Analysis of tumor microenvironment composition in vestibular schwannomas: insights into NF2-associated and sporadic variations and their clinical correlations.

Objective: Vestibular schwannomas (VS), benign tumors stemming from the eighth cranial nerve's Schwann cells, are associated with Merlin gene mutations, inflammation, and the tumor microenvironment (TME), influencing tumor initiation, maintenance, and potential neural dysfunction. Understanding TME composition holds promise for systemic therapeutic interventions, particularly for NF2-related schwannomatosis. Methodology: A retrospective analysis of paraffin-embedded tissue from 40 patients (2013-2020), evenly divided by neurofibromatosis type 2 status, with further stratification based on magnetic resonance imaging (MRI) progression and hearing function. Immunohistochemistry assessed TME components, including T-cell markers (CD4, CD8, CD25), NK cells (CD7), and macrophages (CD14, CD68, CD163, CCR2). Fiji software facilitated image analysis. Results: T-cell markers (CD4, CD8, CD7) exhibited low expression in VS, with no significant NF2-associated vs. sporadic distinctions. Macrophage-related markers (CD14, CD68, CD163, CCR2) showed significantly higher expression (CD14: p = 0.0187, CD68: p < 0.0001, CD163: p = 0.0006, CCR2: p < 0.0001). CCR2 and CD163 significantly differed between NF2-associated and sporadic VS. iNOS, an M1-macrophage marker, was downregulated. CD25, a regulatory T-cell marker, correlated significantly with tumor growth dynamics (p = 0.016). Discussion: Immune cells, notably monocytes and macrophages, crucially contribute to VS pathogenesis in both NF2-associated and sporadic cases. Significant differences in CCR2 and CD163 expression suggest distinct immune responses. Regulatory T-cells may serve as growth dynamic markers. These findings highlight immune cells as potential biomarkers and therapeutic targets for managing VS.

Macrophages influence Schwann cell myelin autophagy after nerve injury and in a model of Charcot-Marie-Tooth disease.

BACKGROUND AND AIMS: The complex cellular and molecular interactions between Schwann cells (SCs) and macrophages during Wallerian degeneration are a prerequisite to allow rapid uptake and degradation of myelin debris and axonal regeneration after peripheral nerve injury. In contrast, in non-injured nerves of Charcot-Marie-Tooth 1 neuropathies, aberrant macrophage activation by SCs carrying myelin gene defects is a disease amplifier that drives nerve damage and subsequent functional decline. Consequently, targeting nerve macrophages might be a translatable treatment strategy to mitigate disease outcome in CMT1 patients. Indeed, in previous approaches, macrophage targeting alleviated the axonopathy and promoted sprouting of damaged fibers. Surprisingly, this was still accompanied by robust myelinopathy in a model for CMT1X, suggesting additional cellular mechanisms of myelin degradation in mutant peripheral nerves. We here investigated the possibility of an increased SC-related myelin autophagy upon macrophage targeting in Cx32def mice. METHODS: Combining ex vivo and in vivo approaches, macrophages were targeted by PLX5622 treatment. SC autophagy was investigated by immunohistochemical and electron microscopical techniques. RESULTS: We demonstrate a robust upregulation of markers for SC autophagy after injury and in genetically-mediated neuropathy when nerve macrophages are pharmacologically depleted. Corroborating these findings, we provide ultrastructural evidence for increased SC myelin autophagy upon treatment in vivo. INTERPRETATION: These findings reveal a novel communication and interaction between SCs and macrophages. This identification of alternative pathways of myelin degradation may have important implications for a better understanding of therapeutic mechanisms of pharmacological macrophage targeting in diseased peripheral nerves.

Presymptomatic macrophage targeting has a long-lasting therapeutic effect on treatment termination in a mouse model of Charcot-Marie-Tooth 1.

Macrophage-mediated inflammation is a potent driver of disease progression in mouse models of Charcot-Marie-Tooth (CMT) 1 diseases. This leads to the possibility to consider these cells as therapeutic targets to dampen disease outcome in the so far non-treatable neuropathies. As a pharmacological proof-of-principle study, long-term targeting of nerve macrophages with the orally applied CSF-1 receptor specific kinase (c-FMS) inhibitor PLX5622 showed a substantial alleviation of the neuropathy in distinct CMT1 mouse models. However, regarding translational options, clinically relevant questions emerged regarding treatment onset, duration and termination. Corroborating previous data, we here show that in a model for CMT1B, peripheral neuropathy was substantially alleviated after early continuous PLX5622 treatment in CMT1B mice, leading to preserved motor function. However, late-onset treatment failed to mitigate histopathological and clinical features, despite a similar reduction in the number of macrophages. Surprisingly, in CMT1B mice, terminating early PLX5622 treatment at six months was still sufficient to preserve motor function at 12 months of age, suggesting a long-lasting, therapeutic effect of early macrophage depletion. This novel and unexpected finding may have important translational implications, since we here show that continuous macrophage targeting appears not to be necessary for disease alleviation, provided that the treatment starts within an early, critical time window.

