BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes.

Patients with asthma experience circadian variations in their symptoms. However it remains unclear how specific aspects of this common airway disease relate to clock genes, which are critical to the generation of circadian rhythms in mammals. Here, we used a viral model of acute and chronic airway disease to examine how circadian clock disruption affects asthmatic lung phenotypes. Deletion of the core clock gene bmal1 or environmental disruption of circadian function by jet lag exacerbated acute viral bronchiolitis caused by Sendai virus (SeV) and influenza A virus in mice. Post-natal deletion of bmal1 was sufficient to trigger increased SeV susceptibility and correlated with impaired control of viral replication. Importantly, bmal1-/- mice developed much more extensive asthma-like airway changes post infection, including mucus production and increased airway resistance. In human airway samples from two asthma cohorts, we observed altered expression patterns of multiple clock genes. Our results suggest a role for bmal1 in the development of asthmatic airway disease via the regulation of lung antiviral responses to common viral triggers of asthma.

Circulating TSLP associates with decreased wheezing in non-atopic preschool children: data from the URECA birth cohort.

BACKGROUND: Mouse models of atopic march suggest that systemic, skin-derived thymic stromal lymphopoietin (TSLP) mediates progression from eczema to asthma. OBJECTIVE: We investigated whether circulating TSLP is associated with eczema, allergic sensitization, or recurrent wheezing in young children. METHODS: A prospective analysis of the relationship between plasma levels of TSLP to allergic sensitization and recurrent wheezing was conducted in the birth cohort from the Urban Environment and Childhood Asthma (URECA) study. Plasma TSLP levels were measured at 1, 2, and 3 years of age and analysed for correlation with clinical parameters in each of the three years. Only those children with consecutive samples for all three years were included in this analysis. RESULTS: We detected TSLP in 33% of 236 children for whom plasma samples were available for all three years. Overall, a consistently significant association was not found between TSLP and eczema or allergic sensitization. With regard to recurrent wheezing, children with detectable TSLP at one year of age were significantly less likely to experience recurrent wheezing by 3 years compared with those children without detectable TSLP, but this was only seen in children without aeroallergen sensitization at 3 years (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to our expectations, circulating TSLP was not significantly associated with eczema, allergen sensitization, or recurrent wheezing during the first three years of life. Early presence of circulating TSLP was significantly associated with reduced incidence of recurrent wheeze in those children not sensitized to aeroallergen. These findings suggest a possible underlying distinction between pathogenesis of developing atopic vs. non-atopic recurrent wheeze.

Consensus guidelines from The American Society of Peritoneal Surface Malignancies on standardizing the delivery of hyperthermic intraperitoneal chemotherapy (HIPEC) in colorectal cancer patients in the United States.

BACKGROUND: The American Society of Peritoneal Surface Malignancies (ASPSM) is a consortium of cancer centers performing cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC). This is a position paper from the ASPSM on the standardization of the delivery of HIPEC. METHODS: A survey was conducted of all cancer centers performing HIPEC in the United States. We attempted to obtain consensus by the modified method of Delphi on seven key HIPEC parameters: (1) method, (2) inflow temperature, (3) perfusate volume, (4) drug, (5) dosage, (6) timing of drug delivery, and (7) total perfusion time. Statistical analysis was performed using nonparametric tests. RESULTS: Response rates for ASPSM members (n = 45) and non-ASPSM members (n = 24) were 89 and 33 %, respectively. Of the responders from ASPSM members, 95 % agreed with implementing the proposal. Majority of the surgical oncologists favored the closed method of delivery with a standardized dual dose of mitomycin for a 90-min chemoperfusion for patients undergoing cytoreductive surgery for peritoneal carcinomatosis of colorectal origin. CONCLUSIONS: This recommendation on a standardized delivery of HIPEC in patients with colorectal cancer represents an important first step in enhancing research in this field. Studies directed at maximizing the efficacy of each of the seven key elements will need to follow.

Motion monitoring in palliative care using unobtrusive bed sensors.

