Enterovirus Persistence in Cardiac Cells of Patients With Idiopathic Dilated Cardiomyopathy Is Linked to 5' Terminal Genomic RNA-Deleted Viral Populations With Viral-Encoded Proteinase Activities.

BACKGROUND: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Immunoglobulin free light chains as an inflammatory biomarker of heart failure with myocarditis.

BACKGROUND: In this study, we measured immunoglobulin free light chains (FLC), a biomarker of inflammation in the sera of patients with heart failure due to myocarditis. METHODS: FLC kappa and FLC lambda were assayed in stored serum samples from patients with heart failure with myocarditis from the US myocarditis treatment trial by a competitive-inhibition multiplex Luminex® assay. RESULTS: The median concentration of circulating FLC kappa/lambda ratio was significantly lower in the sera from patients with heart failure with myocarditis than in healthy controls, and FLC kappa/lambda ratio had good diagnostic ability for identification of heart failure with myocarditis. Further, FLC kappa/lambda ratio was an independent prognostic factor for overall survival, and allowed creation of three prognostic groups by combining with N-terminal pro-B-type natriuretic peptide. CONCLUSIONS: This study suggests that FLC kappa/lambda ratio is a promising biomarker of heart failure with myocarditis.

Creatinine downregulates TNF-α in macrophage and T cell lines.

Creatinine is the breakdown product of creatine, a key participant in the generation of ATP and is traditionally considered to be a biologically inert waste product. Based on our earlier work, we analyzed the effects of creatinine hydrochloride on the expression of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in a human T cell line, as well as human and mouse macrophage cell lines. Exposing cells to creatinine hydrochloride significantly reduced TNF-α mRNA and protein levels compared to control-treated cultures in all cell lines tested. Lipopolysaccharide (LPS), a potent inducer of inflammation, was employed with in mouse macrophage cell lines to induce high levels of TNF-α in order to determine whether creatinine hydrochloride could reduce preexisting inflammation. Cells treated with LPS and creatinine hydrochloride had significantly reduced TNF-α levels compared to cells treated with LPS alone. As the NF-κB signaling pathway represents a major mechanism of TNF-α generation, nuclear extracts were examined for NF-κB pathway activation. Cells exposed to CRN had significantly lower levels of NF-κB in the nucleus compared to control-treated cells. Together, these results support the hypothesis that CRN can alter anti-inflammatory responses by interfering with the activation of the NF-κB pathway.

Three capsid amino acids notably influence coxsackie B3 virus stability.

Coxsackievirus B3 strain 28 (CVB3/28) is less stable at 37 °C than eight other CVB3 strains with which it has been compared, including four in this study. In a variant CVB3/28 population selected for increased stability at 37 °C, the capsid proteins of the stable variant differed from the parental CVB3/28 by two mutations in Vp1 and one mutation in Vp3, each of which resulted in altered protein sequences. Each of the amino acid changes was individually associated with a more stable virus. Competition between CVB3/28 and a more stable derivative of the strain showed that propagation of the less stable virus was favoured in receptor-rich HeLa cells.

Enteroviruses, hygiene and type 1 diabetes: toward a preventive vaccine.

Enteroviruses and humans have long co-existed. Although recognized in ancient times, poliomyelitis and type 1 diabetes (T1D) were exceptionally rare and not epidemic, due in large part to poor sanitation and personal hygiene which resulted in repeated exposure to fecal-oral transmitted viruses and other infectious agents and viruses and the generation of a broad protective immunity. As a function of a growing acceptance of the benefits of hygienic practices and microbiologically clean(er) water supplies, the likelihood of exposure to diverse infectious agents and viruses declined. The effort to vaccinate against poliomyelitis demonstrated that enteroviral diseases are preventable by vaccination and led to understanding how to successfully attenuate enteroviruses. Type 1 diabetes onset has been convincingly linked to infection by numerous enteroviruses including the group B coxsackieviruses (CVB), while studies of CVB infections in NOD mice have demonstrated not only a clear link between disease onset but an ability to reduce the incidence of T1D as well: CVB infections can suppress naturally occurring autoimmune T1D. We propose here that if we can harness and develop the capacity to use attenuated enteroviral strains to induce regulatory T cell populations in the host through vaccination, then a vaccine could be considered that should function to protect against both autoimmune as well as virus-triggered T1D. Such a vaccine would not only specifically protect from certain enterovirus types but more importantly, also reset the organism's regulatory rheostat making the further development of pathogenic autoimmunity less likely.

