Differential Expression of Ovine Innate Immune Genes by Preterm and Neonatal Lung Epithelia Infected with Respiratory Syncytial Virus

Preterm infants have increased susceptibility to severe manifestations of respiratory syncytial virus (RSV) infection. The cause(s) for this age-dependent vulnerability is/are not well-defined, but alterations in innate immune products have been implicated. In sheep, RSV disease severity has similar age-dependent characteristics and sheep have several related innate molecules for study during pulmonary infection including surfactant protein A (SP-A), surfactant protein D (SP-D), sheep beta defensin 1 (SBD1), monocyte chemotactic protein 1 (MCP1), and Toll-like receptor 4 (TLR4). However, the in vivo cellular gene expression as a response to RSV infection is poorly understood. In this study, the effect of RSV infection on expression of these innate immune genes was determined for bovine RSV-infected (bRSV+ fluorescence) epithelial cells, adjacent cells lacking bRSV antigen (adjoining cells lacking fluorescence), and control cells from non-infected lung using laser capture microdissection (LCM) and real-time RT-PCR. Control lambs had increased expression of innate immune molecules in full term (term) compared to preterm epithelia with statistical significance in SBD1, SP-D, and TLR4 mRNA. Infected cells (bRSV+ fluorescent cells) had consistently higher mRNA levels of SP-A (preterm and term), MCP1 (preterm and term), and SP-D (preterm). Interestingly, bRSV- cells of infected term lambs had significantly reduced SP-D mRNA expression compared to bRSV+ and control epithelia, suggesting that RSV infected cells may regulate the adjacent epithelial SP-D expression. This study defines specific innate immune components (e.g., SBD1, SP-D, and TLR4) that have differential age-dependent expression in the airway epithelia. Furthermore, cellular bRSV infection enhanced certain innate immune components while suppressing adjacent cellular SP-D expression in term animals. These in vivo gene expression results provide a framework for future studies on age-dependent susceptibility to RSV and RSV pathogenesis

Mucosal vaccination with a live recombinant rhinovirus followed by intradermal DNA administration elicits potent and protective HIV-specific immune responses

Published: 17 November 2016Mucosal immunity is deemed crucial to control sexual transmission of human immunodeficiency virus (HIV). Herein we report the efficacy of a mucosal HIV vaccine strategy comprising intranasal (IN) vaccination with a cocktail of live recombinant human rhinoviruses (HRVs) encoding overlapping fragments of HIV Gag and full length Tat (rHRV-Gag/Tat) followed by intradermal (ID) vaccination with DNA vaccines encoding HIV Gag and Tat (pVAX-Gag-Tat). This heterologous prime-boost strategy will be referred to hereafter as rHRV-DNA. As a control, IN vaccination with wild type (wt)-HRV-A1 followed by a single ID dose of pVAX (wt-HRV-A1/pVAX vaccination) was included. rHRV-DNA vaccination elicited superior multi-functional CD8(+)T cell responses in lymphocytes harvested from mesenteric lymph nodes and spleens, and higher titres of Tat-specific antibodies in blood and vaginal lavages, and reduced the viral load more effectively after challenge with EcoHIV, a murine HIV challenge model, in peritoneal macrophages, splenocytes and blood compared compared with wt-HRV-A1/pVAX vaccination or administration of 3 ID doses of pVAX-Gag-Tat (3X pVAX-Gag-Tat vaccination). These data provide the first evidence that a rHRV-DNA vaccination regimen can induce HIV-specific immune responses in the gut, vaginal mucosa and systemically, and supports further testing of this regimen in the development of an effective mucosally-targeted HIV-1 vaccine.Khamis Tomusange, Danushka Wijesundara, Jason Gummow, Steve Wesselingh, Andreas Suhrbier, Eric J. Gowans, Branka Grubor-Bau

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead

Monocytic/Macrophagic Pneumonitis after Intrabronchial Deposition of Vascular Endothelial Growth Factor in Neonatal Lambs

Preterm and young neonates are prone to inadequate surfactant production and are susceptible to respiratory distress syndrome characterized by alveolar damage and hyaline-membrane formation. Glucocorticoid therapy is commonly used in preterm and young infants to enhance lung maturation and surfactant synthesis. Recently, vascular endothelial growth factor (VEGF) was suggested to be a novel therapeutic agent for lung maturation that lacked adverse effects in mice. The purpose of this study was to assess the safety of incremental concentration (0.0005, 0.005, and 0.05 mg/ml) and duration (16, 24, and 32 hours) of recombinant human VEGF after bronchoscopic instillation (10 ml) in neonatal lambs. High-dose VEGF caused locally extensive plum-red consolidation that was microscopically characterized by interstitial and alveolar infiltrates of cells that were morphologically and phenotypically (CD68+) consistent with monocytes/macrophages. T cells (CD3+) and B cells (CD79+) were located primarily in bronchus/bronchiole-associated lymphoid tissue and were not consistently altered by treatment with VEGF. The dose of VEGF had significant effects on both gross lesions (P \u3c .0047) and microscopic monocyte/macrophage recruitment scores (P \u3c .0001). Thus, the VEGF dose instilled into the lung greatly influenced cellular recruitment and lesion development. The post-dosing interval of VEGF in this study had minor impact (no statistical significance) on cellular recruitment. This study showed that airway deposition of VEGF in the neonatal lamb induces monocyte/macrophage recruitment to the lung and high doses can cause severe lesions. The cellular recruitment suggests further research is needed to define dosages that are efficacious in enhancing lung maturation while minimizing potential adverse effects

PROBER: oligonucleotide FISH probe design software

PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.ed

«White spleen» in hairy cell leukemia

«White spleen» in hairy cell leukemi

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D

BACKGROUND: Ozone (O(3)), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O(3)-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O(3 )exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O(3 )or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O(3 )exposure. The effect of IL-6, an O(3)-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O(3 )exposure. IL-6, which was highly up-regulated in O(3 )exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O(3 )exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.Tessa Gargett, Branka Grubor-Bauk, Darren Miller, Tamsin Garrod, Stanley Yu, Steve Wesselingh, Andreas Suhrbier, and Eric J Gowan

Inferring tumor progression from genomic heterogeneity

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth. © 2010 by Cold Spring Harbor Laboratory Press