An ultrastructural pathologist's views on fibroblasts, modified smooth muscle cells, wound healing, stenosing arteriopathies, Kawasaki disease, Dupuytren's contracture, and the stroma of carcinomas.

It wasn't until 1960 that the dense bodies of the peripheral actin arrays of fibroblasts were finally visualized, i.e., stress fibers (SFs). Mistakenly assumed that its SFs turned the fibroblast into a unique cell situated somewhere in a continuum between it and a smooth muscle cell (SMC), it was descriptively named a "myofibroblast" (MF). Automatically, spindle cells with SFs and/or smooth muscle actin by SMA IHC-staining, became MFs, although endothelial cells, pericytes, modified SMCs (mSMC), and myoepithelial cells all contain SFs. An invisible "intermediate" cell was hypothesized to exist somewhere between SMA-negative and positive fibroblasts, and named a "proto-myofibroblast". The sub-epithelial spindle cells of normal and malignant tumors of the GI, GU, and respiratory tracts are all fibroblasts with SFs. The second erroneous myofibroblast came from a 1971 rat wound healing study and its 1974 human counterpart. Updated analysis of the papers' TEMs proved that the cells are mSMCs and not fibroblasts (AKA: MFs). The pathognomonic cells of Dupuytren's contracture are mSMCs and fibroblasts and that of the stenosing arteriopathy of Kawasaki Disease and other similar arteriopathies are mSMCs. TEM remains a powerful tool.

The transcriptional profile of coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) can cause potentially life-threatening coronary arteritis in young children, and has a likely infectious etiology. Transcriptome profiling is a powerful approach to investigate gene expression in diseased tissues. RNA sequencing of KD coronary arteries could elucidate the etiology and the host response, with the potential to improve KD diagnosis and/or treatment. METHODS: Deep RNA sequencing was performed on KD (n = 8) and childhood control (n = 7) coronary artery tissues, revealing 1074 differentially expressed mRNAs. Non-human RNA sequences were subjected to a microbial discovery bioinformatics platform, and microbial sequences were analyzed by Metastats for association with KD. RESULTS: T lymphocyte activation, antigen presentation, immunoglobulin production, and type I interferon response were significantly upregulated in KD arteritis, while the tumor necrosis factor α pathway was not differentially expressed. Transcripts from known infectious agents were not specifically associated with KD coronary arteritis. CONCLUSIONS: The immune transcriptional profile in KD coronary artery tissues has features of an antiviral immune response such as activated cytotoxic T lymphocyte and type I interferon-induced gene upregulation. These results provide new insights into the pathogenesis of KD arteritis that can guide selection of new immunomodulatory therapies for high-risk KD patients, and provide direction for future etiologic studies.

Kawasaki Disease has so much to teach us!

Kawasaki Disease (KD) is primarily a childhood vasculitis of mid-size muscular arteries, of which the coronary arteries (CA) are most clinically significant. Although timely treatment with pooled intravenous immunoglobulin (IVIG) has significantly reduced CA pathology, as determined by ECHO cardiology, about 30% of children still develop potentially fatal aneurysms, thrombi, or stenosis. This paper describes several additional pathologies and phenomena of undetermined significance, e.g. myocarditis, endocarditis with fibroelastosis, adrenal medullary necrosis, arterial smooth muscle cell transition into proliferating myofibroblasts, pneumonia in patients dying from KD, and calcification of organizing thrombi.

The so called "malignant fibrous histiocytoma" is actually an unusual fibrosarcoma.

The so-called "malignant fibrous histiocytoma (MFH)" has become progressively more enigmatic since entering the soft tissue tumor field, based on tissue culture studies performed almost 50 years ago. It inexplicably evolved from an exceedingly common soft tissue diagnosis into a problematic diagnosis. Because of the conundrum, clinicians require that "malignant fibrous histiocytoma", the name that they are familiar with, appears somewhere in the diagnosis. A recent review of the electron micrographs from 157 MFHs diagnosed over 32 years appears to explain how it was misnamed and its true identity, and as a consequence requires an updating of its clinico-pathologic correlation (CPC). It is an unusual fibroblastic entity that has the capacity to attract, activate, and stimulate fusion of normal macrophages.