Early targeting of endoneurial macrophages alleviates the neuropathy and affects abnormal Schwann cell differentiation in a mouse model of Charcot-Marie-Tooth 1A.

We have previously shown that targeting endoneurial macrophages with the orally applied CSF-1 receptor specific kinase (c-FMS) inhibitor PLX5622 from the age of 3 months onwards led to a substantial alleviation of the neuropathy in mouse models of Charcot-Marie-Tooth (CMT) 1X and 1B disease, which are genetically-mediated nerve disorders not treatable in humans. The same approach failed in a model of CMT1A (PMP22-overexpressing mice, line C61), representing the most frequent form of CMT. This was unexpected since previous studies identified macrophages contributing to disease severity in the same CMT1A model. Here we re-approached the possibility of alleviating the neuropathy in a model of CMT1A by targeting macrophages at earlier time points. As a proof-of-principle experiment, we genetically inactivated colony-stimulating factor-1 (CSF-1) in CMT1A mice, which resulted in lower endoneurial macrophage numbers and alleviated the neuropathy. Based on these observations, we pharmacologically ablated macrophages in newborn CMT1A mice by feeding their lactating mothers with chow containing PLX5622, followed by treatment of the respective progenies after weaning until the age of 6 months. We found that peripheral neuropathy was substantially alleviated after early postnatal treatment, leading to preserved motor function in CMT1A mice. Moreover, macrophage depletion affected the altered Schwann cell differentiation phenotype. These findings underscore the targetable role of macrophage-mediated inflammation in peripheral nerves of inherited neuropathies, but also emphasize the need for an early treatment start confined to a narrow therapeutic time window in CMT1A models and potentially in respective patients.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

Self-reported quality of life and hope in phase-I trial participants: An observational prospective cohort study.

For advanced cancer patients deliberating early clinical trial participation, adequate information about expected effect on quality of life (HRQoL) and hope, may support decision making. The aim was to assess the potential relation of HRQoL to eligibility for phase-I trial participation, and to observe the variations in patient-reported outcomes. Patients completed questionnaires at preconsent (n = 124), baseline (n = 96), and after first evaluation of a phase-I trial (n = 76). The Mann-Whitney U test was used to test differences between eligible and ineligible patients. Univariate logistic regression was performed for eligibility. Factorial repeated-measures ANOVA compared the outcomes of patients continuing vs. stopping participation after first evaluation over time. Eligibility is associated with significant better global health OR = 0.946, 95% CI [0.918, 0.975], p = 0.001, physical functioning OR = 0.959, 95% CI [0.933, 0.985], p = 0.002, role functioning OR = 0.974, 95% CI [0.957, 0.991] and better appetite OR = 1.114 95% CI [1.035, 1.192]. HRQoL outcomes like global health, social functioning and appetite decline in all patients and differ between patients continuing or having to end participation. Over time, hope and tenacity decline in all patients and coping strategies alter in patients stopping participation. Trial participation influences patient-reported outcomes. Global health may predict for eligibility and trial continuation. Informing patients could affect patients' decision making.

Macrophage Depletion Ameliorates Peripheral Neuropathy in Aging Mice.