Palliative care needs are growing with the aging population. Ambient sensors offer patients comfortable and discreet point-of-care monitoring. In this study, two palliative care participants were monitored in a sensorized bed. Motion monitoring by a two-tier gross and fine movement detector provided accurate detection and classification of movement, compared to annotations by an observer. However, ascribing the motion to the patient rather than caregivers or visitors would require supplemental sensors. Motion was indicative of pain, with 13% of time spent moving while in pain versus 3% while not noted as in pain.

A phase I trial of isolated hepatic perfusion (IHP) using 5-FU and oxaliplatin in patients with unresectable isolated liver metastases from colorectal cancer.

BACKGROUND: Isolated hepatic perfusion (IHP) with melphalan is an established approach for patients with unresectable metastatic liver lesions. This study determined the safety and maximum tolerated dose (MTD) of 5-FU with oxaliplatin via IHP. METHODS: Standard 3 × 3 Phase I design. Subjects with unresectable isolated CRC liver metastases scheduled for HAI pump were eligible. IHP used fixed-dose oxaliplatin with escalating 5-FU doses. Toxicity (CTCAE v 4.0) and response (RECIST), progression-free survival, and overall survival (OS) were assessed. Systemic and IHP plasma PK of 5-FU, anabolites, and platinum were determined. RESULTS: All 12 patients had received ≥ 1 line of pre-IHP chemotherapy. There were 4 grade 3 serious adverse events (33.3 %) and 1 grade 4 event (8.3 %). Also, 2 dose-limiting toxicities occurred at DL2 at 300 mg/m(2), resulting in expansion of DL1 at 200 mg/m(2) 5-FU, the eventual MTD. At 6-month follow-up, 9 patients (82 %) demonstrated partial response, while 2 (18 %) exhibited stable disease. Also, 64 % of patients demonstrated a >50 % decrease in CEA. The 1- and 2-year OS probabilities were 90.9 and 71.6 %, respectively, with median follow-up of 24 months. IHP exposures (AUC0-60 min) were 10.9 ± 4.5 µgPt h/mL, 49.3 ± 30.7 µg h/mL 5-FU (DL1), and 70.5 ± 35.5 µg h/mL 5-FU (DL2). Systemic exposure (AUC0-inf) relative to IHP exposure was negligible for both platinum (1.1 ± 1.5 %) and 5-FU (0.09 ± 0.10 %). CONCLUSIONS: The MTD for IHP was 200 mg/m(2) 5-FU with 40 mg/m(2) oxaliplatin. Systemic exposure to the agents was minimal during IHP. The response and survival observed warrants assessment in a larger phase II trial.

Elastin expression in very severe human COPD.

Alveolar elastic fibres are key targets of proteases during the pathogenesis of chronic obstructive pulmonary disease (COPD). In the current study, we hypothesised that a response to injury leads to enhanced alveolar elastin gene expression in very severe COPD. Lung samples obtained from 43 patients, including 11 with very severe COPD (stage 4), 10 donors, 10 with moderate/severe COPD (stage 2-3) and 12 non-COPD subjects, were analysed for elastin mRNA expression by real-time RT-PCR and in situ hybridisation. Alveolar elastic fibres were visualised using Hart's staining of sections of frozen inflated lungs obtained from 11 COPD stage 4 patients and three donor lungs. Compared with donors, non-COPD and stage 2-3 COPD, elastin mRNA expression was significantly increased in very severe COPD lungs (12-fold change), and localised in situ hybridisation induced elastin expression to alveolar walls. Compared with donors, alveolar elastic fibres also comprised a greater volume fraction of total lung tissue in very severe COPD lungs (p<0.01), but elastic fibre content was not increased per lung volume, and desmosine content was not increased. The present study demonstrates enhanced alveolar elastin expression in very severe COPD. The efficiency of this potential repair mechanism and its regulation remain to be demonstrated.

Surgical management of leiomyosarcoma of the inferior vena cava.

