Paraffin-embedded Heart Tissue Research Articles

Differential Deep RNA Sequencing for Diagnostic Detection of Microbial Infections in Inflammatory Cardiomyopathy.

Inflammatory heart disease can be triggered by a variety of causes, both infectious and noninfectious in nature. We hypothesized that inflammatory cardiomyopathy is potentially related to microbial infection. In this retrospective study, we used deep RNA sequencing on formalin-fixed paraffin-embedded heart tissue specimens to detect pathogenic agents. We first investigated 4 single-sample cases to test the feasibility of this diagnostic protocol and further 3 control-sample paired cases to improve the protocol with differential metatranscriptomics next-generation sequencing (mtNGS) analysis. We demonstrate that differential mtNGS allows identification of various microbials as potentially pathogenic, for example, Cutibacterium acnes, Corynebacterium aurimucosum, and Pseudomonas denitrificans, which are usually commensal in healthy individuals. Differential mtNGS also allows characterization of human host response in each individual by profiling alterations of gene expression, networked pathways, and inferred immune cell compositions, information of which is beneficial for us to understand different etiologies and immunity roles in each case. Additionally, differential mtNGS allows the identification of genetic variants in patients that may contribute to their susceptibility to particular microbial infections. The demonstrated power of differential mtNGS in simultaneous capture of both the infectious microbial(s) and the status of human host immune response could help us better understand the pathogenesis of complex inflammatory cardiomyopathy, if conducted on a larger scale of the population. The developed differential mtNGS method could also shed light on its translation and adoption of such a laboratory test in clinic practice, allowing for a more effective diagnosis to guide therapeutic treatment of the disease.

DNA quality evaluation of formalin-fixed paraffin-embedded heart tissue for DNA methylation array analysis

Archived formalin-fixed and paraffin-embedded (FFPE) heart tissue from autopsied individuals represents an important resource for investigating the DNA methylation of heart tissue of deceased individuals. The DNA quality of FFPE tissue from autopsies may be decreased, affecting the DNA methylation measurements. Therefore, inexpensive screening methods for estimating DNA quality are valuable. We investigated the correlation between the DNA quality of archived FFPE heart tissue examined with the Illumina Infinium HD FFPE QC assay (Infinium QC) and Thermo Fisher’s Quantifiler Trio DNA Quantification kit (QuantifilerTrio), respectively, and the amount of usable DNA methylation data as measured by the probe detection rate (probe DR) obtained with the Illumina Infinium MethylationEPIC array. We observed a high correlation (r2 = 0.75; p < 10−11) between the QuantifilerTrio degradation index, DI, and the amount of usable DNA methylation data analysed with SeSAMe, whereas a much weaker correlation was observed between the Infinium QC and SeSAMe probe DR (r2 = 0.17; p < 0.05). Based on the results, QuantifilerTrio DI seems to predict the proportion of usable DNA methylation data analysed with the Illumina Infinium MethylationEPIC array and SeSAMe by a linear model: SeSAMe probe DR = 0.80–log10(DI) × 0.25.

Detection of early myocardial cell death in owl monkeys (Aotus nancymai) using complement component C9 immunohistochemistry in formalin-fixed paraffin-embedded heart tissues: A retrospective study.

Owl monkeys are commonly used in biomedical research which is affected by the high incidence of cardiomyopathy in this species. Occasionally, owl monkeys with no clinical signs of heart disease are found dead and at necropsy show no, or very mild, cardiomyopathy. A possible explanation for sudden death is acute myocardial infarction; however, early myocardial changes may be difficult to assess by conventional stains and light microscopy. Complement component C9 immunohistochemistry was performed in paraffin-embedded heart tissue samples from owl monkeys who died suddenly, or were euthanized due to sickness, to determine whether these animals suffered from acute myocardial infarcts. C9 deposits were found in the myocardium of 19 out of 20 (95%) animals. The findings in this study suggest owl monkeys suffer from acute myocardial infarcts, and complement component C9 immunohistochemistry may be a useful diagnostic tool.

AdipoRon prevents l-thyroxine or isoproterenol-induced cardiac hypertrophy through regulating the AMPK-related pathway.

