The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase.

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

Ontogeny of the immune response in the ovine lung.

Perinatal lambs are increasingly appreciated as a model to study respiratory infections of premature and newborn human infants. To explore the relationship between developmental age and immunological competence in the respiratory tract, the basal levels of expression of genes involved in innate and adaptive immune functions in the lung were examined in pre-term lambs (115 days and 130 days), at birth (145 days) and post-partum (15 days and 3 years old). Our results show that innate immune genes (TLRs-3, -4, -7, -8; SP-A, SP-D, and SBD1) were differentially expressed through development; cytokines (IFN-γ, IL-6, TNF-α) and chemokines (IL-8, MCP-1) were low during gestation and post-partum but maximal at birth; genes involved in adaptive immunity (PD-1, PD-L1, TGF-ß) were present in pre-term and newborn lung, but were lower in adult lung. The results suggest that pre-term and neonatal lambs may be able to mount an immune response following infection, but that the response may not be optimal. Our studies provide an important set of comparative data on the ontogeny of lung immunity in sheep and set a framework for studies on age-dependent susceptibility to respiratory pathogens.

Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFNγ, IL-8, MCP-1, and PD-L1, and decreased IFNß, IL-10, and TGF-ß. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.

Respiratory syncytial virus infection is associated with an altered innate immunity and a heightened pro-inflammatory response in the lungs of preterm lambs.

INTRODUCTION: Factors explaining the greater susceptibility of preterm infants to severe lower respiratory infections with respiratory syncytial virus (RSV) remain poorly understood. Fetal/newborn lambs are increasingly appreciated as a model to study key elements of RSV infection in newborn infants due to similarities in lung alveolar development, immune response, and susceptibility to RSV. Previously, our laboratory demonstrated that preterm lambs had elevated viral antigen and developed more severe lesions compared to full-term lambs at seven days post-infection. Here, we compared the pathogenesis and immunological response to RSV infection in lungs of preterm and full-term lambs. METHODS: Lambs were delivered preterm by Caesarian section or full-term by natural birth, then inoculated with bovine RSV (bRSV) via the intratracheal route. Seven days post-infection, lungs were collected for evaluation of cytokine production, histopathology and cellular infiltration. RESULTS: Compared to full-term lambs, lungs of preterm lambs had a heightened pro-inflammatory response after infection, with significantly increased MCP-1, MIP-1α, IFN-γ, TNF-α and PD-L1 mRNA. RSV infection in the preterm lung was characterized by increased epithelial thickening and periodic acid-Schiff staining, indicative of glycogen retention. Nitric oxide levels were decreased in lungs of infected preterm lambs compared to full-term lambs, indicating alternative macrophage activation. Although infection induced significant neutrophil recruitment into the lungs of preterm lambs, neutrophils produced less myeloperoxidase than those of full-term lambs, suggesting decreased functional activation. CONCLUSIONS: Taken together, our data suggest that increased RSV load and inadequate immune response may contribute to the enhanced disease severity observed in the lungs of preterm lambs.

Exogenous administration of vascular endothelial growth factor prior to human respiratory syncytial virus a2 infection reduces pulmonary pathology in neonatal lambs and alters epithelial innate immune responses.

Human respiratory syncytial virus (RSV) affects thousands of children every year. Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis, pulmonary maturation, and immunity. In order to test the extent to which VEGF may alter RSV infection, 4 groups of lambs received either human recombinant VEGF (rhVEGF) or phosphate-buffered saline (PBS) pretreatment followed by inoculation with human RSV strain A2 or sterile medium. Lambs in each group were sacrificed at 2, 4, and 6 days post infection. Expression of surfactant protein-A (SP-A), surfactant protein-D (SP-D), sheep ß-defensin-1 (SBD-1), tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-8, interferon ß, and endogenous VEGF were measured to determine effect of rhVEGF pretreatment. RSV lambs pretreated with rhVEGF had reduced viral mRNA and decreased pulmonary pathology at day 6. Pretreatment with rhVEGF increased mRNA expression of SP-A, SBD-1, and TNFα, with alteration of expression in RSV lambs. Endogenous VEGF mRNA levels were increased at day 2 regardless of pretreatment. Pretreatment with rhVEGF increased pulmonary cellular proliferation in RSV lambs at day 4 post infection. Overall, these results suggest that pretreatment with rhVEGF protein may have therapeutic potential to decrease RSV viral load, decrease pulmonary lesion severity, and alter both epithelial innate immune responses and epithelial cell proliferation.

Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory disease in infants and young children worldwide. Currently, treatment is supportive and no vaccines are available. The use of newborn lambs to model hRSV infection in human infants may provide a valuable tool to assess safety and efficacy of new antiviral drugs and vaccines. ALX-0171 is a trivalent Nanobody targeting the hRSV fusion (F) protein and its therapeutic potential was evaluated in newborn lambs infected with a human strain of RSV followed by daily ALX-0171 nebulization for 3 or 5 consecutive days. Colostrum-deprived newborn lambs were infected with hRSV-M37 before being treated by daily nebulization with either ALX-0171 or placebo. Two different treatment regimens were examined: day 1-5 or day 3-5 post-infection. Lambs were monitored daily for general well-being and clinical parameters. Respiratory tissues and bronchoalveolar lavage fluid were collected at day 6 post-inoculation for the quantification of viral lesions, lung viral titers, viral antigen and lung histopathology. Administration by inhalation of ALX-0171 was well-tolerated in these hRSV-infected newborn lambs. Robust antiviral effects and positive effects on hRSV-induced lung lesions and reduction in symptoms of illness were noted. These effects were still apparent when treatment start was delayed and coincided with peak viral loads (day 3 post-infection) and at a time point when signs of RSV disease were apparent. The latter design is expected to have high translational value for planned clinical trials. These results are indicative of the therapeutic potential of ALX-0171 in infants.

