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Lung tumor-promoting environmental exposures and γherpesvirus infections are associated with Type 17 inflammation. To test the effect of γherpesvirus infection in promoting lung tumorigenesis, we infected mutant K-Ras-expressing (K-Ras LA1 ) mice with the murine γherpesvirus MHV68 via oropharyngeal aspiration. After 7 weeks, the infected mice displayed a more than 2-fold increase in lung tumors relative to their K-Ras LA1 uninfected littermates. Assessment of cytokines in the lung revealed that expression of Type 17 cytokines ( Il-6 , Cxcl1 , Csf3 ) peaked at day 7 post-infection. These observations correlated with the post-infection appearance of known immune mediators of tumor promotion via IL-17A in the lungs of tumor-bearing mice. Surprisingly, Cd84 , an immune cell marker mRNA, did not increase in MHV68-infected wild-type mice lacking lung tumors. Csf3 and Cxcl1 protein levels increased more in the lungs of infected K-Ras LA1 mice relative to infected wild-type littermates. Flow cytometric and transcriptomic analyses indicated that the infected K-Ras LA1 mice had increased Ly6G dim /Ly6C hi immune cells in the lung relative to levels seen in uninfected control K-Ras LA1 mice. Selective methylation of adenosines (m 6 A modification) in immune-cell-enriched mRNAs appeared to correlate with inflammatory infiltrates in the lung. These observations implicate γherpesvirus infection in lung tumor promotion and selective accumulation of immune cells in the lung that appears to be associated with m 6 A modification of mRNAs in those cells.

In idiopathic pulmonary fibrosis (IPF), the normal delicate lung architecture is replaced with rigid extracellular matrix (ECM) as a result of the accumulation of activated myofibroblasts and excessive deposition of ECM. Lamins have a role in fostering mechanosignaling from the ECM to the nucleus. Although there is a growing number of studies on lamins and associated diseases, there are no prior reports linking aberrations in lamins with pulmonary fibrosis. Here, we discovered, through analysis of RNA sequencing data, a novel isoform of lamin A/C that is more highly expressed in IPF compared with control lung. This novel LMNA (lamin A/C) splice variant includes retained introns 10 and 11 and exons 11 and 12 as documented by rapid amplification of cDNA ends. We found that this novel isoform is induced by stiff ECM. To better clarify the specific effects of this novel isoform of lamin A/C and how it may contribute to the pathogenesis of IPF, we transduced the lamin transcript into primary lung fibroblasts and alveolar epithelial cells and found that it impacts several biological effects, including cell proliferation, senescence, cell contraction, and the transition of fibroblasts to myofibroblasts. We also observed that type II epithelial cells and myofibroblasts in the IPF lung exhibited wrinkled nuclei, and this is notable because this has not been previously described and is consistent with laminopathy-mediated cellular effects.

The Epstein-Barr virus (EBV) immediate early transactivator Zta plays a key role in regulating the transition from latency to the lytic replication stages of EBV infection. Regulation of Zta is known to be controlled through a number of transcriptional and posttranscriptional events. Here, we show that Zta is targeted for ubiquitin modification and that this can occur in EBV-negative and in EBV-infected cells. Genetic studies show critical roles for both an amino-terminal region of Zta and the basic DNA binding domain of Zta in regulating Zta ubiquitination. Pulse-chase experiments demonstrate that the bulk population of Zta is relatively stable but that at least a subset of ubiquitinated Zta molecules are targeted for degradation in the cell. Mutation of four out of a total of nine lysine residues in Zta largely abrogates its ubiquitination, indicating that these are primary ubiquitination target sites. A Zta mutant carrying mutations at these four lysine residues (lysine 12, lysine 188, lysine 207, and lysine 219) cannot induce latently infected cells to produce and/or release infectious virions. Nevertheless, this mutant can induce early gene expression, suggesting a possible defect at the level of viral replication or later in the lytic cascade. As far as we know, this is the first study that has investigated the targeting of Zta by ubiquitination or its role in Zta function. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen and associated with various human diseases. EBV undergoes latency and lytic replication stages in its life cycle. The transition into the lytic replication stage, at which virus is produced, is mainly regulated by the viral gene product, Zta. Therefore, the regulation of Zta function becomes a central issue regarding viral biology and pathogenesis. Known modifications of Zta include phosphorylation and sumoylation. Here, we report the role of ubiquitination in regulating Zta function. We found that Zta is subjected to ubiquitination in both EBV-infected and EBV-negative cells. The ubiquitin modification targets 4 lysine residues on Zta, leading to both mono- and polyubiquitination of Zta. Ubiquitination of Zta affects the protein's stability and likely contributes to the progression of viral lytic replication. The function and fate of Zta may be determined by the specific lysine residue being modified., (Copyright © 2020 American Society for Microbiology.)

