[Cell cultivation of human plaque tissue following percutaneous and intra-operative coronary atherectomy].
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Cardiac Catheterization↗
Biomedical subjects
R Kandolf
Publications and source records attributed to R Kandolf.
[Increased in vitro motility of human vascular wall myocytes from restenotic lesions of peripheral and coronary vessels].
In this study we report on the successful cultivation of human peripheral and coronary plaque specimens selectively retrieved by percutaneous Simpson atherectomy and obtained by direct operative approach. A total of 32 patients in whom plaque tissue was excised from 22 primary and 10 restenotic lesions comprise the study population. Irrespective of their origin or location, all advanced lesions showed smooth muscle cells (SMC) to be their predominant cell type proven by indirect immunofluorescence technique. Cultured endothelial cells were only identified in 2/6 surgically removed samples. Locomotion analysis of cultured smooth muscle cells was performed with a standardized computer-assisted video system. Cells of all groups exhibited random motility. However, SMC migratory velocity of restenotic origin amounted to 47.4 +/- 3.4 microns/h (n = 10, x +/- SD) and thereby was found 2.4 times (p less than 0.001) increased as compared to primary lesion values of 22.0 +/- 3.7 microns/h (n = 22, x +/- SD). This highly significant difference was seen for both peripheral and coronary lesions. Our data suggest increased SMC migratory activity to represent a basic biological mechanism involved in human accelerated arteriosclerosis and restenosis formation.
Aged↗
A search for the presence of the enteroviral capsid protein VP1 in pancreases of patients with type 1 (insulin-dependent) diabetes and pancreases and hearts of infants who died of coxsackieviral myocarditis.
Using an antiserum raised to a recombinant coxsackie virus B 3 capsid protein, VP1, an immunocytochemical technique was developed which was capable of detecting the presence of all coxsackie B viruses in formalin fixed paraffin embedded infected tissue culture cells. This technique was tested on autopsy heart and pancreas from 21 patients who were thought to have died of acute coxsackievirus B myocarditis. Cardiac myocytes were positive for the VP1 protein in 12 of 20 cases where the heart was available for study. Insulitis was present in the pancreas in seven of these cases and in all seven islet endocrine cells containing VP1 were found. VP1 was only rarely found in exocrine pancreas. In heart and pancreas, cells shown to contain VP1 usually showed signs of necrosis. Autopsy pancreases from 88 patients who had died at clinical presentation of Type 1 (insulin-dependent) diabetes mellitus showed no evidence of the presence of VP1. The continuing destruction of insulin-secreting B cells seen at the time of death in the diabetic pancreas is unlikely to be due to a direct cytopathic effect of a coxsackie B virus. However, this study does not exclude the possibility that a persistent infection of B cells by a defective enterovirus may result in their destruction by an autoimmune mechanism.
Acute Disease↗
Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA: two initial 5' uridine residues are regained during plus-strand RNA synthesis.
A full-length reverse-transcribed, infectious cDNA copy of coxsackievirus B3 (CVB3) was used to determine the nucleotide sequence of this cardiotropic enterovirus. Comparison of the nucleotide sequence and the deduced amino acid sequence of the viral precursor polyprotein with the sequences of other group B coxsackieviruses (CVB1 and CVB4) demonstrates a high degree of genetic identity. They share about 80% homology at the nucleotide level and about 90% when the amino acid sequences of the polyproteins are compared. The potential processing sites of the coxsackievirus polyproteins, as deduced from alignment with the poliovirus sequence, are conserved among these enteroviruses with the exception of the cleavage sites between VP1 and 2Apro and between polypeptides 2B and 2C. Comparison of the 5' termini of the enteroviral genomes reveals a high degree of identity, including the initial 5' consensus UUAAAACAGC, suggesting essential functions in virus replication. An important finding concerning the molecular basis of infectivity was that both recombinant CVB3 cDNA and in vitro-synthesized CVB3 RNA transcripts are infectious, although two initial 5' uridine residues found on the authentic CVB3 RNA were missing. Here, we report that cDNA-generated CVB3, as well as CVB3 generated by in vitro-synthesized RNA transcripts, regains the authentic initial 5' uridine residues during replication in transfected cells, indicating that the picornaviral primer molecule VPg-pUpU may be uridylylated in a template-independent fashion. The generation of virus or virus mutants with infectious recombinant CVB3 cDNA and in vitro-synthesized infectious CVB3 transcripts should provide a valuable means for studying the molecular basis of the pathogenicity of this cardiotropic enterovirus.