The "myofibroblast" that is omnipresent in pathology and key to the EMT concepts does not actually exist, since normal fibroblasts contain stress fibril organelles (SMA bundles with dense bodies) variably detected by TEM and IHC: conclusions by a diagnostic pathologist with decades of ultrastructural experience.

The so-called "enigmatic" unique "myofibroblast" has been erroneously substituted for virtually all things fibroblastic in soft tissue pathology and believed to be the ultimate fibrogenic cell. It is also internationally considered to be the mesenchymal cell in un-proven post-natal EMT, EMT organ/tissue fibrosis, and the assumption that EMT/MET is key to carcinoma/adenocarcinoma invasion and metastasis. However, no such cell exists, having been mistaken for our normal ubiquitous fibrogenic fibroblasts that contain peripheral bundles of actin (SMA) with dense bodies, i.e. stress fibril (SF) organelles variably detectable by TEM and SMA IHC, depending on the degree of activation. The only detectable features distinguishing what are erroneously believed to be two unique fibrogenic spindle cells are the SF. Is the variable detection of SF/SMA in fibroblastic and non-fibroblastic lesions significant? Carcinosarcomas are not bi-phasic malignancies or proof of EMT/MET. What does it mean that the fibroblasts of so-called "carcinoma-associated fibroblasts (CAF)" are not "myofibroblasts"? The true myofibroblast is the ultrastructurally and functionally unique, terminally-differentiated, pathognomonic cell of physiologic wound-healing, which unfortunately has been confused with the activated fibroblast. This study fails to demonstrate any ultrastructural evidence that either normal epithelial (EMT) or carcinoma/adenocarcinoma cells can undergo reversible transition into mesenchymal cells (EMT/MET) under any circumstances. The SF/SMA-positive fibrogenic cell in organ/tissue fibrosis is the genetically up-regulated, activated fibroblast, which has no relationship to EMT. Are any of the innumerable biochemical factors/elements considered to be associated with this non-existent cell and its related processes related to the activated fibroblast? The conclusions are based on review of every electron micrograph taken during a 40-year career in diagnostic and research ultrastructural pathology, and by confirming that the published TEM figures of so-called "myofibroblasts", are actually of fibroblasts.

Integrins α4 and αM, collagen1A1, and matrix metalloproteinase 7 are upregulated in acute Kawasaki disease vasculopathy.

BACKGROUND: Kawasaki disease (KD) can result in fatal coronary artery (CA) aneurysms, especially if left untreated. Our recent studies of its vascular pathology revealed subacute/chronic vasculitis that begins early in the illness with the proliferation of smooth muscle cell-derived myofibroblasts in a complex extracellular matrix (ECM). We hypothesized that a dysregulation of specific ECM and adhesion molecules occurs in KD CAs. METHODS: Gene expression profiling for ECM and adhesion molecules was performed on six acute KD and eight control CAs using a targeted real-time PCR array approach. RESULTS: Integrins α4 and αM (ITGA4, ITGAM), collagen type I, α1 (COL1A1), and matrix metalloproteinase 7 (MMP7) were significantly upregulated in KD CAs as compared with controls. Immunohistochemistry with anti-ITGAM antibodies revealed expression on inflammatory cells within the CA wall in patients with KD but not in controls. CONCLUSION: Integrins ITGA4 and ITGAM are upregulated in KD vasculopathy, probably promoting inflammatory recruitment that stimulates smooth muscle cell transition to myofibroblasts and their proliferation. MMP7 probably enhances myofibroblast proliferation and luminal lesion expansion, and overexpression of COL1A1 may lead to CA stenosis. Identification of the molecular pathogenesis of KD vasculopathy may lead to the development of circulating biomarkers and to directed therapeutic interventions.

Modulation of innate host factors by Mycobacterium avium complex in human macrophages includes interleukin 17.