We used a transcriptomic approach to interrogate the effects of a saline-accommodated fraction from the Macondo 252 well (MC252) oil and Corexit dispersants on lung tissue. Wild-type C57BL/6 male and female mice were exposed on days 0, 7 and 13 by oropharyngeal aspiration to saline accommodated fractions (SAF) of crude oil from the Macondo (MC252) well, Corexit 9500, Corexit 9527, 9500+oil and 9527+oil or a saline solution as the vehicle control. These treatments did not cause overt toxicity, with the exception of the Corexit exposures which caused brief weight loss after the first exposure. On day 14, total RNA was isolated from the left lung for RNA-seq analyses. KEGG-pathway-based differential expression revealed that Corexit 9527 elicited the strongest changes involving the upregulation of 19 KEGG pathways (FDR < 0.10), followed by Corexit 9500 with the upregulation of seven pathways (FDR < 0.10). As an important signature, pathways related to a response to DNA damage (e.g., p53 signaling and mismatch repair) dominate those upregulated by Corexit 9527 and Corexit 9500. In addition, pro-inflammatory pathways (e.g., cytokine-cytokine receptor interaction, IL-17 signaling pathway and TNF signaling pathways) were upregulated selectively in oil-treated male mice. Surprisingly, oil + dispersant combinations caused lesser effects than the individual treatments at the transcriptomic level. Overall, these findings support potential genotoxicity, inflammation and cell death due to dispersant or oil exposures. Similar exposures to lung tumor bearing K-Ras LA1 mice provided evidence for tumor promotion by oil and Corexit dispersant treatments. Our mouse RNA-seq analyses may be relevant to the pulmonary health hazards of MC252 oil and dispersants experienced in exposed populations.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive decline of lung function. Here, we tested the importance of differential proportions of blood immune cells to IPF clinical outcomes. We used Cibersort to deconvolute immune cell components based on PBMCs or whole blood IPF genomics datasets. We found that a higher proportion of resting memory (RM) T cells was associated with a better survival and a higher DLco (diffusing capacity for carbon monoxide) in IPF patients. The association was also found in opposite direction for monocytes. Additionally, in IPF patients as compared to healthy controls, proportions of monocytes were observed to be higher, yet RM T cells were observed to be lower. Taken together, our result suggests a beneficial effect of RM T cells and a detrimental effect of monocytes for IPF. Future genomics studies of IPF should be more focused on these two types of cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Previous investigations proposed a link between the Epstein-Barr virus (EBV) and lung cancer (LC), but the results are highly controversial largely due to the insufficient sample size and the inherent limitation of the traditional viral screening methods such as PCR. Unlike PCR, current next-generation sequencing (NGS) utilizes an unbiased method for the global assessment of all exogenous agents within a cancer sample with high sensitivity and specificity. In our current study, we aim to resolve this long-standing controversy by utilizing our unbiased NGS-based informatics approaches in conjunction with traditional molecular methods to investigate the role of EBV in a total of 1127 LC. In situ hybridization analysis of 110 LC and 10 normal lung samples detected EBV transcripts in 3 LC samples. Comprehensive virome analyses of RNA sequencing (RNA-seq) data sets from 1017 LC and 110 paired adjacent normal lung specimens revealed EBV transcripts in three lung squamous cell carcinoma and one lung adenocarcinoma samples. In the sample with the highest EBV coverage, transcripts from the BamHI A region accounted for the majority of EBV reads. Expression of EBNA-1, LMP-1 and LMP-2 was observed. A number of viral circular RNA candidates were also detected. Thus, we for the first time revealed a type II latency-like viral transcriptome in the setting of LC in vivo. The high-level expression of viral BamHI A transcripts in LC suggests a functional role of these transcripts, likely as long non-coding RNA. Analyses of cellular gene expression and stained tissue sections indicated an increased immune cell infiltration in the sample expressing high levels of EBV transcripts compared to samples expressing low EBV transcripts. Increased level of immune checkpoint blockade factors was also detected in the sample with higher levels of EBV transcripts, indicating an induced immune tolerance. Lastly, inhibition of immune pathways and activation of oncogenic pathways were detected in the sample with high EBV transcripts compared to the EBV-low LC indicating the direct regulation of cancer pathways by EBV. Taken together, our data support the notion that EBV likely plays a pathological role in a subset of LC.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disease, and fibroblast-myofibroblast differentiation (FMD) is thought to be a key event in the pathogenesis of IPF. Histone deacetylase-8 (HDAC8) has been shown to associate with α-smooth muscle actin (α-SMA; a marker of FMD) and regulates cell contractility in vascular smooth muscle cells. However, the role of HDAC8 in FMD or pulmonary fibrosis has never been reported. This study investigated the role of HDAC8 in pulmonary fibrosis with a focus on FMD. We observed that HDAC8 expression was increased in IPF lung tissue as well as transforming growth factor (TGF)β1-treated normal human lung fibroblasts (NHLFs). Immunoprecipitation experiments revealed that HDAC8 was associated with α-SMA in TGFβ1-treated NHLFs. HDAC8 inhibition with NCC170 (HDAC8-selective inhibitor) repressed TGFβ1-induced fibroblast contraction and α-SMA protein expression in NHLFs cultured in collagen gels. HDAC8 inhibition with HDAC8 siRNA also repressed TGFβ1-induced expression of profibrotic molecules such as fibronectin and increased expression of antifibrotic molecules such as peroxisome proliferator-activated receptor-γ (PPARγ). Chromatin immunoprecipitation quantitative PCR using an antibody against H3K27ac (histone H3 acetylated at lysine 27; a known HDAC8 substrate and a marker for active enhancers) suggested that HDAC8 inhibition with NCC170 ameliorated TGFβ1-induced loss of H3K27ac at the PPARγ gene enhancer. Furthermore, NCC170 treatment significantly decreased fibrosis measured by Ashcroft score as well as expression of type 1 collagen and fibronectin in bleomycin-treated mouse lungs. These data suggest that HDAC8 contributes to pulmonary fibrosis and that there is a therapeutic potential for HDAC8 inhibitors to treat IPF as well as other fibrotic lung diseases.