Cardiac hypertrophy is a risk factor which can intrigue heart failure. In the present study, we explored whether AdipoRon attenuates isoprenaline (ISO) or l-thyroxine-induced cardiac hypertrophy in Sprague-Dawley (SD) rats and whether the anti-hypertrophy effect is mediated by AMPK-related pathway. Here, cardiac hypertrophy was induced by injection of l-thyroxine or ISO in SD rats. In the treatment group, AdipoRon was co-administered. We examined the effects of AdipoRon on cardiac hypertrophy and hypertrophy signaling pathway. The weight of SD rats was recorded every day. Rats were killed for collection of blood and heart under anesthesia. The left heart weight and heart weight were weighed. Paraffin-embedded heart tissue regions (4 μm) were stained with hematoxylin and eosin or Masson to detect left heart hypertrophy and myocardial fibrosis. The serum BNP levels were determined by using an enzyme-linked immunosorbent assay. The mRNA levels of ANP, BNP, PGC-1α, and ERRα were evaluated by real-time PCR analysis. The protein expression levels of PGC-1α, ERRα, and pAMPK/AMPK were determined by western blot analysis. The results showed that AdipoRon significantly reversed heart weight (HW)/body weight (BW) ratio, left ventricular (LV)/BW ratio, serum BNP level and the mRNA level of ANP and BNP induced by ISO or l-thyroxine. ISO or l-thyroxine reduced both the mRNA level and protein level of ERRα and PGC-1α, and also reduced the protein level of pAMPK/AMPK. However, AdipoRon reversed ISO or l-thyroxine-induced changes of pAMPK/AMPK, ERRα, and PGC-1α. Our data indicated that the effects of AdipoRon are mediated partly by activating AMPK-related pathway, and AdipoRon plays a potential role in the prevention of cardiac hypertrophy.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

PCR-based diagnosis of cytomegaloviruses in paraffin-embedded heart tissue in cases of suspected sudden infant death syndrome (SIDS)

Immunohistochemical and molecular-pathological techniques have improved the diagnosis, but the incidence of virus-induced lethal myocarditis remains unclear. Therefore, it is of great interest to investigate postmortem myocardial samples in cases of sudden infant death syndrome (SIDS). Cytomegaloviruses are known as possible agents of myocarditis. Viral DNA was specifically isolated and amplified from formaldehyde-fixed material. At autopsy, myocardial samples were taken from 70 SIDS cases from different regions and investigated with PCR. Cytomegalovirus DNA was detected in 2 out of 70 cases. Our results emphasize the importance of modern molecular-pathological methods in cases of sudden unexpected death.

PCR-based diagnosis of adenovirus and Epstein–Barr virus in paraffin-embedded heart tissue

Immunohistochemical and molecular-pathological techniques have improved the diagnosis, but the incidence of virus-induced lethal myocarditis still remains unclear. Therefore, it is of great interest to investigate postmortem myocardial samples in cases of sudden infant death syndrome (SIDS). Adenoviruses and Epstein–Barr viruses are known as possible agents of myocarditis. Viral DNAs were specifically isolated and amplified from formaldehyde-fixed material. At autopsy, myocardial samples were taken from 62 SIDS cases from different regions and investigated with PCR. Adenoviral DNA was detected in 2/62 and Epstein–Barr viruses in 3/62 cases. Our results emphasize the importance of modern molecular-pathological methods in cases of sudden unexpected death.

Chemical characterization of a lambda I amyloid protein isolated from formalin-fixed and paraffin-embedded tissue sections

Amyloid protein was isolated from formalin-fixed paraffin-embedded heart tissue sections from a patient with primary (AL) amyloidosis by extraction with 6 M guanidine HCl. SDS-PAGE analysis of extracted material showed a major band at 16 kDa and a minor band at 18 kDa. Edman degradation analysis before and after pyroglutamate aminopeptidase treatment showed that the amyloid protein contained N-terminal pyroglutamic acid and was derived from an immunoglobulin λ light chain. Analysis of tryptic peptides from the extract identified the amyloid protein as a λI. Of particular interest is that almost the entire amyloid protein amino acid sequence could be obtained from the cardiac sections. These results demonstrate that formalin-fixed paraffin-embedded tissue sections can be used for extensive biochemical characterization of amyloid proteins and will become a valuable source for isolation and extensive biochemical characterization of amyloid proteins as they are now a valuable source for isolation of DNA for genetic analysis.

PCR-Based Diagnosis of Enterovirus and Parvovirus B19 in Paraffin-Embedded Heart Tissue of Children with Suspected Sudden Infant Death Syndrome

The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histologic criteria. Viral cultures and serology are often unrewarding with low yields. The purpose of this study was to analyze the usefulness of PCR in the rapid diagnosis of myocarditis in children. PCR was used to analyze 120 myocardial tissue samples from 60 cases of sudden infant death syndrome (SIDS) and 56 myocardial tissue samples from 36 cases with well-known causes of sudden death (11 children younger than 1 year and 25 children 1–10 years old). The myocardial tissue samples were evaluated for the presence of enteroviruses and parvovirus B19 using PCR primers designed to consensus and unique sequences of these viral genomes. Enteroviruses could be detected in 14 cases of SIDS, whereas the detection of enteroviral nucleic acid within the control group was negative. Seven cases with myocardial infection caused by parvovirus B19 were found in the SIDS group. The detection of parvoviruses in the control group of the 11 children younger than 1 year was negative, whereas 3 positive cases of parvoviruses could be detected in the control group of children from 1 to 10 years old. In the myocardial sample of one SIDS case, both enteroviruses and parvovirus B19 could be detected. Our results emphasize the importance of modern molecular biologic methods in cases of sudden infant death even when conventional histologic examination revealed no serious findings in heart muscle tissue.

