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.

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.

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.

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.

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.

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.

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.

The AIDS and Cancer Specimen Resource.

The AIDS and Cancer Specimen Resource (ACSR) is a cooperative agreement among the United States National Cancer Institute (NCI) (Office of the Director, Office of HIV and AIDS Malignancy (OHAM)) and regional US consortia, University of California, San Francisco (West Coast), George Washington University (East Coast), and The Ohio State University (Mid-Region). The ACSR's main objective is to collect, preserve, and disperse HIV-related tissues and biologic fluids along with clinical data to qualified investigators with a focus on HIV/AIDS-related malignancies. The ACSR biorepository has more than 265,000 human HIV-positive and control samples available from 39 processing types, 16 specimen types, and 52 anatomical site types. These HIV-infected biological fluids and tissues are made available to funded approved investigators at no fee. Technical support such as HIV DNA identification in tissues and tissue microarray (TMA) blocks are available to assist approved investigators. Research needs may be filled through ACSR cooperative arrangements when not met by currently banked material. Those participating with the ACSR are expected to share their research findings with the scientific community. Some 117 abstract/poster and podium reports at national and international scientific meetings and 94 publications have been contributed to the scientific literature (as of 2010). Investigators can browse the ACSR Internet site at http://acsr.ucsf.edu for biospecimens to support their scientific initiatives, including basic, translational, biomarker discovery, and molecular epidemiology studies.

Opportunistic disorders of the gastrointestinal tract in the age of highly active antiretroviral therapy.

Since the 1996 advent of highly active antiretroviral therapy (HAART) for the treatment of HIV/AIDS, the incidence of most opportunistic disorders in the developed world has dramatically declined but definitely has not disappeared. The number of new yearly HIV infections (about 55,000) and the total number of US infections (more than 1.1 million) remain very significant. Post-HAART gastrointestinal (GI) symptoms and biopsy results are still common, especially in large inner-city hospitals. The same opportunistic GI disorders were diagnosed in 442 endoscopies performed since 1996 as before, but at about one half the rate. The esophagus had the highest rate of positive biopsy results (46%), especially due to Candida. Helicobacter pylori infection has become the most common gastric infection. The small bowel still showed cytomegalovirus (CMV), cryptosporidia, and Mycobacterium avium complex (MAC) infections. In decreasing order, the most common large bowel infections were CMV, cryptosporidiosis, MAC, and spirochetosis. Cases of adenovirus, bacterial colitis, Kaposi sarcoma, and lymphoma were still diagnosed. Rectal biopsy specimens were the least productive. Microsporidiosis is now being diagnosed with special stains. Thus, where HIV/AIDS is common, it is important to be able to diagnose these GI processes. In addition to presenting post-HAART incidences, diagnostic features and aids are described for selected entities.

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.

HIV as a risk factor for lung cancer in women: data from the Women's Interagency HIV Study.

PURPOSE: Prior reports of an increased risk of lung cancer in HIV-infected individuals have not always included control groups, nor considered other risk factors such as tobacco exposure. We sought to determine the role of HIV infection and highly active antiretroviral therapy (HAART) on lung cancer incidence in 2,651 HIV-infected and 898 HIV-uninfected women from the Women's Interagency HIV Study (WIHS). METHODS: A prospective study of the incidence rates of lung cancer was conducted, with cases identified through medical records, death certificates, and state cancer registries. Standardized incidence ratios (SIRs) were calculated to compare lung cancer incidence among HIV-infected and uninfected WIHS participants, with population-based expectations using the Surveillance, Epidemiology, and End Results registry. Behavioral characteristics in the WIHS were compared to US women by age and race adjusting the population-based data from the National Health and Nutritional Examination Survey (NHANES) III. RESULTS: Incidence rates of lung cancer were similar among HIV-infected and uninfected WIHS women. Lung cancer SIRs were increased in both HIV-infected and -uninfected women compared with population expectations, but did not differ by HIV status. Among HIV-infected women, lung cancer incidence rates were similar in pre-HAART and HAART eras. All WIHS women with lung cancer were smokers; the risk of lung cancer increased with cumulative tobacco exposure. WIHS women were statistically more likely to smoke than US women studied in NHANES III. CONCLUSION: HIV infection is strongly associated with smoking behaviors that increase lung cancer risk. The role of HIV itself remains to be clarified.

Evaluation of the pathogenesis of decreasing CD4(+) T cell counts in human immunodeficiency virus type 1-infected patients receiving successfully suppressive antiretroviral therapy.

Most human immunodeficiency virus (HIV)-infected individuals experience increases in peripheral CD4(+) T cell counts with suppressive antiretroviral therapy (ART) that achieves plasma HIV RNA levels that are less than the limit of detection. However, some individuals experience decreasing CD4(+) T cell counts despite suppression of plasma viremia. We evaluated 4 patients with a history of CD4(+) T cell decline despite successfully suppressive ART, from a median of 719 cells/mm(3) (range, 360-1141 cells/mm(3)) to 227 cells/mm(3) (range, 174-311 cells/mm(3)) over a period of 18-24 months; 3 of the patients were receiving tenofovir and didanosine, which may have contributed to this decrease. There was no evidence of HIV replication, nor of antiretroviral drug resistance in the blood or lymphoid tissue, or increased proliferation or decreased thymic production of naive CD4(+) T cells. All 4 patients had significant fibrosis of the T cell zone of lymphoid tissue, which appeared to be an important factor in the failure to reconstitute T cells.