Linda M. Fox

Three Linked Vasculopathic Processes Characterize Kawasaki Disease: A Light and Transmission Electron Microscopic Study

Background Kawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course. Methodology/Principal Findings Specimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an “eosinophilic-type” myocarditis. Conclusions/Significance NA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.

Immunocytochemical and electron-microscopic characterization of macrophage/microglia cells and expression of class II major histocompatibility complex in the pineal gland of the rat

Interstitial cells in the pineal gland of the rat were characterized immunocytochemically using the monoclonal antibodies MRC OX-42 and ED1 for macrophages/microglia, and MRC OX-6, which recognizes major histocompatibility complex (MHC) class II antigen. A polyclonal antibody against GFAP was used to identify astrocytes. Cells immunopositive for OX-42 and/or ED1 were distributed throughout the gland; they extended processes primarily along the perivascular spaces and occasionally within the parenchyma of the gland. Ultrastructurally, these OX-42-positive cells were characterized by a nucleus with sparse heterochromatin and cytoplasmic vacuoles/lysosomes. Cells expressing MHC class II antigen had a distribution and morphology similar to OX-42-immunopositive cells, suggesting that pineal macrophages/microglia play a role as antigen-presenting cells. GFAP-positive astrocytes were concentrated at the proximal end of the pineal where the pineal stalk enters the gland. The occurrence of antigenpresenting cells in the circumventricular neuroendocrine gland has important functional implications as these cells may be mediators of neuroimmunomodulatory mechanisms, and involved in certain disease states such as autoimmune pinealitis.

Cytoplasmic Inclusion Bodies Are Detected by Synthetic Antibody in Ciliated Bronchial Epithelium during Acute Kawasaki Disease

Abstract BackgroundIn developed nations, Kawasaki disease (KD) is the most common cause of acquired heart disease in children. An infectious etiology is likely but has not yet been identified. We have previously reported that oligoclonal immunoglobulin A plasma cells infiltrate acute KD tissues and that synthetic KD antibodies detect a distinctive spheroidal antigen in acute KD ciliated bronchial epithelium MethodsTo further characterize the antigen in acute KD bronchi, we examined paraffin-embedded ciliated bronchial epithelium using light microscopy (LM) and transmission electron microscopy (TEM) ResultsThe spheroids observed by immunohistochemistry (IHC) are visualized as inclusion bodies with hematoxylin-eosin and nucleic acid stains and in methylene blue/azure II/basic fuchsin trichrome–stained plastic sections, suggesting the presence of both protein and nucleic acid. The structures visualized by LM correspond to homogeneous electron-dense perinuclear inclusion bodies (up to 1.4 microns in diameter) in ciliated bronchial epithelium from 4 patients with acute KD examined by TEM. Inclusion bodies were not present in control bronchial epithelium or in nonciliated cells ConclusionsThe antigen detected in acute KD ciliated bronchial epithelium by IHC with synthetic KD antibodies resides in cytoplasmic inclusion bodies that are consistent with aggregates of viral proteins and associated nucleic acid and may derive from the etiologic agent of KD

RNA-Containing Cytoplasmic Inclusion Bodies in Ciliated Bronchial Epithelium Months to Years after Acute Kawasaki Disease

Background Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common. Methods and Principal Findings Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9–84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI. Significance These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD.

Enriched immune-environment of blood-brain barrier deficient areas of normal adult rats.

The circumventricular organs (CVOs) in the brain are without a blood-brain barrier (BBB) and as such directly exposed to blood plasma constituents and blood-borne pathogens. In light of previous studies showing discrepancies regarding the immunocompetence of these organs, we initiated the present study to provide a comprehensive immunohistochemical analysis of the cellular expression of immune-associated antigens within the pineal gland, area postrema and the subfornical organ. In all CVOs, subpopulations of cells morphologically similar to complement receptor type 3 immunoreactive microglial/macrophage cells expressed major histocompatibility complex (MHC) class II antigen, leucocyte common antigen (LCA/CD45), as well as CD4 and ED1 antigen. Based on morphological criteria the MHC class II antigen expressing cells could be grouped into a major population of classical parenchymal and perivascular ramified microglial cells and a minor population presenting itself as scattered or small groups of rounded macrophage-like cells. CD4 and ED1 antigen were expressed by both cell types. CD45 was preferentially expressed by macrophage-like cells. MHC class I antigen was expressed by the vascular endothelium in both BBB-protected and BBB-deficient areas and was additionally present as a lattice-like network throughout the BBB-deficient parenchyma in all CVOs. The results suggest that the BBB-free areas of the brain besides being constantly surveyed by blood-borne macrophages, possess an intrinsic immune surveillance system based on resting and activated microglial cells, which may function as a non-endothelial, cellular barrier against blood-borne pathogens.