Both cigarette smoke (CS) and asbestos cause lung inflammation and lung cancer, and at high asbestos exposure levels, populations exposed to both of these carcinogens display a synergistic increase in the development of lung cancer. The mechanisms through which these two toxic agents interact to promote lung tumorigenesis are poorly understood. Here, we begin to dissect the inflammatory signals induced by asbestos in combination with CS using a rodent inhalation model and in vitro cell culture. Wild-type C57BL/6 mice were exposed to room air as a control, CS, and/or asbestos (4 days per week to CS and 1 day per week to asbestos for 5 weeks). Bronchoalveolar lavage (BAL) fluid was collected following exposure and analyzed for inflammatory mediators. Asbestos-exposed mice displayed an increased innate immune response consistent with NLRP3 inflammasome activation. Compared to mice exposed only to asbestos, animals coexposed to CS + asbestos displayed attenuated levels of innate immune mediators and altered inflammatory cell recruitment. Histopathological changes in CS + asbestos-exposed mice correlated with attenuated fibroproliferative lesion development relative to their counterparts exposed only to asbestos. In vitro experiments using a human monocyte cell line (THP-1 cells) supported the in vivo results in that coexposure to cigarette smoke extract repressed NLRP3 inflammasome markers in cells treated with asbestos. These observations indicate that CS represses central components of the innate immune response to inhaled asbestos., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Recent findings demonstrate that inhaled cigarette smoke, the predominant lung carcinogen, elicits a T helper 17 (Th17) inflammatory phenotype. Interleukin-17A (IL-17), the hallmark cytokine of Th17 inflammation, displays pro- and antitumorigenic properties in a manner that varies according to tumor type and assay system. To investigate the role of IL-17 in lung tumor growth, we used an autochthonous tumor model (K-Ras(LA1) mice) with lung delivery of a recombinant adenovirus that expresses IL-17A. Virus-mediated expression of IL-17A in K-Ras(LA1) mice at 8-10 wk of age doubled lung tumor growth in 3 wk relative to littermates that received a green fluorescent protein-expressing control adenovirus. IL-17 induced matrix metalloproteinase-9 (MMP-9) expression in vivo and in vitro. In accord with this finding, selective and specific inhibitors of MMP-9 repressed the increased motility and invasiveness of IL-17-treated lung tumor cells in culture. Knockdown or mutation of p53 promoted the motility of murine lung tumor cells and abrogated the promigratory role of IL-17. Coexpression of siRNA-resistant wild-type, but not mutant, human p53 rescued both IL-17-mediated migration and MMP-9 mRNA induction in p53 knockdown lung tumor cells. IL-17 increased MMP-9 mRNA stability by reducing interaction with the mRNA destabilizing serine/arginine-rich splicing factor 1 (SRSF1). Taken together, our results indicate that IL-17 stimulates lung tumor growth and regulates MMP-9 mRNA levels in a p53- and SRSF1-dependent manner., (Copyright © 2014 the American Physiological Society.)

Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17-92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

Composition of extracellular matrix (ECM) is crucial to the establishment and maintenance of epithelial apical-basolateral polarity. Increased ECM rigidity caused by deposition of fibrillar collagen, for example, collagen type I (Col-1), promotes loss of epithelial polarity and tumor progression. microRNAs are small non-coding RNAs that regulate gene expression and fundamental cellular processes. The current study explored a link between microRNAs and Col-1 using organotypic three-dimensional culture in which epithelial cells are embedded within Matrigel, a mimic of basement membrane matrix (Matrigel 3-D). Matrigel 3-D culture of A549, MCF-7, and mK-ras-LE cells (lung and mammary epithelial cell lines) gave rise to acinus, an in vitro equivalent of apical-basolateral polarity that consists of a polarized monolayer of epithelial cells facing a central lumen. Supplementation of Col-1 disrupted acinus. Moreover, Col-1 up-regulated the expression of miR-21, a well-documented oncogenic microRNA, via a post-transcriptional mechanism. Similar post-transcriptional up-regulation of miR-21 correlated with deposition of Col-1 in a murine model of lung fibrogenesis. In summary, our findings link altered ECM composition/rigidity and the expression of oncogenic microRNAs. The current study also suggests a novel post-transcriptional mechanism for regulation of miR-21 expression at maturation from pre-miR-21 to mature miR-21., (Copyright © 2011 Wiley-Liss, Inc.)

Several studies have implicated gamma-herpesviruses, particularly Epstein-Barr virus (EBV), in the progression of idiopathic pulmonary fibrosis. The data presented here examine the possible role that EBV plays in the potentiation of this disease by evaluating the pulmonary response to expression of the EBV lytic transactivator protein Zta. Expression of Zta in the lungs of mice via adenovirus-mediated delivery (Adv-Zta) produced profibrogenic inflammation that appeared most pronounced by day 7 postexposure. Relative to mice exposed to control GFP-expressing adenovirus (Adv-GFP), mice exposed to Adv-Zta displayed evidence of lung injury and a large increase in inflammatory cells, predominantly neutrophils, recovered by bronchoalveolar lavage (BAL). Cytokine and mRNA profiling of the BAL fluid and cells recovered from Adv-Zta-treated mice revealed a Th2 and Th17 bias. mRNA profiles from Adv-Zta-infected lung epithelial cells revealed consistent induction of mRNAs encoding Th2 cytokines. Coexpression in transient assays of wild-type Zta, but not a DNA-binding-defective mutant Zta, activated expression of the IL-13 promoter in lung epithelial cells, and detection of IL-13 in Adv-Zta-treated mice correlated with expression of Zta. Induction of Th2 cytokines in Zta-expressing mice corresponded with alternative activation of macrophages. In cell culture and in mice, Zta repressed lung epithelial cell markers. Despite the profibrogenic character at day 7, the inflammation resolves by 28 days postexposure to Adv-Zta without evidence of fibrosis. These observations indicate that the EBV lytic transactivator protein Zta displays activity consistent with a pathogenic role in pulmonary fibrosis associated with herpesvirus infection.

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1-) subsets of mouse embryonic fibroblasts (MEF). TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK) only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

This Commentary discusses the role of NFkappaB signaling in lung inflammation and fibrosis.

Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.

The chemotherapeutic drug bleomycin causes DNA damage and apoptosis in the lungs of mice within hours of endotracheal instillation followed by inflammation and fibrosis weeks later. The p53 tumor suppressor protein mediates cellular responses to DNA damage, including induction of apoptosis, but the effects of p53 activation in the various cell types of the lung during bleomycin-induced pulmonary fibrosis remain unclear. We show here that a transgene with a dominant-negative mutant form of human p53 expressed from the surfactant protein C promoter sensitizes mice to bleomycin-induced lung injury. The bleomycin-exposed transgenic animals display more severe lung pathology with associated collagen deposition and more pronounced lung eosinophilia than simultaneously exposed nontransgenic littermates. These observations suggest that compromising p53 function in the alveolar epithelium impairs recovery of the lung from bleomycin-induced injury.

Cricothyrotomy is indicated for patients who require an immediate airway and in whom orotracheal or nasotracheal intubation is unsuccessful or contraindicated. Cricothyrotomy is considered safe with cervical spine (c-spine) injury; however, the amount of c-spine movement that occurs during the procedure has not been determined. In this experimental study, an established cadaver model of c-spine injury was used to quantify movement during cricothyrotomy. A complete C5--6 transection was performed by using an osteotome on 13 fresh-frozen cadavers. Standard open cricothyrotomy was performed on each cadaver, with c-spine images recorded in real time on fluoroscopy, then transferred to video and Kodachrome still images. Outcome measures included movement across the C5--6 site with regard to angulation expressed in degrees of rotation and linear measures of axial distraction and anterior-posterior (AP) displacement expressed as a proportion of C5 body width. Data were analyzed by using descriptive statistics to determine mean change from baseline in each of three planes of movement. Significance was assumed if 95% confidence intervals did not include zero. A significant amount of movement was observed with regard to AP displacement (6.3% of C5 width) and axial distraction (-4.5% of C5 width, indicating narrowing of the intervertebral space). These correspond to 1--2 mm AP displacement and less than 1 mm axial compression. No significant angular displacement was observed. In conclusion, cricothyrotomy results in a small but significant amount of movement across an unstable c-spine injury in a cadaver model. This degree of movement is less than the threshold for clinical significance.

