.
Martin AB, Webber S, Fricker FJ, Jaffe R, Demmler G,
Kearney D, Zhang Y-H, Bodurtha J, Gelb B, Ni J, Bricker T, Towbin JA.
Acute myocarditis: rapid diagnosis by PCR in children.
Circulation. 1994;90:330–339.
8. Griffin LD, Kearney D, Ni J, Jaffe R, Fricker FJ, Webber S, Demmler G, Gelb BD, Towbin JA. Analysis of formalin-fixed and frozen myocardial autopsy samples for viral genome in childhood myocarditis and dilated cardiomyopathy with endocardial fibroelastosis using polymerase chain reaction (PCR). Cardiovasc Pathol. 1995;4:3–11.
9.
Schowengerdt KO, Ni J, Denfield SW, Gajarski RJ,
Bowles NE, Rosenthal G, Kearney DL, Price JK, Rogers BB, Schauer GM,
Chinnock RE, Towbin JA. Association of parvovirus B19 genome in
children with myocarditis and cardiac allograft rejection: diagnosis
using the polymerase chain reaction. Circulation. 1997;96:3549–3554.
10. Bowles NE, Towbin JA. Viral heart muscle disease in children. Newsletter Sci Council Cardiomyopathies. 1996;11:4–5.
11. Schonian U, Crombach M, Maser S, Maisch B. Cytomegalovirus-associated heart muscle disease. Eur Heart J. 1995;16(suppl O):46–49.
12. McCarthy RE III, Hare JM, Chen C-L, Hruban RH, Ni J, Kasper EK, Baughman KL, Towbin JA. Adenovirus may play a significant role as a primary or co-infectious agent in adult myocarditis. Circulation. 1997;96(suppl I):I-321. Abstract.
13. Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, Fenoglio JJ, Olsen EG, Schoen FJ. Myocarditis. A histopathologic definition and classification. Am J Cardiovasc Pathol. 1987;1:3–14.[Medline] [Order article via Infotrieve]
14. Olsen EG. Endomyocardial biopsies. Int J Cardiol. 1983;3:240–243.[Medline] [Order article via Infotrieve]
15. Dodge HT, Sheehan FH. Quantitative contrast angiography for assessment of ventricular performance in heart disease. J Am Coll Cardiol. 1983;1:73–81.[Abstract]
16. Ng SY, Gunning P, Eddy R, Ponte P, Leavitt J, Shows T, Kedes L. Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol. 1995;5:2720–2732.
17. Chomczynski P, Sacchi N. Single step method of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
18. Rotbart HA. PCR amplification of enteroviruses. In: PCR Protocols: A Guide to Methods and Applications. New York, NY: Academic Press, Inc; 1990:372–377.
19.
Kühl U, Noutsias M, Seeberg B, Schultheiss H-P.
Immunohistological evidence for a chronic
intramyocardial inflammatory process in dilated
cardiomyopathy. Heart. 1996;75:295–300.
20. Towbin JA, Griffin LD, Martin AB, Nelson S, Siu B, Ayres NA, Demmler G, Moise KJ, Zhang Y-H. Intrauterine adenoviral myocarditis presenting as non-immune hydrops fetalis: diagnosis by polymerase chain reaction. Pediatr Infect Dis J. 1994;13:144–150.[Medline] [Order article via Infotrieve]
21. Lozinski GM, Davis GG, Krous HF, Billman GF, Shimizu H, Burns JC. Adenovirus myocarditis: retrospective diagnosis by gene amplification from formalin-fixed, paraffin-embedded tissues. Hum Pathol. 1994;25:831–834.[Medline] [Order article via Infotrieve]
22. Shimizu C, Rambaud C, Cheron G, Rouzioux C, Lozinski GM, Rao A, Stanway G, Krous HF, Burns JC. Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis. Pediatr Infect Dis J. 1995;14:584–588.[Medline] [Order article via Infotrieve]
23. Chow LH, Radio SJ, Sears TD, McManus BM. Insensitivity of right ventricular endomyocardial biopsy in the diagnosis of myocarditis. J Am Coll Cardiol. 1989;14:915–920.[Abstract]
24.
Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA,
Krithvas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a
common receptor for coxsackie B viruses and adenoviruses 2 and 5.
Science. 1997;275:1320–1323.
25.
Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human
and mouse cellular receptors for subgroup C adenoviruses and group B
coxsackieviruses. Proc Natl Acad Sci U S A. 1997;94:3352–3356.
26. Hayder H, Müllbacher. Molecular basis of immune evasion strategies by adenoviruses. Immunol Cell Biol. 1996;74:504–512.[Medline] [Order article via Infotrieve]
27. Gooding LR, Elmore LW, Tollefson AE, Brady HA, Wold WS. A 14 700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell. 1988;53:341–346.[Medline] [Order article via Infotrieve]
28.