INTRODUCTION: Leiomyosarcoma of the inferior vena cava (IVC) is a rare tumor for which en bloc resection offers the only chance of cure. Due to its rarity, however, optimal strategies for the management of the primary tumor and subsequent recurrences are not well defined. METHODS: We performed a retrospective review of patients who underwent surgical resection of IVC leiomyosarcoma. We evaluated clinical presentations, operative techniques, patterns of recurrence and survival. RESULTS: From 1990 to 2008, nine patients (four females) were identified. Median age was 55 years (40-76). Presentations included abdominal pain (n = 5), back pain (n = 2), leg swelling (n = 4) and abdominal mass (n = 2). Pre-operative imaging studies showed tumor location to be from the right atrium to renal veins (n = 1), retrohepatic (n = 5), and from hepatic veins to the iliac bifurcations (n = 3). En bloc resection included right nephrectomy (n = 5), right adrenalectomy (n = 4), pancreaticoduodenectomy (n = 1), right hepatic trisectionectomy (n = 1) and right hemicolectomy (n = 1). The IVC was ligated in six patients, and a prosthetic graft was used for IVC reconstruction in three patients. Resection margins were negative in seven cases. Median length of stay was 12 days (range, 6-22 days). Major morbidity included renal failure (n = 1) and there was one post-operative mortality. Five patients had leg edema post-operatively, four of whom had IVC ligation. Median survival was 47 months (range, 1-181 months). Four patients had recurrence and the median time to recurrence was 14 months (range, 3-25 months). Two patients underwent successful resection of recurrence. CONCLUSIONS: Curative resection of IVC leiomyosarcoma can lead to long-term survival. However, recurrence is common, and effective adjuvant treatments are needed. In selected cases, aggressive surgical treatment of recurrence should be considered.

Interferon-gamma induces p11 gene and protein expression in human epithelial cells through interferon-gamma-activated sequences in the p11 promoter.

The effect of interferon (IFN)-gamma on p11 expression was studied in two human epithelial cell lines (BEAS-2B and HeLa). Treatment with IFN-gamma resulted in increased steady-state levels of p11 mRNA and protein expression, with a time-dependent and dose-dependent effect. Transient transfection experiments of a reporter gene construct containing 1498 bp of the 5'-flanking region of the p11 promoter demonstrated that IFN-gamma induced p11 gene expression at the transcriptional level. These effects were inhibited at the promoter and protein levels by a specific JAK-2 kinase inhibitor, AG-490. Functional analysis of the p11 promoter indicates that two gamma-activated sequence elements (GAS) located at positions 1219 and 1090 are important for the induction of the p11 promoter by IFN-gamma. Transfection of mutated reporter constructs demonstrated that the mutation at the GAS-2 site (1090) inhibited the p11 promoter activity, with a reduction of about approximately 73% and mutation at the GAS-3 site (1219) eliminated about 26% of the p11 promoter activity. A STAT1 dominant negative mutant vector at Tyr-701 (JAK kinase phosphorylation site) blocked the effect of IFN-gamma on the p11 promoter activity. IFN-gamma induced a rapid tyrosine phosphorylation and nuclear translocation of STAT1 protein, which is involved in the binding to the GAS-2 site in the p11 promoter by EMSA analysis. These data suggest that IFN-gamma-induced p11 expression is mediated through the binding of STAT1 to GAS sites in the p11 promoter. Inhibition of p11 expression by inhibitory antisense RNAs (iRNA) treatment resulted in enhanced IFN-gamma and calcium ionophore-stimulated arachidonic acid release suggesting that at least in part IFN-gamma-stimulated p11 expression may serve a counterregulatory role.

Effect of mechanical deformation of neutrophils on their CD18/ICAM-1-dependent adhesion.

Mechanical deformation of polymorphonuclear leukocytes (PMN) changes their expression of the surface adhesion molecule CD11b/CD18. We tested the hypothesis that mechanical deformation of PMN enhances their adhesiveness. Purified human PMN were deformed through either 5- or 3-microm polycarbonate membrane filters and allowed to adhere to 96-well plates coated with human recombinant intercellular adhesion molecule-1 (ICAM-1). Flow cytometric studies showed that deformation of PMN increased CD11b/CD18 expression (P < 0.01). PMN adhesion to ICAM-1-coated plates was dependent on the magnitude of cell deformation (5 microm, 63.8 +/- 8.1%, P < 0.04; 3 microm, 232.4 +/- 20.9%, P < 0.01). Priming of PMN (0.5 nM N-formyl-methionyl-leucyl-phenylalanine) before deformation (5 microm) increased PMN adhesion (63.8 +/- 8.1 vs. 105.3 +/- 16.4%; P < 0.04). Stimulation (5% zymosan-activated plasma) of PMN after deformation resulted in increased adhesion, and the degree of increase was dependent on the magnitude of PMN deformation (stimulation, 50.6 +/- 4%; 5-microm filtration and stimulation, 62.9 +/- 6.6%; 3-microm filtration and stimulation, 249.9 +/- 24.2%; P < 0.01). This study shows that mechanical deformation of PMN causes an increase in PMN adhesiveness to ICAM-1 that was enhanced by both priming of PMN before deformation and stimulation after cell deformation.