Amino Acid Sequence↗
[Patterns of acute and persistent infections in enteroviral heart diseases].
Enteroviruses are considered as the major etiologic agents of myocarditis in humans. Recent in situ hybridization studies on endomyocardial biopsies indicate that not only in acute myocarditis (pattern of acute infection), but also in chronic dilated cardiomyopathy enterovirus RNA can be detected (pattern of persistent infection). Our experimental studies on murine coxsackievirus B3 myocarditis provided evidence that persistent infection occurs also in mice. Quantitative in-situ hybridization and immunohistochemistry as well as electron microscopic in situ hybridization experiments were performed on ACA/SnJ mice three to thirty days after infection. The pattern of acute infection (days 3-9 p.i.) is characterized by rapid progression of myocardial lesions, an increasing number of inflammatory cells and a high number of infected myocytes. Hallmarks of the persistent pattern (day 15-30 p.i.) are reduced inflammation, reduced numbers of persistently infected cells and a slow progression of myocardial lesions. Infection is primarily restricted to degenerated, atrophic myocytes and to fibroblasts.
Acute Disease↗
Viral heart disease.
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Cardiomyopathy, Dilated↗
Detection of parvovirus in a patient with "reactive arthritis" by in situ hybridization.
We used in situ hybridization to search for the presence of viruses in synovial fluid cell preparations obtained from patients with various forms of knee arthropathies. One patient, presenting with acute reactive arthritis, was found to replicate parvovirus DNA in cells from synovial fluid whereas six other patients with various other forms of arthritis were negative for parvovirus infection. Five patients with osteoarthritis, constituting a control group in which an infectious etiology would not be expected, were consistently negative. In addition, no hybridization was found when synovial cell preparations of all patients were hybridized in situ with DNA probes specific for Epstein-Barr virus, cytomegalovirus, or enteroviruses.
Adult↗
Hybridization techniques provide improved sensitivity for HCMV detection and allow quantitation of the virus in clinical samples.
Hybridization techniques (slot-blot and in-situ hybridization assays) and immunostaining using murine monoclonal antibodies directed against different proteins of the human cytomegalovirus (HCMV) were compared for their sensitivity and specificity for detection of HCMV. A model system with HCMV infected human embryonic lung fibroblasts and lung biopsy specimens obtained from patients with culture positive HCMV interstitial pneumonia were used for evaluation of these techniques. The hybridization techniques were found to provide an improved sensitivity compared to immunostaining. Additionally a good correlation was found between the virus dose determined by TCID50 and the amount of viral DNA detected by slot-blot hybridization and by the number of autoradiographic silver grains per 100 cells per 2 weeks exposure time detected in the infected fibroblasts by in-situ hybridization. Thus, at least in the model system quantification of the virus was achieved by hybridization assays.
Cells, Cultured↗
Isolated pericardial relapse following allogeneic bone marrow transplantation for acute myelogenous leukemia.
A 34-year-old male patient developed an isolated pericardial relapse of an acute myelogenous leukemia (M3) 11 months after marrow grafting from his HLA-identical brother. Alloenzyme pattern analysis revealed recipient type of the myeloblasts obtained from the pericardial effusion. Recurrence of the original leukemia was preceded by a reactivation of latent cytomegalovirus (CMV) infection which, in spite of a systemic humoral immune response to the virus, persisted in the pericardium as shown by dot-blot hybridization using CMV-specific DNA fragments. Activated T cells propagated with IL-2 from the pericardial effusion did not reveal any cytotoxic or restimulation capacity on the original or relapse myeloblasts, nor on other donor, recipient or NK target cells. Local coincidence of virus persistence and leukemic relapse suggested CMV-mediated modulation of the immune response in the pericardium with consequent induction of a proliferation of the original malignant cell clone. After local chemotherapy and one course of systemic treatment the patient is still in complete remission--longer than after the marrow grafting.
Adult↗
[The study of intra-articular infectious agents in reactive arthritis].
Reactive arthritis is differentiated from infectious arthritis by the lack of intraarticular infectious agents. Recently 2 groups, using different techniques have demonstrated intra-articular antigens in cases of reactive arthritis associated with Chlamydia and Yersinia infections. In this article we report the preliminary results of screening cells from synovial fluid for DNA of certain microorganisms by in situ hybridization. Our findings provide complementary evidence of the intra-articular presence of at least parts of microbes.
Antigens, Bacterial↗
Expression of coxsackievirus B3 capsid proteins in Escherichia coli and generation of virus-specific antisera.