Coxsackievirus can persist in murine pancreas by deletion of 5' terminal genomic sequences.

Enterovirus infections are generally acute and rapidly cleared by the host immune response. Enteroviruses can at times persist in immunologically intact individuals after the rise of the type-specific neutralizing immune response. The mechanism of enterovirus persistence was shown in group B coxsackieviruses (CVB) to be due to naturally-occurring deletions at the 5' terminus of the genome which variably impact the stem-loop secondary structure called domain I. These deletions result in much slower viral replication and a loss of measurable cytopathic effect when such 5' terminally deleted (TD) viruses are assayed in cell culture. The existence and persistence of CVB-TD long after the acute phase of infection has been documented in hearts of experimentally inoculated mice and naturally infected humans but to date, the existence of TD enteroviral populations have not been documented in any other organ. Enteroviral infections have been shown to impact type 1 diabetes (T1D) onset in humans as well as in the non-obese diabetic mouse model of T1D. The first step to studying the potential impact of CVB-TD on T1D etiology is to determine whether CVB-TD populations can arise in the pancreas. After inoculation of NOD diabetic mice with CVB, viral RNA persists in the absence of cytopathic virus in pancreas weeks past the acute infectious period. Analysis of viral genomic 5' termini by RT-PCR showed CVB-TD populations displace the parental population during persistent replication in murine pancreata.

Complete sequence of the closed circular extrachromosomal element of Naegleria pringsheimi De Jonckheere (strain Singh).

The DNA encoding the ribosomal RNA in Naegleria is encoded on closed circular extrachromosomal ribosomal DNA-containing elements (CERE) in the nucleolus. In this report, we describe the sequence of the CERE of Naegleria pringsheimi De Jonckheere (strain Singh).

Complete Sequence of the Closed Circular Extrachromosomal Element of Naegleria jadini Willaert and Ray (Strain ITMAP400).

Amoebae of the Naegleria genus carry all ribosome-encoding DNA on closed circular extrachromosomal elements (CERE). We report the sequence of the CERE of Naegleria jadini (strain Willaert and Ray).

Complete sequence of the closed circular extrachromosomal element (CERE) of Naegleria australiensis De Jonckheere (strain PP 397).

Ribosomal RNA is not encoded in chromosomal DNA in amoebae of the Naegleria genus but the rRNA genes are located on closed circular extrachromosomal ribosomal DNA (rDNA)-containing elements (CERE). In this report, we describe the sequence of the CERE of Naegleria australiensis De Jonckheere (strain PP397).

Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment.

While group B coxsackieviruses (CVB) use the coxsackievirus and adenovirus receptor (CAR) as the receptor through which they infect susceptible cells, some CVB strains are known for their acquired capacity to bind other molecules. The CVB3/RD strain that emerged from a CVB3/Nancy population sequentially passaged in the CAR-poor RD cell line binds decay-accelerating factor (DAF) (CD55) and CAR. A new strain, CVB3/RDVa, has been isolated from RD cells chronically infected with CVB3/RD and binds multiple molecules in addition to DAF and CAR. The capsid proteins of CVB3/RD differ from those of CVB3/28, a cloned strain that binds only CAR, by only four amino acids, including a glutamate/glutamine dimorphism in the DAF-binding region of the capsid. The capsid proteins of CVB3/RD and CVB3/RDVa differ by seven amino acids. The ability of CVB3/RDVa to bind ligands in addition to CAR and DAF may be attributed to lysine residues near the icosahedral 5-fold axes of symmetry. Considered with differences in the stability of the CVB3 strains, these traits suggest that in vitro selection in a CAR-limited environment selects for virus populations that can associate with molecules on the cell surface and survive until CAR becomes available to support infection.