Interleukin 12 p40 production by barrier epithelial cells during airway inflammation.

Human airway epithelial cells appear specially programmed for expression of immune response genes implicated in immunity and inflammation. To better determine how this epithelial system operates in vivo, we analyzed its behavior in mouse models that allow for in vitro versus in vivo comparison and genetic modification. Initial comparisons indicated that tumor necrosis factor alpha induction of epithelial intercellular adhesion molecule 1 required sequential induction of interleukin (IL)-12 (p70) and interferon gamma, and unexpectedly localized IL-12 production to airway epithelial cells. Epithelial IL-12 was also inducible during paramyxoviral bronchitis, but in this case, initial IL-12 p70 expression was followed by 75-fold greater expression of IL-12 p40 (as monomer and homodimer). Induction of IL-12 p40 was even further increased in IL-12 p35-deficient mice, and in this case, was associated with increased mortality and epithelial macrophage accumulation. The results placed epithelial cell overgeneration of IL-12 p40 as a key intermediate for virus-inducible inflammation and a candidate for epithelial immune response genes that are abnormally programmed in inflammatory disease. This possibility was further supported when we observed IL-12 p40 overexpression selectively in airway epithelial cells in subjects with asthma and concomitant increases in airway levels of IL-12 p40 (as homodimer) and airway macrophages. Taken together, these results suggest a novel role for epithelial-derived IL-12 p40 in modifying the level of airway inflammation during mucosal defense and disease.

Cross-talk between interleukin 1beta (IL-1beta) and IL-6 signalling pathways: IL-1beta selectively inhibits IL-6-activated signal transducer and activator of transcription factor 1 (STAT1) by a proteasome-dependent mechanism.

Interleukin 1beta (IL-1beta) suppresses the IL-6-dependent induction of type II acute-phase response genes, but the underlying mechanism for this suppression remains uncertain. Here we report that treatment of human hepatocullular carcinoma HepG2 cells with IL-1beta inhibited the IL-6-dependent binding of signal transducer and activator of transcription factor (STAT)1, but not that of STAT3, to the high-affinity serum-inducible element ('SIE'). Furthermore, IL-1beta selectively down-regulated the IL-6-induced tyrosine phosphorylation of STAT1 without affecting the level of STAT1 or tyrosine phosphorylation of STAT3. Kinase assays in vitro indicated that the inhibition of STAT1 phosphorylation by IL-1beta was not due to an upstream blockade of Janus kinase (JAK1 or JAK2) activation. However, pretreatment with the proteasome inhibitor MG132 under conditions that prevented the IL-1beta-dependent activation of the nuclear factor NF-kappaB also blocked the inhibitory effect of IL-1beta on IL-6-activated STAT1. In related experiments, the protein tyrosine phosphatase inhibitor Na(3)VO(4) also antagonized the inhibitory effect of IL-1beta on the activation of STAT1 by IL-6. Taken together, these findings indicate that, by using a proteasome-dependent mechanism, IL-1beta concomitantly induces NF-kappaB activation and dephosphorylates IL-6-activated STAT1; the latter might partly account for the inhibition by IL-1beta of the IL-6-dependent induction of type II acute-phase genes.

Regulation of T cell apoptosis.

Proliferative expansion of lymphoid cells is required for effective immune responses against invading microorganisms, but after the infection is controlled, the expanded effector cells must be eliminated to prevent non-adaptive accumulation of cells. Higher vertebrates have developed extensive networks of signal transduction pathways to ensure controlled activation and expansion of cells during immune responses and apoptotic deletion of lymphoid cells that are no longer needed at the end of immune responses. Extracellular signals received by cell surface receptors that trigger intracellular signaling cascades are essential elements that control both processes. These signal transduction pathways converge to regulate cell fate at both transcriptional and post-transcriptional levels. Here we review the role of pathways, especially those triggered by TNF receptor-related molecules, that determine the fate of T cells during development and activation. In addition, we introduce the possibility that these same pathways may be abnormally programmed and so lead to immune cell accumulation during inflammatory diseases such as asthma.

Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization.

Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the beta-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11-389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.

Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma.

Cytokine effects on immunity and inflammation often depend on the transcription factors termed signal transducers and activators of transcription (STATs), so STAT signaling pathways are candidates for influencing inflammatory disease. We reasoned that selective IFN responsiveness of the first STAT family member (Stat1) and Stat1-dependent immune-response genes such as intercellular adhesion molecule-1 (ICAM-1), IFN regulatory factor-1 (IRF-1), and Stat1 itself in airway epithelial cells provides a basis for detecting cytokine signaling abnormalities in inflammatory airway disease. On the basis of nuclear localization and phosphorylation, we found that epithelial Stat1 (but not other control transcription factors) was invariably activated in asthmatic compared with normal control or chronic bronchitis subjects. Furthermore, epithelial levels of activated Stat1 correlated with levels of expression for epithelial ICAM-1, IRF-1, and Stat1, and in turn, ICAM-1 levels correlated with T-cell accumulation in tissue. However, only low levels of IFN-gamma or IFN-gamma-producing cells were detected in airway tissue in all subjects. The results therefore provide initial evidence linking abnormal behavior of STAT pathways for cytokine signaling to the development of an inflammatory disease. In that context, the results also change the current scheme for asthma pathogenesis to one that must include a localized gain in transcriptional signal ordinarily used for a T helper 1-type cytokine (IFN-gamma) in combination with allergy-driven overproduction of T helper 2-type cytokines.

Resistance to Fas-mediated T cell apoptosis in asthma.

Over activation of CD4+ T cells in the peripheral blood and airway tissues is characteristic of asthma; therefore, we investigated whether activated T cells from asthmatic subjects have altered apoptotic potential through the Fas death receptor. We found that mitogen-stimulated peripheral blood T cells of asthmatic subjects expressed cell surface Fas, but failed to undergo the normal degree of apoptosis after Fas receptor ligation. T cells from asthmatics exhibited normal apoptotic responses to gamma-irradiation (dependent on IL-1 converting enzyme family proteases), ceramide, and mitogen challenge, suggesting functional integrity of the apoptotic pathway. Furthermore, the defect in Fas-dependent apoptosis was overcome by prestimulation with allogeneic accessory cells instead of mitogen. Taken together, the findings suggest that selective resistance to Fas-dependent apoptosis reflects altered Ag-driven, accessory cell-dependent signaling and that ineffective activation of Fas signal transduction may contribute to T cell-dependent immunoinflammation in asthma.

Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo.

Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon gamma [IFN-gamma] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived beta-chemokines indicated that IFN-gamma treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial-T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-gamma treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.

Control of epithelial immune-response genes and implications for airway immunity and inflammation.

A major goal of our research is to understand how immune cells (especially T cells) infiltrate the pulmonary airway during host defense and inflammatory disease (especially asthma). In that context, we have proposed that epithelial cells lining the airway provide critical biochemical signals for immune-cell influx and activation and that this epithelial-immune cell interaction is a critical feature of airway inflammation and hyperreactivity. In this brief report, we describe our progress in defining a subset of epithelial immune-response genes the expression of which is coordinated for viral defense both directly in response to replicating virus and indirectly under the control of a specific interferon-gamma signal transduction pathway featuring the Stat1 transcription factor as a critical relay signal between cytoplasm and nucleus. Unexpectedly, the same pathway is also activated during asthmatic airway inflammation in a setting where there is no apparent infection and no increase in interferon-gamma levels. The findings provide the first evidence of an overactive Stat1-dependent gene network in asthmatic airways and a novel molecular link between mucosal immunity and inflammation. The findings also offer the possibility that overactivity of Stat1-dependent genes might augment a subsequent T helper cell (Th1)-type response to virus or might combine with a heightened Th2-type response to allergen to account for more severe exacerbations of asthma.