Electrophysiology and ultrastructure of eustachian ridge from cat right atrium: a comparison with SA node *

Intracellular microelectrode and electron microscopic techniques were used to investigate and correlate the electrophysiology of subsidiary pacemaker activity with the presence of cells having ultrastructural characteristics of pacemaker cells i.e. P cells, in Eustachian ridge tissue isolated from cat right atrium. In addition, the electrophysiological characteristics of subsidiary pacemaker activity and the ultrastructural characteristics of P cells in Eustachian ridge were compared to those of SA node obtained from the same hearts. Action potential recordings and morphological analysis were restricted to the endocardial site of earliest activation. Electrophysiological recordings revealed that spontaneously active Eustachian ridge tissues generate slow response action potentials with pacemaker characteristics similar, although not identical, to those of SA node. These included a relatively steep diastolic slope, low maximum diastolic potential (-70 mV), rate of rise (5.5 V/s), take-off potential (-52.5 mV), a relatively large overshoot potential (+7.7 mV) and a spontaneous cycle length (948 ms) about twice as long as SA node (434 ms). Morphological analysis revealed cells with ultrastructural characteristics of P cells, that were restricted to the endocardial site of earliest pacemaker activation. Morphological measurements indicate that Eustachian ridge P cells are not significantly different from P cells in SA node of the same hearts. However, Eustachian ridge P cells exhibit a unique apposition of subsarcolemmal cisternae between cells not seen in SA node. We conclude that pacemaker cells within the Eustachian ridge generate stable, spontaneous activity via slow response pacemaker action potentials. Cells responsible for this subsidiary pacemaker activity are most likely P cell types that are similar, although not identical, to P cells in SA node.

Pinealocyte Dense‐Cored Vesicles and Synaptic Ribbons: A Correlative Ultrastructural‐Biochemical Investigation in Rats and Mice

Dense‐cored vesicles (DCV) and synaptic ribbons (SR) were quantified in the pineal gland of the rat (Sprague‐Dawley) and mouse (Sasco/ ICR strain), and day/ night differences in frequency of these organelles correlated with levels of indoles determined by high performance liquid chromatography (HPLC). There were significant day/night differences in levels of serotonin (5HT), 5‐hydroxyindole acetic acid (5HIAA), N‐acetyl‐5HT, and melatonin in the rat gland. Melatonin and N‐acetyl‐5HT were not detectable in the mouse gland sampled every 4 h over the light:dark cycle. The concentrations of 5HT and 5HIAA (ng/μg protein) were similar in light‐adapted rats and mice, but these indoles did not exhibit a circadian rhythm in the mouse gland. Correlative ultrastructural/biochemical results suggest that DCV do not contain physiologically important stores of 5HT since 1) the mouse gland contains the same number of DCV as the rat during the daytime, but only one‐tenth the levels of 5HT, 2) day/night 5HT levels do not vary in the mouse gland, but there is a significant nocturnal decline in DCV numbers, and 3) 5HT levels in the rat gland decline at night when DCV numbers increase. Numbers of SR were significantly elevated at night in the rat and mouse, and the frequency of this organelle was similar in both species. However, ribbon‐type SR predominated in rat pinealocytes, whereas SR in the mouse were almost exclusively spherical in shape. Day/night diffferences in SR numbers in the mouse gland suggest that cellular mechanisms regulating the frequency of this organelle do not involve factors related to indole metabolism. Because of the lack of photoperiodic effects on indole metabolism in the mouse pineal gland, this species is a potentially important model to study the functional relationship of pinealocyte organelles to cyclical changes in pineal products other than indoles (e.g., peptide/ protein factors).

Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.

Abstract Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.

Synaptic ribbon populations in the pineal gland of the rhesus monkey (Macaca mulatta).

SummaryPinealocytes of rhesus monkeys that had been ovariectomized and given intramuscular injections of 250 μg estradiol-benzoate for 3 consecutive days tended to have more synaptic ribbons (SR) and exhibited a significantly greater size of ribbon fields (RF) compared to untreated animals. These data are consistent with hypotheses that pinealocyte function in primates is altered by hormonal imbalances and that the SR participates in this response. RF were positioned in various parts of the cytoplasm and along the plasma membrane. Participation of SR in direct cell-to-cell contacts was suggested by the formation of densities along the plasma membrane. It is postulated that large RF serve as storage organelles and that the formation of RF results from division of pre-existing SR in each field. Reconstructions made from serial thin sections revealed that profiles of RF comprised separate SR that were not folded and sectioned along various planes.

Immunocytochemical demonstration of nerve growth factor (NGF) receptor in the pineal gland: effect of NGF on pinealocyte neurite formation

Nerve growth factor receptor immunoreactivity (NGFRI) in the pineal gland was examined both light and electron microscopically using the monoclonal antibody 192IgG. NGFRI was located on sympathetic fibers and on perivascular cells resembling macrophage/microglia. A pineal gland dispersed cell culture model confirmed the presence of NGFRI in cells that exhibited processes of varying lengths and were distributed among pinealocytes and other flat cells. Pinealocytes in dispersed cell culture were identified immunocytochemically by their expression of S-antigen, their round shape and small size and their tendency to extend neurites in the direction of the flat cells in culture. The length of pinealocyte neurites showed a significant increase when cultured in the presence of NGF (25 ng/ml), suggesting that trophic factors, mediated by these macrophage/microglial cells, are important to the morphogenesis of these neuroendocrine cells. Neurotrophic activation of these neuroendocrine macrophage/microglia may have neuro-immunomodulatory implications leading to expression of proteins encoded by the major histocompatibility complex.