Enteroviruses and type 1 diabetes.

BACKGROUND: Human enteroviruses, which are transmitted via a faecal-oral route, have long been associated with type 1 diabetes onset. Increased hygiene in the 20th century may now be responsible for a decreased chance of enterovirus exposure from an early age onward. Infections with enteroviruses may also be more likely to occur at a later age; the recurrent poliomyelitis epidemics in the 20th century were linked to increased hygiene, consistent with this hypothesis. The association of fewer enterovirus exposures and increased diabetes rates may seem at first non-intuitive but may be explained using a combination of human observations and data from experimental coxsackie B virus infections in nonobese diabetic mice. METHODS: Network for Pancreatic Organ Donors with Diabetes samples were examined for the presence of detectable enteroviral RNA by RT-PCR. RESULTS: Viral RNA was not detected. CONCLUSIONS: A role for enteroviruses in the aetiology of human type 1 diabetes is hard to refute but in order to definitively link enteroviruses in general, and specific viruses in particular, with the disease, pancreas biopsy tissue must become available at the time of disease diagnosis.

The Role of Creatine in the Development and Activation of Immune Responses.

The use of dietary supplements has become increasingly common over the past 20 years. Whereas supplements were formerly used mainly by elite athletes, age and fitness status no longer dictates who uses these substances. Indeed, many nutritional supplements are recommended by health care professionals to their patients. Creatine (CR) is a widely used dietary supplement that has been well-studied for its effects on performance and health. CR also aids in recovery from strenuous bouts of exercise by reducing inflammation. Although CR is considered to be very safe in recommended doses, a caveat is that a preponderance of the studies have focused upon young athletic individuals; thus there is limited knowledge regarding the effects of CR on children or the elderly. In this review, we examine the potential of CR to impact the host outside of the musculoskeletal system, specifically, the immune system, and discuss the available data demonstrating that CR can impact both innate and adaptive immune responses, together with how the effects on the immune system might be exploited to enhance human health.

Complete Genome Sequence of the Naegleria fowleri (Strain LEE) Closed Circular Extrachromosomal Ribosomal DNA Element.

The circular extrachromosomal ribosomal DNA (rDNA) element of Naegleria fowleri strain LEE was molecularly cloned and fully sequenced. The element comprises 15,786 bp and contains a single copy of the organism's rDNA cistron. The nonribosomal sequence contains five potential open reading frames, two large direct repeat sequences, and numerous smaller repeated-sequence regions.

Mapping the Single Origin of Replication in the Naegleria gruberi Extrachromosomal DNA Element.

The genes encoding the ribosomal RNA (rRNA) subunits of the amoeba Naegleria gruberi are encoded in a relatively uncommon arrangement: on a circular extrachromosomal DNA element with each organism carrying about 4,000 copies of the element. As complete sequence analysis of the N. gruberi chromosomal DNA revealed no copy of the rRNA genes, these extrachromosomal elements must therefore replicate autonomously. We reported elsewhere the molecular cloning and the complete sequence analysis of the entire rRNA gene-containing element of N. gruberi (strain EGB). Using neutral/neutral two-dimensional agarose electrophoresis, the region in the element enclosing the single replication origin using DNA from asynchronous and axenically propagated N. gruberi populations was localized within a 2.1 kbp fragment located approximately 2,300bp from the 18S rRNA gene and 3,700bp from the 28S rRNA gene. The results indicate that replication occurs from a single origin via a theta-type mode of replication rather than by a rolling circle mode. Further, G-quadruplex elements, often located near DNA replication origins, occur in and near this fragment in a repeated sequence.