PCR-based diagnosis of enterovirus and parvovirus B19 in paraffin-embedded heart tissue

Although immunohistochemical and molecular biological techniques have improved the diagnosis, the incidence of virus-induced lethal courses of myocarditis is still unclear. Therefore, it is desirable to investigate postmortem myocardial samples in cases of unknown cause of death. While enteroviruses are the most common agents of myocarditis, parvovirus B19 is also known to be highly cardiotropic. The enteroviral genome consists of a single-stranded RNA molecule. Parvovirus B19 is the only known human pathogen virus of the family Parvoviridae and consists of a linear single-stranded DNA molecule. In our investigation, RNA and DNA were specifically isolated and demonstrated from formalin-fixed material. Myocardial samples from 60 autopsy cases with unknown cause of death after autopsy were taken from different regions and investigated with a nested polymerase-chain-reaction (PCR). Enteroviruses could be detected in 14 cases. PCR revealed eight cases with myocardial infection due to parvovirus B19. In the myocardial sample of one case, both enteroviruses and parvovirus B19 were found. Our results emphasize the importance of modern molecular biological methods in cases of sudden death even when histological examination revealed no serious findings in heart muscle tissue.

Codeposition of Apolipoprotein A-IV and Transthyretin in Senile Systemic (ATTR) Amyloidosis

Protein material was extracted from amyloid-rich sections of formalin-fixed and paraffin-embedded heart tissue from an individual with senile systemic amyloidosis, known to contain wild-type transthyretin as major amyloid fibril protein. Amino acid sequence analysis of tryptic peptides of this material revealed in addition to transthyretin sequences, also amino acid sequence corresponding to an N-terminal fragment of apolipoprotein A-IV. In immunohistochemistry, an antiserum to a synthetic apolipoprotein A-IV peptide labeled amyloid specifically. This peptide formed spontaneously amyloid-like fibrils in vitro and enhanced fibril formation from wild-type transthyretin. We conclude that several apolipoproteins, including apolipoprotein A-IV, may be important minor amyloid constituents, promoting fibril formation.

Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS)

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 29:79-84 (1997) - doi:10.3354/dao029079 Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS) Grotmol S, Totland GK, Kryvi H The present study shows that a nodavirus-like agent is associated with the lesions of cardiac myopathy syndrome (CMS), a disease of unknown etiology which affects reared adult Atlantic salmon Salmo salar. In archive paraffin-embedded heart tissue from Atlantic salmon diagnosed as suffering from CMS, a distinct immunohistochemical reaction was observed when using a primary antibody against striped jack nervous necrosis virus (Nodaviridae). Immunolabeling was detected in the mesothelium and hypercellular lesions of the epicardium and in endothelial cells and myocytes within mild multifocal lesions of the atrial and ventricular trabeculae. Transmission electron microscopy, performed on deparaffinized samples of the same tissue blocks, revealed substantial amounts of virus-like particles (VLP) in the cytoplasm of endocardial endothelium, in myocytes and in mesothelial cells of the epicardium. The VLP were isometric, spherical and unenveloped, with mean capsid diameters of approximately 25 nm, resembling a virus belonging to the Nodaviridae. Cardiac myopathy syndrome (CMS) · Fish · Atlantic salmon · Nodavirus · Immunohistochemistry Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 29, No. 2. Publication date: May 22, 1997 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1997 Inter-Research.

Detection of the 4977 Base Pair Mitochondrial DNA Deletion in Paraffin-Embedded Heart Tissue Using the Polymerase Chain Reaction—A New Method to Probe Sudden Cardiac Death Molecular Mechanisms?

Detection of mitochondrial DNA deletions is performed in fresh or frozen material. At our institute, however, heart samples from subjects referred for autopsy are systematically processed for histologic examination (that is, paraffin-embedded). We were interested to know if mtDNA deletions can be detected in such material. Our data indicate that: 1) the most frequently observed deletion--the 4977 base pair deletion--can easily be detected in paraffin-embedded heart tissue; 2) this assay is sufficiently sensitive, since very low levels of the deletion can be found in normal heart tissue from young adults; and 3) buffered formalin appears to be the fixative of choice. Recent literature shows that repeated episodes of ischemia result in the accumulation of mtDNA deletions in myocardial cells. Because ischemic heart disease is a major cause of sudden cardiac death, a sensitive method for the detection of mtDNA damage in myocardial cells will be an important tool to facilitate understanding of unexpected cardiac arrest mechanisms.