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.

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.

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.

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.

HIV enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in HIV/Microsporidia-infected jejunal mucosa.

OBJECTIVES: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. DESIGN: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. METHODS: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. RESULTS: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. CONCLUSION: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.

Mycobacterium avium-induced SOCS contributes to resistance to IFN-gamma-mediated mycobactericidal activity in human macrophages.

Mycobacterium avium is an opportunistic pathogen that commonly infects individuals colonized with HIV-1, although it is less frequent in the post-HAART era. These microorganisms invade macrophages after interacting with TLR2 and/or CD14 co-receptors, but signaling pathways promoting survival in macrophages are not well defined. Although IFN-gamma plays an important role in protective immunity against bacterial infections, IFN-gamma responses are compromised in AIDS patients and evidence suggests that exogenous IFN-gamma is inadequate to clear the mycobacteria. To determine the mechanism by which M. avium survives intracellularly, even in the presence of IFN-gamma, we studied the effect of mycobacteria infection in macrophages during early IFN-gamma signaling events. M. avium infected cells exhibited a reduced response to IFN-gamma, with suppressed phosphorylation of STAT-1 compared with uninfected cells. Interaction of M. avium with macrophage receptors increased gene expression of the suppressors of cytokine signaling (SOCS) to diminish IFN responsiveness. Specifically, we observed an increase in mRNA for both SOCS-3 and SOCS-1, which correlates with elevated levels of SOCS protein and positive immunostaining in M. avium/HIV-1 co-infected tissues. We also linked the p38 MAPK signaling pathway to mycobacterial-induced SOCS gene transcription. The induction of SOCS may be part of the strategy that allows the invader to render the macrophages unresponsive to IFN-gamma, which otherwise promotes clearance of the infection. Our data provide new insights into the manipulation of the host response by this opportunistic pathogen and the potential for modulating SOCS to influence the outcome of M. avium infection in immunocompromised hosts.

The cooperative prostate cancer tissue resource: a specimen and data resource for cancer researchers.

PURPOSE: The Cooperative Prostate Cancer Tissue Resource (CPCTR) is a National Cancer Institute-supported tissue bank that provides large numbers of clinically annotated prostate cancer specimens to investigators. This communication describes the CPCTR to investigators interested in obtaining prostate cancer tissue samples. EXPERIMENTAL DESIGN: The CPCTR, through its four participating institutions, has collected specimens and clinical data for prostate cancer cases diagnosed from 1989 onward. These specimens include paraffin blocks and frozen tissue from radical prostatectomy specimens and paraffin blocks from prostate needle biopsies. Standardized histopathological characterization and clinical data extraction are performed for all cases. Information on histopathology, demography (including ethnicity), laboratory data (prostate-specific antigen values), and clinical outcome related to prostate cancer are entered into the CPCTR database for all cases. Materials in the CPCTR are available in multiple tissue formats, including tissue microarray sections, paraffin-embedded tissue sections, serum, and frozen tissue specimens. These are available for research purposes following an application process that is described on the CPCTR web site (www.prostatetissues.org). RESULTS: The CPCTR currently (as of October 2003) contains 5135 prostate cancer cases including 4723 radical prostatectomy cases. Frozen tissues, in some instances including patient serum samples, are available for 1226 cases. Biochemical recurrence data allow identification of cases with residual disease, cases with recurrence, and recurrence-free cases. CONCLUSIONS: The CPCTR offers large numbers of highly characterized prostate cancer tissue specimens, including tissue microarrays, with associated clinical data for biomarker studies. Interested investigators are encouraged to apply for use of this material (www.prostatetissues.org).

Viral and host cofactors facilitate HIV-1 replication in macrophages.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T lymphocytes leads to their progressive loss, whereas HIV-1-infected macrophages appear to resist HIV-1-mediated apoptotic death. The differential response of these two host-cell populations may be critical in the development of immunodeficiency and long-term persistence of the virus. Multiple contributing factors may favor the macrophage as a resilient host, not only supporting infection by HIV-1 but also promoting replication and persistence of this member of the lentivirus subfamily of primate retroviruses. An encounter between macrophages and R5 virus engages a signal cascade eventuating in transcriptional regulation of multiple genes including those associated with host defense, cell cycle, nuclear factor-kappaB regulation, and apoptosis. It is important that enhanced gene expression is transient, declining to near control levels, and during this quiescent state, the virus continues its life cycle unimpeded. However, when viral replication becomes prominent, an increase in host genes again occurs under the orchestration of viral gene products. This biphasic host response must fulfill the needs of the parasitic virus as viral replication activity occurs and leads to intracellular and cell surface-associated viral budding. Inroads into understanding how HIV-1 co-opts host factors to generate a permissive environment for viral replication and transmission to new viral hosts may provide opportunities for targeted interruption of this lethal process.

Pityriasis rosea is associated with systemic active infection with both human herpesvirus-7 and human herpesvirus-6.

Pityriasis rosea is a common skin disease that has been suspected to have a viral etiology. We performed nested polymerase chain reaction to detect human herpesvirus-7, human herpesvirus-6, and cytomegalovirus DNA in lesional skin, nonlesional skin, peripheral blood mononuclear cells, serum, and saliva samples isolated from 14 pityriasis rosea patients. Viral mRNA expression and virion visualization within lesional skin were studied by in situ hybridization and transmission electron microscopy, respectively. By nested polymerase chain reaction, human herpesvirus-7 DNA was present in lesional skin (93%), nonlesional skin (86%), saliva (100%), peripheral blood mononuclear cells (83%), and serum (100%) samples, whereas human herpesvirus-6 DNA was detected in lesional skin (86%), nonlesional skin (79%), saliva (80%), peripheral blood mononuclear cells (83%), and serum (88%) samples. By contrast, cytomegalovirus DNA was not detected in these tissues. Control samples from 12 healthy volunteers and 10 psoriasis patients demonstrated rare positivity for either human herpesvirus-7 or human herpesvirus-6 DNA in skin or serum. By in situ hybridization, infiltrating mononuclear cells expressing human herpesvirus-7 and human herpesvirus-6 mRNA were identified in perivascular and periappendageal areas in 100% and 75% pityriasis rosea skin lesions, respectively, compared to herpesviral mRNA positivity in only 13% normal skin and psoriasis skin controls. Transmission electron microscopy failed to reveal herpesviral virions in pityriasis rosea lesional skin. Nested polymerase chain reaction and in situ hybridization enabled detection of human herpesvirus-7 and human herpesvirus-6 in skin and other tissues isolated from patients with pityriasis rosea. These results suggest that pityriasis rosea is associated with systemic active infection with both human herpesvirus-7 and human herpesvirus-6.