BACKGROUND: Although opportunistic infections due to Mycobacterium avium complex (MAC) have been less common since the introduction of highly active antiretroviral therapy, globally, human immunodeficiency virus-1 (HIV-1)-positive patients remain predisposed to these infections. Absence of a properly functioning acquired immune response allows MAC persistence within macrophages localized in lymph nodes coinfected with HIV and MAC. Although a deficiency in interferon γ appears to play a part in the ability of MAC to deflect the macrophage-associated antimicrobial attack, questions about this process remain. Our study examines the ability of MAC to regulate interleukin 17 (IL-17), a proinflammatory cytokine involved in host cell recruitment. METHODS: Coinfected lymph nodes were examined for IL-17 by immunohistochemical analysis. In vitro, macrophages exposed to mycobacteria were evaluated for transcription activities, proteins, and signaling pathways responsible for IL-17 expression. Infected macrophages were also analyzed for expression of interleukin 21 (IL-21) and negative regulators of immune responses. RESULTS: Infection of macrophages triggered synthesis of IL-17, correlating with IL-17 expression by macrophages in coinfected lymph nodes. Infected macrophages exposed to exogenous IL-17 expressed CXCL10, which favors recruitment of new macrophages as targets for infection. Blockade of nuclear factor κ-light-chain-enhancer of activated B cells and mitogen-activated protein kinase pathways suppressed mycobacteria-induced IL-17 expression. MAC triggered expression of IL-21, IRF4, and STAT3 genes related to IL-17 regulation, as well as expression of the negative immunoregulators CD274(PD-L1) and suppressors of cytokine signaling. CONCLUSIONS: MAC-infected macrophages can provide an alternative source for IL-17 that favors accumulation of new targets for perpetuating bacterial and viral infection while suppressing host antimicrobial immune responses.

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

BACKGROUND: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. METHODS: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls. RESULTS: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. CONCLUSIONS: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.

Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a "new" virus associated with Kawasaki disease.

BACKGROUND: Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent. METHODS: Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung. RESULTS: An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available. CONCLUSIONS: KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.

Primary hepatocyte culture supports hepatitis C virus replication: a model for infection-associated hepatocarcinogenesis.

UNLABELLED: Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. CONCLUSION: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease.

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells.

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.

PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity.

Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY-->SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.

Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.

Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings have implications for pathogenesis of both HIV disease and atherosclerosis, because they reveal the role of cholesterol efflux impairment in HIV infectivity and suggest a possible mechanism by which HIV infection of macrophages may contribute to increased risk of atherosclerosis in HIV-infected patients.

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

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.

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1.

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.

Cerebellar medulloblastoma in the elderly.

Although medulloblastoma is the most common central nervous system malignancy in children, cases are much less common in adults. Moreover, this tumor is exceedingly rare in patients older than 65 years. Analysis of previous case reports reveals that medulloblastoma in the elderly is more commonly seen in males in a lateral location; histologically, medulloblastomas in aged individuals usually belong to the classic subtype. During intraoperative consultation, the pathologist should consider medulloblastoma in the differential diagnosis of a cerebellar mass in the elderly because cytologic features may overlap with metastatic small cell carcinoma or lymphoma. We present a case of medulloblastoma in a 66-year-old man and review the literature on the subject.

Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity.

Intestinal macrophages, which are thought to orchestrate mucosal inflammatory responses, have received little investigative attention compared with macrophages from other tissues. Here we show that human intestinal macrophages do not express innate response receptors, including the receptors for LPS (CD14), Fcalpha (CD89), Fcgamma (CD64, CD32, CD16), CR3 (CD11b/CD18), and CR4 (CD11c/CD18); the growth factor receptors IL-2 (CD25) and IL-3 (CD123); and the integrin LFA-1 (CD11a/CD18). Moreover, resident intestinal macrophages also do not produce proinflammatory cytokines, including IL-1, IL-6, IL-10, IL-12, RANTES, TGF-beta, and TNF-alpha, in response to an array of inflammatory stimuli but retain avid phagocytic and bacteriocidal activity. Thus, intestinal macrophages are markedly distinct in phenotype and function from blood monocytes, although intestinal macrophages are derived from blood monocytes. To explain this paradox, we show that intestinal stromal cell-derived products downregulate both monocyte receptor expression and, via TGF-beta, cytokine production but not phagocytic or bacteriocidal activity, eliciting the phenotype and functional profile of intestinal macrophages. These findings indicate a mechanism in which blood monocytes recruited to the intestinal mucosa retain avid scavenger and host defense functions but acquire profound "inflammatory anergy," thereby promoting the absence of inflammation characteristic of normal intestinal mucosa despite the close proximity of immunostimulatory bacteria.