Aging is known as a major risk factor for the structure and function of the nervous system. There is urgent need to overcome such deleterious effects of age-related neurodegeneration. Here we show that peripheral nerves of 24-month-old aging C57BL/6 mice of either sex show similar pathological alterations as nerves from aging human individuals, whereas 12-month-old adult mice lack such alterations. Specifically, nerve fibers showed demyelination, remyelination and axonal lesion. Moreover, in the aging mice, neuromuscular junctions showed features typical for dying-back neuropathies, as revealed by a decline of presynaptic markers, associated with α-bungarotoxin-positive postsynapses. In line with these observations were reduced muscle strengths. These alterations were accompanied by elevated numbers of endoneurial macrophages, partially comprising the features of phagocytosing macrophages. Comparable profiles of macrophages could be identified in peripheral nerve biopsies of aging persons. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by applying an orally administered CSF-1R specific kinase (c-FMS) inhibitor. The 6-month-lasting treatment started before development of degenerative changes at 18 months and reduced macrophage numbers in mice by ∼70%, without side effects. Strikingly, nerve structure was ameliorated and muscle strength preserved. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may pave the way for treating degeneration in the aging peripheral nervous system by targeting macrophages, leading to reduced weakness, improved mobility, and eventually increased quality of life in the elderly.SIGNIFICANCE STATEMENT Aging is a major risk factor for the structure and function of the nervous system. Here we show that peripheral nerves of 24-month-old aging mice show similar degenerative alterations as nerves from aging human individuals. Both in mice and humans, these alterations were accompanied by endoneurial macrophages. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by blocking a cytokine receptor, essential for macrophage survival. The treatment strongly reduced macrophage numbers and substantially improved nerve structure and muscle strength. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may be helpful for treatment weakness and reduced mobility in the elderly.

Understanding how coping strategies and quality of life maintain hope in patients deliberating phase I trial participation.

OBJECTIVE: This study aimed to understand how hope and motivation of patients considering phase I trial participation are affected by psychological factors such as coping strategies and locus of control (LoC) and general well-being as measured by the quality of life (QoL). METHODS: An exploratory cross-sectional study was performed in patients with incurable cancer (N = 135) referred to our phase I unit for the first time. Patients were potentially eligible for phase I trial participation and participated in our study while deliberating phase I trial participation. We used questionnaires on hope, motivation to participate, coping, LoC, and QoL. To investigate the nature and magnitude of the relationships between the scales, a structural equation modeling (SEM) was fitted to the data. RESULTS: Hope significantly predicted the motivation to participate in phase I trials. Predictors of hope were a combination of flexible and tenacious goal pursuit (both P < .01), internal LoC (P < .01), and QoL (P < .01). The SEM showed an exact fit to the data, using a null hypothesis significance test: chi-square (8) = 9.30, P = .32. CONCLUSIONS: Patients considering phase I trial participation seem to use a pact of tenacious and flexible coping and control to stay hopeful. Furthermore, hope and QoL positively affected each other. The psychological pact may promote an adaptation enabling them to adjust to difficult circumstances by unconsciously ignoring information, called dissonance reduction. This mechanism may impair their ability to provide a valid informed consent. We suggest including a systematic exploration of patients' social context and values before proposing a phase I trial.

Rare Variants in MME, Encoding Metalloprotease Neprilysin, Are Linked to Late-Onset Autosomal-Dominant Axonal Polyneuropathies.

Axonal polyneuropathies are a frequent cause of progressive disability in the elderly. Common etiologies comprise diabetes mellitus, paraproteinaemia, and inflammatory disorders, but often the underlying causes remain elusive. Late-onset axonal Charcot-Marie-Tooth neuropathy (CMT2) is an autosomal-dominantly inherited condition that manifests in the second half of life and is genetically largely unexplained. We assumed age-dependent penetrance of mutations in a so far unknown gene causing late-onset CMT2. We screened 51 index case subjects with late-onset CMT2 for mutations by whole-exome (WES) and Sanger sequencing and subsequently queried WES repositories for further case subjects carrying mutations in the identified candidate gene. We studied nerve pathology and tissue levels and function of the abnormal protein in order to explore consequences of the mutations. Altogether, we observed heterozygous rare loss-of-function and missense mutations in MME encoding the metalloprotease neprilysin in 19 index case subjects diagnosed with axonal polyneuropathies or neurodegenerative conditions involving the peripheral nervous system. MME mutations segregated in an autosomal-dominant fashion with age-related incomplete penetrance and some affected individuals were isolated case subjects. We also found that MME mutations resulted in strongly decreased tissue availability of neprilysin and impaired enzymatic activity. Although neprilysin is known to degrade ß-amyloid, we observed no increased amyloid deposition or increased incidence of dementia in individuals with MME mutations. Detection of MME mutations is expected to increase the diagnostic yield in late-onset polyneuropathies, and it will be tempting to explore whether substances that can elevate neprilysin activity could be a rational option for treatment.

Myelin and macrophages in the PNS: An intimate relationship in trauma and disease.

