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R Kandolf

Publications and source records attributed to R Kandolf.

At least 91 records · Page 5Linked to original sources
PubMed1995

Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.

Amino Acid Sequence↗
PubMed1995

Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts.

In heart transplantation, long-term engraftment success is severely limited by the rapid development of obliterative disease of the coronary arteries. Data from various groups have been suggestive of a pathogenetic role of herpesviruses, particularly human cytomegalovirus, in accelerated allograft coronary artery disease; however, results are not yet conclusive. This study examines the hypothesis that human cytomegalovirus infection of allograft tissues is related pathogenetically and directly to accelerated coronary artery disease. Using in situ DNA hybridization and polymerase chain reaction, we examined particular coronary artery segments from 41 human heart allografts (ranging from 4 days to greater than 4 years after transplantation; mean, 457 days) and 22 donor age- and gender-comparable, coronary site-matched trauma victims for presence of human cytomegalovirus DNA. Human cytomegalovirus genome was detected in 8 of 41 (19.5%) allografts and in 1 of 22 (4.5%) control hearts. This difference in positivity was not statistically significant (P = 0.10). In the human cytomegalovirus-positive hearts, viral genome was localized to perivascular myocardium or coronary artery media or adventitia. Human cytomegalovirus genome was not detected in arterial intima of any allograft or control heart, although human cytomegalovirus genome was readily identified within intima of small pulmonary arteries from lung tissue with human cytomegalovirus pneumonitis. By statistical analyses, the presence of human cytomegalovirus genome was not associated with the nature or digitized extent of transplant arteriopathy, evidence of rejection, allograft recipient or donor serological data suggestive of human cytomegalovirus infection, duration of allograft implantation, or causes of death or retransplantation. Thus, our data indicate a low frequency of detectable human cytomegalovirus genome in accelerated coronary artery disease and do not support a direct role for human cytomegalovirus in vascular wall infection or in the development of accelerated coronary artery disease.

Adolescent↗
PubMed1995

The inhibitory effect of astragalus membranaceus on coxsackie B-3 virus RNA replication.

Using mice infected with coxsackie B-3 virus (CVB3) as a viral myocarditis model, we observed the inhibitory effect of Astragalus membranaceus (AM) on CVB3-RNA replication in myocardial tissue of mice by RNA-RNA in situ hybridization with negative-strand RNA probes labelled with 35S and quantitative imaging analysis of positive signals. The mechanism of its effect on CVB3-RNA replication has been investigated by detection of beta-interferon (beta-IFN) as well. Results showed that the copy numbers of CVB3-RNA as well as the histologic scores (necrosis) in myocardial tissues of infected-AM treated mice were significantly lower than those in infected and normal saline treated mice, suggesting that AM could inhibit the replication of CVB3-RNA, but its effect on CVB3-RNA replication had no correlation with induction of beta-IFN.

Animals↗
PubMed1994

Enterovirus-induced myocarditis: hemodynamic deterioration with immunosuppressive therapy and successful application of interferon-alpha.

In 1985, myocarditis was diagnosed by endomyocardial biopsy in a 53-year-old woman who was free of symptoms of heart insufficiency. Immunosuppressive therapy with azathioprine and prednisolone was prescribed as the patient had deteriorated to NYHA II heart insufficiency in March 1986. When application of immunosuppressive agents was terminated in 1987, serial endomyocardial biopsies revealed no signs of myocarditis, thus indicating effectiveness of immunosuppression. Nevertheless, the patient had worsened clinically and hemodynamically. At follow-up in 1991, the patient had progressed to dilated cardiomyopathy resulting in NYHA III heart insufficiency. Enterovirus infection of the heart was detected by in situ nucleic acid hybridization. In addition, retrospective serology indicated a significant increase of coxsackievirus B2 antibodies between 1985 and 1991. Investigational antiviral therapy with recombinant interferon alpha-2a was tolerated well and a favorable clinical, hemodynamic, and virologic response was observed. Thus, progression of biopsy-proven myocarditis to dilated cardiomyopathy may have been facilitated by immunosuppressive therapy in enterovirus infection. Antiviral therapy might be advantageous in patients with enterovirus myocarditis and dilated cardiomyopathy.

Azathioprine↗
PubMed1994

Investigation of the coxsackievirus B3 nonstructural proteins 2B, 2C, and 3AB: generation of specific polyclonal antisera and detection of replicating virus in infected tissue.

The coxsackievirus B3 (CVB3) nonstructural proteins 2B and 3AB were synthesized as beta-galactosidase fusion proteins in E. coli in order to generate specific polyclonal antisera. 2B and 3AB fusion proteins were purified by preparative SDS-polyacrylamide gel electrophoresis and inoculated into rabbits. Protein 2C-specific antiserum was produced using synthetic oligopeptides which were defined by computer based amino acid sequence analysis. Specificity of the generated antisera was analysed by immunoblotting, immunofluorescence, immunohistochemistry and immunoelectron microscopy. All antisera allowed specific detection of the viral proteins 2B, 2C, and 3AB in CVB3-infected cells as well as in myocardial and pancreatic tissue of CVB3-infected mice. In addition, the CVB3 2C-specific antiserum was shown to be highly cross-reactive with the analogous protein of other picornaviruses, including cardiotropic enterovirus serotypes such as coxsackievirus A9, coxsackievirus B (types 1-5), and echovirus 11. Moreover, the immunological detection of nonstructural proteins enables diagnosis of replicating virus in infected tissue. These results demonstrate that the generated antisera are valuable tools for diagnostic approaches. Furthermore, they may help to elucidate the role of the nonstructural proteins 2B, 2C, and 3AB in enteroviral replication and pathogenesis.