Burgert H-G, Maryanski JL, Kvist S. "E3/19 k"
protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes
by blocking cell-surface expression of histocompatibility class I
antigens. Proc Natl Acad Sci U S A. 1987;84:1356–1360.
29. White E. Regulation of apoptosis by the transforming genes of the DNA tumor virus adenovirus. Proc Soc Exp Biol Med. 1993;204:30–39.[Medline] [Order article via Infotrieve]
30. Janaswami PM, Kalvakolanu DVR, Zhang Y, Sen GC. Transcriptional repression of interleukin-6 gene by adenoviral E1A proteins. J Biol Chem. 1992;267:886–891.
31. Takeda T, Nakajima K, Kojima H, Hirano T. E1A repression of IL-6-induced gene activation by blocking the assembly of IL-6 response element binding complexes. J Immunol. 1994;153:4573–4582.[Abstract]
32.
Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD,
Hajjar RJ, Schmidt U, Semigran MJ, Dec GW, Khaw B-A. Apoptosis
in myocytes in end-stage heart disease. N Engl J
Med. 1996;335:1182–1189.
33. Bowles NE, Kearney D, Ni J, Towbin JA. Association of adenovirus infection with apoptosis in the pathogenesis of myocarditis and dilated cardiomyopathy. Pediatr Res.. 1997;41:19a.
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L. T. Cooper, K. L. Baughman, A. M. Feldman, A. Frustaci, M. Jessup, U. Kuhl, G. N. Levine, J. Narula, R. C. Starling, J. Towbin, et al. The Role of Endomyocardial Biopsy in the Management of Cardiovascular Disease: A Scientific Statement From the American Heart Association, the American College of Cardiology, and the European Society of Cardiology Circulation, November 6, 2007; 116(19): 2216 - 2233. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hausler, B. Sellhaus, S. Scheithauer, B. Gaida, S. Kuropka, K. Siepmann, A. Panek, W. Berg, A. Teubner, K. Ritter, et al. Myocarditis in newborn wild-type BALB/c mice infected with the murine gamma herpesvirus MHV-68 Cardiovasc Res, November 1, 2007; 76(2): 323 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kobayashi-Miura, K. Shioji, Y. Hoshino, H. Masutani, H. Nakamura, and J. Yodoi Oxygen sensing and redox signaling: the role of thioredoxin in embryonic development and cardiac diseases Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2040 - H2050. [Abstract] [Full Text] [PDF]
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|
 |
|
 G. F. Cox, L. A. Sleeper, A. M. Lowe, J. A. Towbin, S. D. Colan, E. J. Orav, P. R. Lurie, J. E. Messere, J. D. Wilkinson, and S. E. Lipshultz Factors Associated With Establishing a Causal Diagnosis for Children With Cardiomyopathy Pediatrics, October 1, 2006; 118(4): 1519 - 1531. [Abstract] [Full Text] [PDF]
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J. W. Magnani and G. W. Dec Myocarditis: Current Trends in Diagnosis and Treatment Circulation, February 14, 2006; 113(6): 876 - 890. [Full Text] [PDF]
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K. L. Baughman Diagnosis of Myocarditis: Death of Dallas Criteria Circulation, January 31, 2006; 113(4): 593 - 595. [Full Text] [PDF]
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K. B. Vallbracht, P. L. Schwimmbeck, U. Kuhl, U. Rauch, B. Seeberg, and H.-P. Schultheiss Differential Aspects of Endothelial Function of the Coronary Microcirculation Considering Myocardial Virus Persistence, Endothelial Activation, and Myocardial Leukocyte Infiltrates Circulation, April 12, 2005; 111(14): 1784 - 1791. [Abstract] [Full Text] [PDF]
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U. Kuhl, M. Pauschinger, M. Noutsias, B. Seeberg, T. Bock, D. Lassner, W. Poller, R. Kandolf, and H.-P. Schultheiss High Prevalence of Viral Genomes and Multiple Viral Infections in the Myocardium of Adults With "Idiopathic" Left Ventricular Dysfunction Circulation, February 22, 2005; 111(7): 887 - 893. [Abstract] [Full Text] [PDF]
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K. B. Vallbracht, P. L. Schwimmbeck, U. Kuhl, B. Seeberg, and H.-P. Schultheiss Endothelium-Dependent Flow-Mediated Vasodilation of Systemic Arteries Is Impaired in Patients With Myocardial Virus Persistence Circulation, November 2, 2004; 110(18): 2938 - 2945. [Abstract] [Full Text] [PDF]
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|
 A. Dorner, D. Xiong, K. Couch, T. Yajima, and K. U. Knowlton Alternatively Spliced Soluble Coxsackie-adenovirus Receptors Inhibit Coxsackievirus Infection J. Biol. Chem., April 30, 2004; 279(18): 18497 - 18503. [Abstract] [Full Text] [PDF]