Study Objective: Orotracheal intubation (OTI) is commonly used to establish a definitive airway in major trauma victims, with several different cervical spine immobilization techniques and laryngoscope blade types used. This experimental, randomized, crossover trial evaluated the effects of manual in-line stabilization and cervical collar immobilization and 3 different laryngoscope blades on cervical spine movement during OTI in a cadaver model of cervical spine injury., Methods: A complete C5-C6 transection was performed by using an osteotome on 14 fresh-frozen cadavers. OTI was performed in a randomized crossover fashion by using both immobilization techniques and each of 3 laryngoscope blades: the Miller straight blade, the Macintosh curved blade, and the Corazelli-London-McCoy hinged blade. Intubations were recorded in real time on fluoroscopy and then transferred to video and color still images. Outcome measures included movement across C5-C6 with regard to angulation expressed in degrees of rotation and axial distraction and anteroposterior displacement with values expressed as a proportion of C5 body width. Cormack-Lehane visualization grades were also recorded as a secondary outcome measure. Data were analyzed by using multivariate analysis of variance to test for differences between immobilization techniques and between laryngoscope blades and to detect for interactions. Significance was assumed for P values of less than.05., Results: Manual in-line stabilization resulted in significantly less movement than cervical collar immobilization during OTI with regard to anteroposterior displacement. Use of the Miller straight blade resulted in significantly less movement than each of the other 2 blades with regard to axial distraction. The Cormack-Lehane grade was significantly better with manual in-line stabilization versus cervical collar immobilization; no differences were observed between blades., Conclusion: Manual in-line stabilization results in less cervical subluxation and allows better vocal cord visualization during OTI in a cadaver model of cervical spine injury. The Miller laryngoscope blade allowed less axial distraction than the Macintosh or Corzelli-London-McCoy blades. The clinical significance of this degree of movement is unclear.

Introduction: Intensive care treatment of patients with severe head injury is aimed at preventing secondary injury. One of the cornerstones in this treatment is sedation and ventilation. Use of Neuromuscular Blocking Agents (NBA) has gained widespread use as part of the protocol for maintaining normal intracranial pressure values, without class 1 evidence for the efficacy of the treatment., Methods: We examined data of the use of NBA as infusion during ventilator treatment, and IntraCranial Pressure (ICP) measurements in the database from the international multicenter randomized double blind trial of the NMDA receptor antagonist Selfotel. No specific mode of sedation was recommended in the study protocol., Results: Of the 427 patients enrolled in the study 326 had a full data set, 138 received NBA during their stay in the ICU. There were no statistical difference in demographic data between the two groups. During their stay in the ICU, patients who received NBA had a median of 13.5 hours with a recorded ICP above 20 mm Hg, patients who did not receive NBA had a median of 6.5 hours with ICP above 20 mm Hg (p < 0.05)., Conclusion: Our data challenges the concept of using NBA as part of a routine sedation strategy in treatment of patients with severe head injury.

Object: Recently, a renewed emphasis has been placed on managing severe head injury by elevating cerebral perfusion pressure (CPP), which is defined as the mean arterial pressure minus the intracranial pressure (ICP). Some authors have suggested that CPP is more important in influencing outcome than is intracranial hypertension, a hypothesis that this study was designed to investigate., Methods: The authors examined the relative contribution of these two parameters to outcome in a series of 427 patients prospectively studied in an international, multicenter, randomized, double-blind trial of the N-methyl-D-aspartate antagonist Selfotel. Mortality rates rose from 9.6% in 292 patients who had no clinically defined episodes of neurological deterioration to 56.4% in 117 patients who suffered one or more of these episodes; 18 patients were lost to follow up. Correspondingly, favorable outcome, defined as good or moderate on the Glasgow Outcome Scale at 6 months, fell from 67.8% in patients without neurological deterioration to 29.1% in those with neurological deterioration. In patients who had clinical evidence of neurological deterioration, the relative influence of ICP and CPP on outcome was assessed. The most powerful predictor of neurological worsening was the presence of intracranial hypertension (ICP > or = 20 mm Hg) either initially or during neurological deterioration. There was no correlation with the CPP as long as the CPP was greater than 60 mm Hg., Conclusions: Treatment protocols for the management of severe head injury should emphasize the immediate reduction of raised ICP to less than 20 mm Hg if possible. A CPP greater than 60 mm Hg appears to have little influence on the outcome of patients with severe head injury.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