Animals↗
PubMed1994

Characterization of the N-terminal part of the neutralizing antigenic site I of coxsackievirus B4 by mutation analysis of antigen chimeras.

Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Ag1 of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar.

Amino Acid Sequence↗
PubMed1994

[Effect and mechanism of Astragalus membranaceus on coxsackie B3 virus RNA in mice].

Using Balb/c mice infected with Coxsackie virus B3 (CVB3) as a model, the effect of Astragalus membranaceus (AM) on CVB3-RNA has been observed in myocardial tissues of mice by RNA-RNA in situ hybridization with negative-strand RNA probes leballing with 35S and quantitative imaging analysis of positive hybridization signals. The mechanism of its effect on CVB3-RNA has also been investigated by induction with AM and detection of beta-interferon (beta-IFN). Results showed that the copy numbers of CVB3-RNA as well as the histologic necrotic sizes in myocardial tissues of AM treated infected mice were significantly smaller than that in infected normal saline treated mice (P < 0.01, P < 0.05) respectively, suggesting that AM could inhibit the replication of CVB3-RNA, but its effect on CVB3-RNA was not correlated with induction of beta-IFN.

Animals↗
PubMed1994

Characterization of a cardiac-selective and developmentally upregulated promoter in transgenic mice.

Transcriptional regulatory mechanisms which mediate cardiac-specific gene expression have not yet been completely understood. Potential cardiac-specific promoter sequences, sharing similar protein binding motives, show either coexpression in skeletal muscle, local restriction to the atrium or late onset of expression during fetogenesis. Based on in situ hybridization studies that indicated the expression of the cardiac myosin-light-chain-2 (MLC-2) gene in ventricular myocardium and in the lower outflow tract, a model system for selective targeting of foreign genes to the heart of transgenic mice has been developed. The regulatory promoter element was derived from the rat cardiac MLC-2 gene. 2100 bp of the 5' regulatory MLC-2 sequences were found to drive constitutive cardiac expression of a firefly luciferase reporter gene from early tubular heart formation. During ventricular loop and septum formation luciferase activity was 10-fold upregulated in comparison to steady-state levels observed 10 days after birth. No luciferase activity was detectable in any other muscle or non-muscle tissue of transgenic mice. These data suggest that the 2.1 kb DNA sequences of the 5' flanking region of the cardiac MLC-2 gene contain sufficient regulatory elements for a selective gene expression in cardiac myocytes from embryogenesis. The transgenic model should aid in determining the influences of pathogenic gene products on developing and mature heart muscle to elucidate the etiology of myocardial diseases such as cardiomyopathies.

Animals↗
PubMed1994

[Mechanisms of re-stenosis after angioplasty].

Restenosis post angioplasty is a segmentally limited, wound healing response to a circumscript traumatization of the vascular wall associated with the therapeutic intervention, which also comprises residual or recoiling plaque components at the time of initial revascularization. Studies with animal models and the analysis of human plaque tissue harvested by autopsy or atherectomy indicate a cascade-like course of this wound healing reaction, in which initially different cell types such as thrombocytes, endothelial cells, monocytes/macrophages and smooth muscle cells (SMCs), later predominantly SMCs are involved. In the first phase of inflammation, angioplasty as a multifactorial stimulus induces a sequence of (a) destruction of endothelial and subendothelial structures, (b) traumatization of medial regions with rupture of the internal elastic lamina, (c) exposition of thrombogenic factors such as collagen or tissue factor, (d) stretching of smooth muscle cells with subsequent expression of proto-oncogens (c-fos, c-myc, c-myb), (e) release of growth factors from cells of the bloodstream, endothelial cells and SMCs by direct traumatization and segmental thrombus formation, and (f) thrombin production with autocatalytic activation of the SMC thrombin receptor. Overlapping the inflammation period, granulation begins 3 days after angioplasty. Proteinases such as plasmin as well as collagenases induce the disintegration of extracellular matrix structures, thereby modulating plaque formation, and lead to an organelle-rich SMC phenotype within the intima and media. The phenotypic alteration of SMCs is considered to be the prerequisite for mitogenic and migratory stimulation. This stage shows different expression patterns of growth factors and their receptors; however, there is only limited knowledge about spatiotemporal and maximal expression as well as their coordination for human vascular wall tissue (PDGF, PDGF-R, EGF-R, FGF, FGF-R, TGF-beta). Overlapping with the granulation period, induction of different components of the extracellular matrix occurs 1-2 weeks after angioplasty, possibly mediated by TGF-beta (phase of matrix formation). Smooth muscle cells produce and secrete matrix proteins such as tenascin, fibronectin, collagens and proteoglycans, and thereby induce a marked increase of the neointimal plaque volume. Angiographic restenoses of coronary and peripheral arteries histologically exhibit tissue with high cellularity (> 500 cells/mm2), associated with SMC activity markers such as PCNA or NMMHC-B. Proliferative and migratory activities of these cells in vitro are augmented by a factor of 2 to 3 as compared to those from chronic primary lesions. Transmission electron microscopic analysis proves that within the media a nearly complete re-differentiation of SMCs occurs, whereas intimal SMCs persist in the intermediate phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Angioplasty, Balloon, Coronary↗
PubMed1993

Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5'-nontranslated region does not contain major attenuation determinants.

To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full-length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or early-passage virus, although disintegration of the monolayers was observed with late-passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggests that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of > 10(4). Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V-1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5'-terminus of the genome of attenuant p14V-1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5'-nontranslated region (5'NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5'-terminal 891 nucleotides of the wild-type genome with the corresponding region of the attenuant p14V-1. This chimeric virus, CB3/p14V-1/1, produced wild-type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Pathogenetic differences between coxsackie A and B virus infections in newborn mice.

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

Animals↗
PubMed1993

Myocardial indium-111 antimyosin uptake in patients with idiopathic dilated cardiomyopathy: its relation to haemodynamics, histomorphometry, myocardial enteroviral infection, and clinical course.

The myocardial uptake of indium-111 antimyosin indicates the presence of ongoing myocyte damage. To evaluate the role of this finding in patients with idiopathic dilated cardiomyopathy (IDC), 36 patients were studied by planar and SPECT antimyosin imaging. The diagnosis of IDC was based on coronary angiography and left ventricular endomyocardial biopsy. The antimyosin scan was evaluated qualitatively from SPECT images and assessed quantitatively by a count density index (CDI) which measured the tracer activity over the heart relative to the lung and sternal region (normal value less than 1.20). Group 1 consisted of 13 patients (36%) with an increased myocardial antimyosin uptake, while 23 patients had a normal antimyosin scan (group 2). Clinical data, pulmonary artery pressures, gated blood pool ejection fraction and histomorphometry of endomyocardial biopsies were similar in both groups. During a follow-up of 21 +/- 12 months there were two cardiac deaths in group 1 and 10 deaths in group 2 (P = 0.12). The 2-year survival rate was 81% and 59%, respectively. During follow-up, there was no significant change in haemodynamic parameters in either group, but there was a slight improvement in functional NYHA class in group 1 (P < 0.05). No association was found between the presence of myocardial enterovirus infection, determined in 17 patients by in situ hybridization and the antimyosin scan (P = 0.5 g). Myocardial antimyosin uptake was found in a high percentage of patients with IDC, indicating ongoing myocyte damage. This finding was not related to any clinical, haemodynamic, morphological parameter, or enterovirus infection. Myocyte damage is a distinct feature in a subgroup of patients with IDC unrelated to any known causes of myocellular destruction. This subgroup showed a trend towards a more favourable clinical outcome.

Actuarial Analysis↗
PubMed1993

Molecular pathogenesis of enterovirus-induced myocarditis: virus persistence and chronic inflammation.

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, for example interferons, may lead to the development of new therapeutic strategies capable of providing protection against myocardial enterovirus infection.

Animals↗
PubMed1993

Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice.

Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2-luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene, no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice. This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular mechanisms of myocardial diseases such as cardiomyopathies.

Animals↗
PubMed1993

The role of enterovirus replication in the development of acute and chronic heart muscle disease in different immunocompetent mouse strains.

In order to study interrelationships between virus replication and the development of acute and chronic heart disease, different immunocompetent mouse strains (A.CA/SnJ, A.BY/SnJ, SWR/J, DBA/1J) were infected with coxsackievirus B3 (CVB3). Based on in situ hybridization, patterns of acute and persistent infections were quantitated in the representative mouse strain A.CA/SnJ by application of computer-assisted digital image processing and correlated with the extent of myocardial injury and cellular immune response. Here we show that the acute infection is characterized by virus-induced myocytolysis with increasing densities of inflammatory mononuclear cells, whereas persistent infection reveals a decreased number of infected myocardial cells in the presence of a reduced but ongoing inflammation. The major role of viral replication in the pathogenesis of acute and chronic heart disease is substantiated by the finding that tissue lesions as well as inflammation consistently developed at the sites of virus replication. No inflammatory lesions were observed to evolve independently of virus replication, indicating that persistent infection is essential for the development of chronic disease. The availability of a murine model of ongoing CVB3-induced myocarditis should prove to be useful for further studies on molecular mechanisms of enterovirus persistence, e.g. with regard to myocyte metabolism in persistently infected cells or identification of host genes involved in the pathogenesis of chronic enterovirus-induced cardiomyopathy.

Animals↗