Subgenomic fragments of cloned infectious coxsackievirus B3 (CVB3) cDNA up to the size of the complete coding sequence of the viral polyprotein were inserted into the prokaryotic expression vector pPLc24 and expressed in Escherichia coli. Fusion proteins, containing 54 amino acids of MS2 replicase at their amino terminus followed by different parts of the CVB3 structural proteins, were expressed from several constructs. The expression product of a plasmid encoding the capsid proteins VP4, VP2, and the amino-terminal part of VP3 was obtained in high amounts. However, primary expression products containing the complete viral capsid precursor VP4-VP1 were completely degraded, indicating the presence of domains downstream from VP3 that are accessible to E. coli proteases. This finding is consistent with the observation that the structural intact expression product of the separately subcloned VP1 gene is also extremely unstable and consequently obtained only in low amounts. Two fusion proteins of non-overlapping parts of the viral structural proteins containing VP4, VP2, and VP3 or VP1, respectively, were isolated and used for the generation of antisera in rabbits. The antisera obtained recognize distinct CVB3 structural proteins in infected cell cultures as well as from purified CVB3 preparations. In addition, significant cross-reactivity of the described antisera with the corresponding structural proteins of other enteroviruses was observed, indicating that these antisera provide a valuable tool for an improved broad spectrum diagnosis of enteroviral infections.
Capsid↗
In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease.
We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.
Animals↗
[Primary and secondary chylopericardium].
Primary chylopericardium proved to be the (rare) cause of chronic, symptom-poor pericardial effusion in a 19-year-old man. Echocardiography and pericardiocentesis provided a firm diagnosis. Lymphography demonstrated extravasation into the mediastinum but no direct connection into the pericardial sac. Ligation of the thoracic duct at diaphragmatic level and pericardiostomy achieved a cure. Secondary chylopericardium requires treatment of the underlying disease, e.g. mediastinal tumor. Chylopericardium after thoracic or cardiac operation without signs of tamponade is preferably treated conservatively by pericardiocentesis and diet.
Adult↗
Coxsackie B3 virus can replicate in cultured human foetal heart cells and is inhibited by interferon.
Coxsackie B viruses (types 1 to 5) are the most frequent reported cause of acute viral myocarditis. To study the pathogenesis of the disease at the cellular level, we simulated an infectious situation by infecting cultured human foetal heart cells with Coxsackie B3 (CB3) virus. Successful replication of this virus could be demonstrated by the presence of virus particles inside cultivated foetal myocytes together with high titres of progeny virus of 10(8) plaque-forming units (PFU) per millilitre culture medium. Within 9 h of infection networks of myocytes lost their ability to contract spontaneously followed by disintegration and replacement by overgrowing fibroblasts which survived the infection. These cells produced CB3 virus continuously over several months, indicating carrier state infection of human myocardial fibroblasts. Human fibroblasts interferon (IFN-beta) was found to act as a potent inhibitor of the replication of this virus. Virus yields could be reduced from 1.2 x 1.8 x 10(5) PFU/ml culture medium when human heart cells were incubated with IFN-beta 20 h prior to challenge with a high input multiplicity of 50 PFU of CB3 virus per cell, demonstrating the major protective role of IFN-beta in CB3 viral infection. It thus appear that IFN-beta might become useful as an antiviral agent in the treatment of Coxsackie myocarditis.
Cells, Cultured↗
Molecular cloning of the genome of a cardiotropic Coxsackie B3 virus: full-length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.
The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is characterized by restriction enzyme analysis and partial nucleotide sequencing of the 5' end. The Coxsackie B3 virus cDNA described can now be used to study the molecular basis of human enteroviral heart disease, and it provides a valuable diagnostic means for patients with suspected viral heart disease.
Animals↗
Decrease of myocardial mRNA in adriamycin-treated rats.
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Amino Acyl-tRNA Synthetases↗
Isolation and characterization of structurally and functionally intact polyribosomes and mRNA from rat heart muscle.
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Age Factors↗
Immunosuppressive therapy of chronic myocarditis in children: three cases and the design of a randomized prospective trial of therapy.
Three infants, each with a clinical picture of dilated cardiomyopathy, underwent endomyocardial biopsy. Immunohistologic analysis revealed chronic myocarditis. In one infant, a postviral etiology of chronic myocarditis could be assessed on the basis of molecular techniques. Therapy with azathioprine and prednisone resulted in the normalization of echocardiographic findings. Based on these observations, a randomized, multicenter treatment study of chronic myocarditis in children (TCMC) has been initiated.
Anti-Inflammatory Agents↗