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M. Vatta, B. Mohapatra, S. Jimenez, X. Sanchez, G. Faulkner, Z. Perles, G. Sinagra, J.-H. Lin, T. M. Vu, Q. Zhou, et al. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction J. Am. Coll. Cardiol., December 3, 2003; 42(11): 2014 - 2027. [Abstract] [Full Text] [PDF]
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J. W Mason Myocarditis and dilated cardiomyopathy: An inflammatory link Cardiovasc Res, October 15, 2003; 60(1): 5 - 10. [Abstract] [Full Text] [PDF]
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F. Calabrese and G. Thiene Myocarditis and inflammatory cardiomyopathy: microbiological and molecular biological aspects Cardiovasc Res, October 15, 2003; 60(1): 11 - 25. [Abstract] [Full Text] [PDF]
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 |
|
 B. J. Maron Sudden Death in Young Athletes N. Engl. J. Med., September 11, 2003; 349(11): 1064 - 1075. [Full Text] [PDF]
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U. Kuhl, M. Pauschinger, T. Bock, K. Klingel, C. P. L. Schwimmbeck, B. Seeberg, L. Krautwurm, W. Poller, H.-P. Schultheiss, and R. Kandolf Parvovirus B19 Infection Mimicking Acute Myocardial Infarction Circulation, August 26, 2003; 108(8): 945 - 950. [Abstract] [Full Text] [PDF]
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N. E. Bowles, J. Ni, D. L. Kearney, M. Pauschinger, H.-P. Schultheiss, R. McCarthy, J. Hare, J. T. Bricker, K. R. Bowles, and J. A. Towbin Detection of viruses in myocardial tissues by polymerase chain reaction: evidence of adenovirus as a common cause of myocarditis in children and adults J. Am. Coll. Cardiol., August 6, 2003; 42(3): 466 - 472. [Abstract] [Full Text] [PDF]
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C. Baboonian and W. McKenna Eradication of viral myocarditis: Is there hope? J. Am. Coll. Cardiol., August 6, 2003; 42(3): 473 - 476. [Full Text] [PDF]
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 |
|
 M. Noutsias, M. Pauschinger, H.-P. Schultheiss, and U. Kuhl Cytotoxic perforin+ and TIA-1+ infiltrates are associated with cell adhesion molecule expression in dilated cardiomyopathy Eur J Heart Fail, August 1, 2003; 5(4): 469 - 479. [Abstract] [Full Text] [PDF]
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U. Kuhl, M. Pauschinger, P. L. Schwimmbeck, B. Seeberg, C. Lober, M. Noutsias, W. Poller, and H.-P. Schultheiss Interferon-{beta} Treatment Eliminates Cardiotropic Viruses and Improves Left Ventricular Function in Patients With Myocardial Persistence of Viral Genomes and Left Ventricular Dysfunction Circulation, June 10, 2003; 107(22): 2793 - 2798. [Abstract] [Full Text] [PDF]
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A. W. Nugent, P. E.F. Daubeney, P. Chondros, J. B. Carlin, M. Cheung, L. C. Wilkinson, A. M. Davis, S. G. Kahler, C.W. Chow, J. L. Wilkinson, et al. The Epidemiology of Childhood Cardiomyopathy in Australia N. Engl. J. Med., April 24, 2003; 348(17): 1639 - 1646. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 K. Klingel, H.-C. Selinka, M. Sauter, C.-T. Bock, G. Szalay, and R. Kandolf Molecular mechanisms in enterovirus and parvovirus B19 associated myocarditis and inflammatory cardiomyopathy Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I8 - I12. [Abstract] [PDF]
|
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|
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W. Poller, H. Fechner, M. Noutsias, C. Tschoepe, M. Pauschinger, and H.-P. Schultheiss The molecular basis of cardiotropic viral infections Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I18 - I30. [Abstract] [PDF]
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M. Noutsias, M. Pauschinger, H.-P. Schultheiss, and U. Kuhl Advances in the immunohistological diagnosis of inflammatory cardiomyopathy Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I54 - I62. [Abstract] [PDF]
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S.B. Felix, A. Staudt, and G. Baumann Immunoadsorption as a new therapeutic principle for treatment of dilated cardiomyopathy Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I63 - I68. [Abstract] [PDF]
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A. Frustaci, M. Pieroni, and C. Chimenti Immunosuppressive therapy in inflammatory cardiomyopathy Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I69 - I72. [Abstract] [PDF]
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|