Object: Excessive activity of excitatory amino acids released after head trauma has been demonstrated to contribute to progressive injury in animal models and human studies. Several pharmacological agents that act as antagonists to the glutamate receptor have shown promise in limiting this progression. The efficacy of the N-methyl-D-aspartate receptor antagonist Selfotel (CGS 19755) was evaluated in two parallel studies of severely head injured patients, defined as patients with post resuscitation Glasgow Coma Scale scores of 4 to 8., Methods: A total of 693 patients were prospectively enrolled in two multicenter double-blind studies. Comparison between the treatment groups showed no significant difference with regard to demographic data, previous incidence of hypotension, and severity of injury. As the study progressed, the Safety and Monitoring Committee became concerned about possible increased deaths and serious brain-related adverse events in the treatment arm of the two head injury trials, as well as deaths in the two stroke trials being monitored concurrently. The Selfotel trials were stopped prematurely because of this concern and because an interim efficacy analysis indicated that the likelihood of demonstrating success with the agent if the studies had been completed was almost nil., Conclusions: Subsequently, more complete data analysis revealed no statistically significant difference in mortality rates in all cases between the two treatment groups in the head injury trials. In this report the authors examine the studies in detail and discuss the potential application of the data to future trial designs.

Because of its expression pattern and its potent effects on mesenchymal cells, platelet-derived growth factor (PDGF) has been implicated as an important factor in epithelial-mesenchymal cell interactions during normal lung development and in the pathogenesis of fibrotic lung disease. To further explore the role of PDGF in these processes, we have developed transgenic mice that express the PDGF-B gene from the lung-specific surfactant protein C (SPC) promoter. Adult SPC-PDGFB transgenic mice exhibited lung pathology characterized by enlarged airspaces, inflammation, and fibrosis. Emphysematous changes frequently occurred throughout the lung, but inflammation and fibrotic lesions were usually confined to focal areas. The severity of this phenotype varied significantly among individual mice within the same SPC-PDGFB transgenic lineage. A pathology similar to that observed in adult mice was noted in lungs from transgenic mice as young as 1 week of age. Neonatal transgenic mice exhibited enlarged saccules and thickened primary septa. Results of these studies indicated that overexpression of PDGF-B induced distinct abnormalities in the developing and adult lung and led to a complex phenotype that encompassed aspects of both emphysema and fibrotic lung disease.

Human immunodeficiency virus type 1 (HIV-1) Tat transcriptionally activates expression from a number of viral and cellular promoters. Recent studies demonstrate the ability of Tat to differentially modulate cellular responses to apoptotic signaling. The antiapoptotic effects of Tat appear to correlate with increased expression of Bcl-2, a cellular protein that enhances cellular survival. Here, endogenous expression of HIV-1 Tat in HeLa and Jurkat cells elevates levels of Bcl-2. Transient expression assays performed in HeLa cells demonstrate that Tat directly or indirectly enhances Bcl-2 promoter-directed gene expression by more than 10-fold. Analyses of Tat mutants demonstrate that two noncontiguous regions in the N- and C-termini of Tat mediate maximal transactivation of the Bcl-2 promoter. The requirement for C-terminal sequences contrasts with transactivation of the HIV-1 long terminal repeat in which the N-terminal 57 amino acids are required but downstream residues are not. Bcl-2 promoter analyses suggest that sequences required for Tat responsiveness are located upstream of P1 and between the P1 and P2 promoter units. Results from these studies reveal effects of HIV-1 Tat on Bcl-2 expression and provide a putative mechanism by which endogenously expressed Tat affects cellular survival through the up-regulation of Bcl-2., (Copyright 1999 Academic Press.)

The proliferating cell nuclear antigen (PCNA) is a highly conserved cellular protein that functions both in DNA replication and in DNA repair. Exposure of a rat embryo fibroblast cell line (CREF cells) to gamma radiation induced simultaneous expression of PCNA with the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). PCNA mRNA levels transiently increased in serum-starved cells exposed to ionizing radiation, an observation suggesting that the radiation-associated increase in PCNA expression could be dissociated from cell cycle progression. Irradiation of CREF cells activated a transiently expressed PCNA promoter chloramphenicol acetyltransferase construct through p53 binding sequences via a mechanism blocked by a dominant negative mutant p53. Electrophoretic mobility shift assays with nuclear extracts prepared from irradiated CREF cells produced four p53-specific DNA-protein complexes with the PCNA p53 binding site. Addition of monoclonal antibody PAb421 (p53-specific) or AC238 (specific to the transcriptional coactivator p300/CREB binding protein) to the mobility shift assay distinguished different forms of p53 that changed in relative abundance with time after irradiation. These findings suggest a complex cellular response to DNA damage in which p53 transiently activates expression of PCNA for the purpose of limited DNA repair. In a population of nongrowing cells with diminished PCNA levels, this pathway may be crucial to survival following DNA damage.