Macrophages of the peripheral nervous system belong to the so-called tissue macrophages, with multiple functions during injury and disease. Their origin during ontogeny has not yet been completely resolved, but it is clear that upon injury and disease conditions, they are supplemented by hematopoietic derivatives. In the peripheral nervous system, the most abundantly investigated scenario in which resident and infiltrating macrophages are involved is the so-called "Wallerian degeneration", a complex degenerative process where macrophages exhibit mostly beneficial functions by phagocytosing myelin and axonal remnants. Of special interest is the implication of macrophages in inflammatory nerve diseases, like acute Guillain-Barré syndromes and its permanent variant, chronic inflammatory demyelinating polyneuropathy, where macrophages are supposed to be substantial (co-)mediators of the diseases. In inherited peripheral neuropathies nerve macrophages possess a clear disease-amplifying function. In the corresponding animal models, a coordinated interplay between mutant Schwann cells, macrophages, endoneurial fibroblasts and the target structure, myelin, emerged. Along this process, a newly discovered disease mechanism mediated by macrophages is the dedifferentiation of myelinating Schwann cells. As macrophages are amplifiers of the genetically-mediated, non-curable diseases, targeting the mechanisms of their activation might be a promising strategy to treat these disorders. This article is part of a Special Issue entitled SI: Myelin Evolution.

Targeting the colony stimulating factor 1 receptor alleviates two forms of Charcot-Marie-Tooth disease in mice.

See Scherer (doi:10.1093/awv279) for a scientific commentary on this article.Charcot-Marie-Tooth type 1 neuropathies are inherited disorders of the peripheral nervous system caused by mutations in Schwann cell-related genes. Typically, no causative cure is presently available. Previous preclinical data of our group highlight the low grade, secondary inflammation common to distinct Charcot-Marie-Tooth type 1 neuropathies as a disease amplifier. In the current study, we have tested one of several available clinical agents targeting macrophages through its inhibition of the colony stimulating factor 1 receptor (CSF1R). We here show that in two distinct mouse models of Charcot-Marie-Tooth type 1 neuropathies, the systemic short- and long-term inhibition of CSF1R by oral administration leads to a robust decline in nerve macrophage numbers by ∼70% and substantial reduction of the typical histopathological and functional alterations. Interestingly, in a model for the dominant X-linked form of Charcot-Marie-Tooth type 1 neuropathy, the second most common form of the inherited neuropathies, macrophage ablation favours maintenance of axonal integrity and axonal resprouting, leading to preserved muscle innervation, increased muscle action potential amplitudes and muscle strengths in the range of wild-type mice. In another model mimicking a mild, demyelination-related Charcot-Marie-Tooth type 1 neuropathy caused by reduced P0 (MPZ) gene dosage, macrophage blockade causes an improved preservation of myelin, increased muscle action potential amplitudes, improved nerve conduction velocities and ameliorated muscle strength. These observations suggest that disease-amplifying macrophages can produce multiple adverse effects in the affected nerves which likely funnel down to common clinical features. Surprisingly, treatment of mouse models mimicking Charcot-Marie-Tooth type 1A neuropathy also caused macrophage blockade, but did not result in neuropathic or clinical improvements, most likely due to the late start of treatment of this early onset disease model. In summary, our study shows that targeting peripheral nerve macrophages by an orally administered inhibitor of CSF1R may offer a highly efficacious and safe treatment option for at least two distinct forms of the presently non-treatable Charcot-Marie-Tooth type 1 neuropathies.

Endogenous antibodies contribute to macrophage-mediated demyelination in a mouse model for CMT1B.

BACKGROUND: We could previously identify components of both the innate and the adaptive immune system as disease modifiers in the pathogenesis of models for Charcot-Marie-Tooth (CMT) neuropathies type 1B and 1X. As part of the adaptive immune system, here we investigated the role of antibodies in a model for CMT1B. METHODS: Antibodies were localized and characterized in peripheral nerves of the CMT1B model by immunohistochemistry and Western blot analysis. Experimental ablation of antibodies was performed by cross breeding the CMT1B models with mutants deficient in B-lymphocytes (JHD-/- mutants). Ameliorated demyelination by antibody deficiency was reverted by intravenous injection of mouse IgG fractions. Histopathological analysis was performed by immunocytochemistry and light and quantitative electron microscopy. RESULTS: We demonstrate that in peripheral nerves of a mouse model for CMT1B, endogenous antibodies strongly decorate endoneurial tubes of peripheral nerves. These antibodies comprise IgG and IgM subtypes and are preferentially, but not exclusively, associated with nerve fiber aspects nearby the nodes of Ranvier. In the absence of antibodies, the early demyelinating phenotype is substantially ameliorated. Reverting the neuropathy by reconstitution with murine IgG fractions identified accumulating antibodies as potentially pathogenic at this early stage of disease. CONCLUSIONS: Our study demonstrates that in a mouse model for CMT1B, endogenous antibodies contribute to early macrophage-mediated demyelination and disease progression. Thus, both the innate and adaptive immune system are mutually interconnected in a genetic model for demyelination. Since in Wallerian degeneration antibodies have also been shown to be involved in myelin phagocytosis, our study supports our view that inherited demyelination and Wallerian degeneration share common mechanisms, which are detrimental when activated under nonlesion conditions.