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U. Kuhl, M. Pauschinger, M. Noutsias, J.-F. Kapp, and H.-P. Schultheiss Diagnosis and treatment of patients with virus induced inflammatory cardiomyopathy Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I73 - I80. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Afanasyeva and N. R. Rose Cardiomyopathy Is Linked to Complement Activation Am. J. Pathol., August 1, 2002; 161(2): 351 - 357. [Full Text] [PDF]
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|
|
|
T. P. Zwaka, D. Manolov, C. Ozdemir, N. Marx, Z. Kaya, M. Kochs, M. Hoher, V. Hombach, and J. Torzewski Complement and Dilated Cardiomyopathy: A Role of Sublytic Terminal Complement Complex-Induced Tumor Necrosis Factor-{alpha} Synthesis in Cardiac Myocytes Am. J. Pathol., August 1, 2002; 161(2): 449 - 457. [Abstract] [Full Text] [PDF]
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A. Frustaci, L. Cuoco, C. Chimenti, M. Pieroni, G. Fioravanti, N. Gentiloni, A. Maseri, and G. Gasbarrini Celiac Disease Associated With Autoimmune Myocarditis Circulation, June 4, 2002; 105(22): 2611 - 2618. [Abstract] [Full Text] [PDF]
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|
|
N. E. Bowles, J. Ni, F. Marcus, and J. A. Towbin The detection of cardiotropic viruses in the myocardium of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy J. Am. Coll. Cardiol., March 6, 2002; 39(5): 892 - 895. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 N G Mahon, B Zal, G Arno, P Risley, J Pinto-Basto, W J McKenna, M J Davies, and C Baboonian Absence of viral nucleic acids in early and late dilated cardiomyopathy Heart, December 1, 2001; 86(6): 687 - 692. [Abstract] [Full Text] [PDF]
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|
|
|
M. Noutsias, H. Fechner, H. de Jonge, X. Wang, D. Dekkers, A.B. Houtsmuller, M. Pauschinger, J. Bergelson, R. Warraich, M. Yacoub, et al. Human Coxsackie-Adenovirus Receptor Is Colocalized With Integrins {alpha}v{beta}3 and {alpha}v{beta}5 on the Cardiomyocyte Sarcolemma and Upregulated in Dilated Cardiomyopathy: Implications for Cardiotropic Viral Infections Circulation, July 17, 2001; 104(3): 275 - 280. [Abstract] [Full Text] [PDF]
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|
|
|
G. S. Shirali, J. Ni, R. E. Chinnock, J. K. Johnston, G. L. Rosenthal, N. E. Bowles, and J. A. Towbin Association of Viral Genome with Graft Loss in Children after Cardiac Transplantation N. Engl. J. Med., May 17, 2001; 344(20): 1498 - 1503. [Abstract] [Full Text] [PDF]
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J.M. Hare and K.L. Baughman Fulminant and acute lymphocytic myocarditis: the prognostic value of clinicopathological classification Eur. Heart J., February 2, 2001; 22(4): 269 - 270. [PDF]
|
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|
|
|
|
S. Fujioka, Y. Kitaura, A. Ukimura, H. Deguchi, K. Kawamura, T. Isomura, H. Suma, and A. Shimizu Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy J. Am. Coll. Cardiol., November 15, 2000; 36(6): 1920 - 1926. [Abstract] [Full Text] [PDF]
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A. M. Feldman and D. McNamara Myocarditis N. Engl. J. Med., November 9, 2000; 343(19): 1388 - 1398. [Full Text] [PDF]
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 |
|
 G.-H. Lee, C. Badorff, and K. U. Knowlton Dissociation of Sarcoglycans and the Dystrophin Carboxyl Terminus From the Sarcolemma in Enteroviral Cardiomyopathy Circ. Res., September 15, 2000; 87(6): 489 - 495. [Abstract] [Full Text] [PDF]
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A. Dorner, M. Pauschinger, P. L. Schwimmbeck, U. Kuhl, and H.-P. Schultheiss The shift in the myocardial adenine nucleotide translocator isoform expression pattern is associated with an enteroviral infection in the absence of an active T-cell dependent immune response in human inflammatory heart disease J. Am. Coll. Cardiol., June 1, 2000; 35(7): 1778 - 1784. [Abstract] [Full Text] [PDF]
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 |
|
 K. Höfling, S. Tracy, N. Chapman, K.-S. Kim, and J. Smith Leser Expression of an Antigenic Adenovirus Epitope in a Group B Coxsackievirus J. Virol., May 15, 2000; 74(10): 4570 - 4578. [Abstract] [Full Text]
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R. E. McCarthy, J. P. Boehmer, R. H. Hruban, G. M. Hutchins, E. K. Kasper, J. M. Hare, and K. L. Baughman Long-Term Outcome of Fulminant Myocarditis as Compared with Acute (Nonfulminant) Myocarditis N. Engl. J. Med., March 9, 2000; 342(10): 690 - 695. [Abstract] [Full Text] [PDF]
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