Neurologic deterioration observed following head injury is recognized as having a deleterious effect on outcome. The present study examines this occurrence in detail to determine the frequency of these episodes, their antecedent events and causal relationships in order to identify patients who are at risk. Data was collected prospectively from a consecutive series of 427 patients entered into the international trial of the NMDA receptor antagonist Selfotel. Using a definition of neurologic worsening based upon objective criteria, 117 patients were identified who suffered 164 episodes of deterioration. The occurrence of a single episode of neurologic worsening increased mortality by more than five-fold and reduced favorable outcomes (good or moderate on the Glasgow Outcome Scale), by more than 50%. Increased intracranial volume resulting in intracranial hypertension was the single most frequent cause of neurologic worsening. This serves to emphasize the importance of more adequate treatments of intracranial hypertension in improving the outcome of patients with severe head injury.

Objective: A recently improved understanding of the pathophysiological features of head injuries has led to the development of new drug therapies. Accurate human clinical trials remain necessary to document the efficacy and safety of new agents. It would be helpful to decrease the time from drug development to clinical use and general availability for drugs found to be effective. Conversely, ineffective agents could be abandoned in a timely fashion., Rationale: A new endpoint measure, defined as neuroworsening (NW), is an objective observable event that is identifiable during hospitalization. This may enable the efficacy of drugs to be demonstrated or disproved much earlier than with 6-month outcome assessments. The prospective, double-blind, multicenter trial of the N-methyl-D-aspartate receptor antagonist Selfotel was used to acquire data on the efficacy of NW in predicting neurological outcomes. The 6-month Glasgow Outcome Scale scores, which were the primary endpoints of that trial, were compared with the frequency of NW. NW was an observable event that could be objectively defined after head injuries. Patients who suffered one or more episodes of NW demonstrated significantly higher morbidity and mortality rates than did patients who did not., Conclusion: Future trials should consider the use of NW as an outcome measure that can be included with more traditional measures in the study design. If the strong correlation demonstrated between NW and 6-month Glasgow Outcome Scale scores can be prospectively demonstrated in a successful trial, the time to approval of future agents could be decreased.

Object: The authors prospectively studied the efficacy of tirilazad mesylate, a novel aminosteroid, in humans with head injuries., Methods: A cohort of 1120 head-injured patients received at least one dose of study medication (tirilazad or placebo). Eighty-five percent (957) of the patients had suffered a severe head injury (Glasgow Coma Scale [GCS] score 4-8) and 15% (163) had sustained a moderate head injury (GCS score 9-12). Six-month outcomes for the tirilazad- and placebo-treated groups for the Glasgow Outcome Scale categories of both good recovery and death showed no significant difference (good recovery in the tirilazad-treated group was 39% compared with the placebo group in which it was 42% [p=0.461]; death in the tirilazad-treated group occurred in 26% of patients compared with the placebo group, in which it occurred in 25% [p=0.750]). Subgroup analysis suggested that tirilazad mesylate may be effective in reducing mortality rates in males suffering from severe head injury with accompanying traumatic subarachnoid hemorrhage (death in the tirilazad-treated group occurred in 34% of patients; in the placebo group it occurred in 43% [p=0.026]). No significant differences in frequency or types of serious adverse events were shown between the treatment and placebo groups., Conclusions: Striking problems with imbalance concerning basic prognostic variables were observed in spite of the large population studied. These imbalances concerned pretreatment hypotension, pretreatment hypoxia, and the incidence of epidural hematomas. In future trials of pharmacological therapy for severe head injury, serious consideration must be given to alternative randomization strategies. Given the heterogeneous nature of head injury and the identification of populations that do relatively well with standard therapy, target populations with a higher risk for mortality and morbidity may be more suitable for clinical trials of such agents.

Object: Congenital spinal hamartomas are defined as tumors of well-differentiated mature elements situated in an abnormal location. In this report, the authors document the clinical and pathological features of spinal hamartomas in 10 patients., Methods: Ten patients presented with midline dorsal malformations at birth, initially diagnosed as teratomas or myelomeningoceles. The locations of the masses were variable: two were located in the thoracic region, four at the thoracolumbar junction, two in the lumbar region, one at the lumbosacral junction, and one in the sacral region. The results of the neurological examination were normal in nine patients. All but one mass had intact skin and seven had palpable bone components. Neuroimaging studies revealed widening of the spinal canal, heterotopic bone located dorsally in some patients, and varying degrees of involvement of the intraspinal contents. During surgery, six patients were found to have involvement of the spinal cord or cauda equina. The pathological characteristics of the masses included three or more of the following: bone, cartilage, synovial membrane, urinary tract tissue, cyst wall, yellow or brown fat, and nerves. The well-differentiated cellular elements, which formed mature structures, along with the absence of primitive cellular components and neoplastic characteristics are more consistent with a diagnosis of hamartoma than teratoma., Conclusions: In this series, the authors describe a lesion that is overt on physical examination, yet can have occult spinal canal involvement. Complete neurosurgical evaluation is essential to provide appropriate treatment and prognosis.