Complete Genome Sequence of the Circular Extrachromosomal Element of Naegleria gruberi Strain EGB Ribosomal DNA.

The circular extrachromosomal element of Naegleria gruberi strain EGB was linearized, molecularly cloned, and fully sequenced. The sequence comprises 14,007 bp and encodes the organism's rRNA genes, two potential open reading frames, and numerous repeated sequence regions.

Coxsackievirus infections and NOD mice: relevant models of protection from, and induction of, type 1 diabetes.

Human enteroviruses (HEVs) like the group B coxsackieviruses (CVBs) are prime candidates for infectious, environmental causes of human type 1 diabetes (T1D). Non-obese diabetic (NOD) female mice are well protected from T1D onset if inoculated with CVB when young. Older, prediabetic NOD mice can rapidly develop T1D following inoculation with CVB, mimicking clinical reports of disease-associated T1D onset. The ability to induce rapid T1D in NOD mice is linked to the rate of replication of the CVB strain in beta cell cultures and pancreatic tissue, indicating that any CVB strain is potentially diabetogenic under the correct conditions. Rapid T1D onset is preceded by CVB replication in islet cells including beta cells. Although CVB strains do not productively infect healthy islets of young mice, CVBs can replicate in healthy islets in the presence of murine IL-4. These models expand much of what is known or suspected regarding the etiologic role of HEVs in human T1D.

Establishment of a model to examine the early events involved in the development of virus-induced demyelinating lesions.

The ability to study the immediate, early events in the demyelinating process has been difficult on account of the lack of model systems that address this phase of lesion development. The vast majority of animal models used to study multiple sclerosis (MS) focuses on the later events of myelin destruction. To address this deficiency, we have modified the currently used Theiler's murine encephalomyelitis virus (TMEV)-induced model of demyelination to precisely identify the area where virus-induced demyelination first occurs. Following surgical exposure of the spinal cord, we directly injected TMEV into the spinal cord of female SJL/J mice. Characterization of the events in the spinal cord in the days following injection of virus support the use of this model to dissect the pathways triggered in the host in the early phases of demyelination. A complete understanding of the genesis of the sclerotic plaque may provide insights into enhanced treatment for patients with central nervous system (CNS) demyelination.

Myocarditis and heart failure associated with hepatitis C virus infection.

BACKGROUND: The aim of study is to determine the prevalence of hepatitis C virus (HCV) infection and myocardial injury among patients enrolled in the Myocarditis Treatment Trial. HCV infection has recently been noted in patients with cardiomyopathies and myocarditis. However, prevalence of HCV infection in myocarditis and heart failure remains to be clarified. METHODS AND RESULTS: Patients with heart failure up to 2 years in duration without a distinct cause were enrolled in the trial between 1986 and 1990. Frozen blood samples were available from 1355 among 2233 patients enrolled and examined for presence of anti-HCV antibodies, circulating cardiac troponins I and T, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Anti-HCV antibodies were identified in 59 of 1355 patients (4.4%). This higher prevalence of HCV infection than that observed in the general US population (1.8%), varied widely (0-15%) among the different medical centers and regions. The concentrations of circulating cardiac troponin (cTn) I were elevated in 17 of 56 patients (30%), and cTnT was detectable in 28 of 59 patients (48%) with HCV antibodies, suggesting the persistence of ongoing myocardial injury. The concentrations of NT-proBNP were elevated in 42 of 42 patients (100%) with HCV antibodies, (10,000 +/- 5860 pg/mL), a mean value significantly greater than in 1276 patients without HCV antibody (2508 +/- 160 pg/mL, P < .0001). CONCLUSION: Anti-HCV antibodies were identifiable in sera stored for 13 to 17 years and were more prevalent in patients with myocarditis and HF than in the general population. In regions where its prevalence is high, HCV infection may be an important cause of myocarditis and HF. NT-proBNP is a more sensitive marker of myocardial injury than cardiac troponins in patients with heart failure from HCV myocarditis.