CSF-1-activated macrophages are target-directed and essential mediators of Schwann cell dedifferentiation and dysfunction in Cx32-deficient mice.

We investigated connexin 32 (Cx32)-deficient mice, a model for the X-linked form of Charcot-Marie-Tooth neuropathy (CMT1X), regarding the impact of low-grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not-yet addressed disease-related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32-positive and -negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32-positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM- and L1-positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32-deficient mice additionally deficient for the macrophage-directed cytokine colony-stimulating factor (CSF)-1. In the absence of CSF-1, Cx32-deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF-1-activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32-deficient mice.

Nonuniform molecular features of myelinating Schwann cells in models for CMT1: distinct disease patterns are associated with NCAM and c-Jun upregulation.

We investigated three models for Charcot-Marie-Tooth type 1 (CMT1) neuropathy, comprising mice lacking connexin 32 (Cx32def), mice with reduced myelin protein zero (P0) expression (P0het) and transgenic mouse mutants overexpressing peripheral myelin protein 22 (PMP22tg), with regard of the expression of the developmentally regulated molecules NCAM, L1, the low-affinity NGF-receptor p75 (p75(NTR) ) and the transcription factor component c-Jun. We found that all molecules were uniformly expressed by myelin deficient and supernumerary Schwann cells. The mutant myelinating Schwann cells of PMP22tg mice showed a robust NCAM-immunoreactivity in Schmidt-Lanterman incisures (SLI) that accompanies other early onset abnormalities, such as the presence of supernumerary Schwann cells and impaired myelin formation in some fibers. In line with this, Cx32def and P0het mice, which represent demyelinating models, only rarely express NCAM in SLI. Surprisingly, c-Jun immunoreactivity displayed a mosaic-like pattern with mostly negative and some weakly or moderately positive nuclei both in myelinating Schwann cells and Remak cells of wildtype (wt), P0het and PMP22tg mice. However, c-Jun expression was substantially upregulated in myelinating Schwann cells of Cx32def mice and spatially associated with axon perturbation, a typical predemyelinating feature of Cx32 deficiency. Additionally, c-Jun upregulation was correlated with an elevated level of GDNF, possibly causally linked to the typical compensatory sprouting of axons in Cx32def mice and CMT1X patients. Our findings suggest that in myelinating Schwann cells of distinct models of CMT1, c-Jun upregulation is a marker for predemyelinating axonal perturbation while myelin-related NCAM expression is indicative for early Schwann cell abnormalities.

Neuroinflammation by cytotoxic T-lymphocytes impairs retrograde axonal transport in an oligodendrocyte mutant mouse.

Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.

Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis.

Paraffin tissue blocks from 27 cases with sporadic myocarditis were collected during a 12-year period at a single medical examiner's office. Blocks were studied by using histopathology; immunohistochemistry for viruses (adenovirus, enterovirus, influenza A and B, and human herpes types 4 and 5), bacteria (Neisseria meningitidis, Ehrlichia sp, spotted fever group Rickettsia) and parasites (Toxoplasma gondii and Trypanosoma cruzi); and polymerase chain reaction (PCR)/RT-PCR for adenovirus and enterovirus. We identified enterovirus in 5 (18.5%) cases and Sarcocystis in a 36-year-old woman who had focal inflammation and myocyte necrosis. Immunohistochemical evidence of enteroviruses was found in the myocytes of 2 patients less than 6 months old who had diffuse mononuclear myocardial inflammation, interstitial pneumonitis; one also had encephalitis. In these 2 patients, the presence of enterovirus was confirmed by RT-PCR targeting the 5' nontranslated region and was serotyped as coxsackievirus B2 by sequencing the VP1 capsid region. In another 3 cases (ages 12, 47, and 54), enterovirus was detected by the 5' nontranslated region region; VP1 sequencing identified these as echoviruses 6, 13, and 7, respectively. Accurately identifying an infectious agent is the foundation for clinical and public health interventions. Despite using multiple diagnostic methods, an organism could only be detected in a small proportion of sporadic myocarditis cases.