Lung cancer is the most frequent cause of cancer deaths in the United States. A strong correlation exists between mutations in the gene encoding the p53 tumor suppressor protein and lung malignancies. Our goal is to prepare a transgenic mouse model with disrupted p53 function in the epithelial cells of the peripheral lung. To achieve this goal, a "dominant negative" mutant form of p53 was expressed from the human surfactant protein C (SPC) promoter. The dominant negative p53 expressed from the SPC promoter will antagonize wild-type p53 functions in alveolar type II pneumocytes and some bronchiolar cells of the transgenic animals and thereby promote development of carcinoma of the lung. This animal model should prove useful to the study of lung carcinogenesis and to the identification of agents that contribute to neoplastic conversion in the lung.

Objective: To assess the efficacy and safety of a new atraumatic, self-retaining cranial nerve electrode for direct cochlear nerve monitoring during cerebellopontine angle surgery., Study Design: Prospective clinical investigation., Setting: The Skull Base Surgery Center at Kaiser Permanente, San Diego, a tertiary referral center for neurotologic and skull-base surgery within Southern California Permanente Medical Group., Patients: Eighteen patients, with aidable preoperative hearing, underwent direct cochlear nerve monitoring with this new electrode during cerebellopontine angle surgery for a variety of diagnoses., Methods: Intraoperative observations of cochlear nerve action potential amplitude and latency were recorded. Preoperative and 1-month postoperative audiograms were compared to assess the degree of hearing preservation. Postoperative facial nerve function was assessed using the House-Brackmann method., Results: Good auditory function was preserved in four of eight acoustic tumors, with poor hearing preserved in two additional patients. Good auditory function was preserved in the remaining ten patients. Cochlear nerve action potential amplitudes between 5 and 70 microV were recorded. Postoperative facial nerve function was House-Brackmann class I-II in all 18 patients., Conclusion: The authors find this new electrode to be safe and effective for monitoring cochlear nerve function during cerebellopontine angle surgery.

Objective: This study investigated the use of a continuous parasacral sciatic nerve block for anesthesia and postoperative analgesia for lower extremity surgery., Methods: A continuous parasacral sciatic nerve block was performed in two patients (triple ankle arthrodesis and below-knee amputation). The sacral plexus was identified using an insulated Tuohy needle and a nerve stimulator. A catheter was placed near the elements of the sacral plexus via the Tuohy needle., Results: In both patients, surgical anesthesia was successfully established through the parasacral catheter with lidocaine 1% (1/200,000 epinephrine), and postoperative analgesia was successfully established with a bolus of bupivacaine 0.375% (1/200,000 epinephrine) and maintained with a continuous infusion of bupivacaine 0.1% (8 mL/h) for 48 hours., Conclusion: We conclude that continuous parasacral sciatic nerve block can provide anesthesia and long-term analgesia for operative procedures of the foot and leg.

Tat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kappa B elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of I kappa B alpha, I kappa B alpha delta N, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-kappa B to kappa B enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and -distal transcripts derived from TAR-deleted and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the lavel of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-kappa B-binding sites in the HIV-1 LTR.

The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to approximately 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.

HIV-1 Tat is a potent transactivator that stimulates expression from the HIV-1 LTR, from certain cellular gene promoters and from several heterologous viral promoters. Previous reports show that HIV-1 Tat transactivates tumor necrosis factor-beta (TNF-beta) promoter-directed gene expression in lymphocytic and monocytic cell lines and further demonstrate that a 'TAR-like structure' downstream of the TNF-beta promoter is essential for Tat activity. The ability of Tat to activate TNF-beta may have profound effects as TNF has been shown to be a potent activator of HIV-1 gene expression and an important immunomodulatory and growth regulatory factor. The studies presented herein demonstrate a novel finding where HIV-1 Tat specifically represses (> 10-fold) TNF-beta promoter-directed gene expression in central nervous system-derived glial cells. Amino acid residues 2 to 36 of HIV-1 Tat are required for TNF-beta repression. Tat repression of TNF-beta, a factor which upregulates HIV-1 gene expression, suggests a novel mechanism whereby HIV-1 is able to establish latent infection of glial cells that present no detectable virions and/or viral antigens.