Neonatal ovine pulmonary dendritic cells support bovine respiratory syncytial virus replication with enhanced interleukin (IL)-4 And IL-10 gene transcripts.

The lung microenvironment is constantly exposed to microorganisms and particulate matter. Lung dendritic cells (DCs) play a crucial role in the uptake and processing of antigens found within the respiratory tract. Respiratory syncytial virus (RSV) is a common respiratory tract pathogen in children that induces an influx of DCs to the mucosal surfaces of the lung. Using a neonatal lamb model, we examined the in vivo permissiveness of DCs to RSV infection, as well as overall cell surface changes and cytokine responses of isolated lung DCs after bovine RSV (BRSV) infection. We report that isolated lung DCs and alveolar macrophages support BRSV replication. Isolated lung DCs were determined to be susceptible to BRSV infection as demonstrated by quantification of BRSV non-structural protein 2 mRNA. BRSV infection induced an initial upregulation of CD14 expression on lung DCs, but by 5 d postinfection expression was similar to that on control cells. No significant changes in CD80/86 or MHC class I expression were seen on lung DCs after BRSV infection. Low to moderate expression of MHC class II and DEC-205 was detected by day 5 postinfection. Initially, on day 3 postinfection, lung DCs from BRSV-infected lambs had decreased endocytosis of fluorescein isothiocyanate (FITC)-ovalbumin (OVA). The amount of FITC-OVA endocytosed by lung DCs isolated on day 5 postinfection was similar to that of controls. The most interesting observation was the induction of immunomodulatory interleukin (IL)-4 and IL-10 cytokine gene transcription in lung DCs and alveolar macrophages after in vivo infection with BRSV. Overall, these findings are the first to demonstrate that neonatal lung DCs support in vivo BRSV replication and produce type II cytokines after viral infection.

Pretreatment with recombinant human vascular endothelial growth factor reduces virus replication and inflammation in a perinatal lamb model of respiratory syncytial virus infection.

Vascular endothelial growth factor (VEGF) is increasingly recognized as a perinatal regulator of lung maturation and surfactant protein expression. Preterm and young infants are at increased risk for pulmonary immaturity characterized by insufficient surfactant production as well as increased risk for severe manifestations of respiratory syncytial virus (RSV) infection. Innate immune components including surfactant proteins A and D, and beta-defensins have putative antimicrobial activity against pulmonary pathogens including RSV. Our hypothesis was that recombinant human VEGF (rhVEGF) pretreatment therapy would decrease RSV disease in the perinatal lamb RSV model. Newborn lambs were pretreated with rhVEGF, betamethasone, or saline and then inoculated with bovine RSV or sterile medium. Tissues were collected 5 d postinoculation, corresponding to the initiation of severe lesions and peak viral replication. In RSV-infected lambs, rhVEGF therapy increased the mean daily body temperature, decreased airway neutrophil exudate, and reduced RSV replication compared with betamethasone or saline pretreatment. Furthermore, rhVEGF therapy significantly mitigated the RSV-induced increase in surfactant protein A mRNA expression and decrease in surfactant protein D mRNA expression. In control (non-RSV-infected) lambs, pretreatment with rhVEGF increased sheep beta-defensin-1 (SBD1) mRNA expression, but no alteration in surfactant proteins A and D was detected. This novel study demonstrates that rhVEGF pretreatment mitigates RSV disease and, in addition, rhVEGF regulation of innate immune genes is dependent on RSV infection status.

Maternal alcohol ingestion reduces surfactant protein A expression by preterm fetal lung epithelia.

In addition to neurodevelopmental effects, alcohol consumption at high levels during pregnancy is associated with immunomodulation and premature birth. Premature birth, in turn, is associated with increased susceptibility to various infectious agents such as respiratory syncytial virus (RSV). The initial line of pulmonary innate defense includes the mucociliary apparatus, which expels microorganisms trapped within the airway secretions. Surfactant proteins A and D (SP-A and SP-D, respectively) are additional components of pulmonary innate immunity and have an important role in pulmonary defense against inhaled pathogens. The purpose of this study was to determine if chronic alcohol consumption during the third trimester of pregnancy alters the function of the mucociliary apparatus and expression of SP-A and SP-D of fetal lung epithelia. Sixteen, date-mated ewes were assigned to two different groups; an ethanol-exposed group in which ewes received ethanol through surgically implanted intra-abomasal cannula during the third trimester of pregnancy, and a control group in which ewes received the equivalent amount of water instead of ethanol. Within these two groups, ewes were further randomly assigned to a full-term group in which the lambs were naturally delivered, and a preterm group in which the lambs were delivered prematurely via an abdominal incision and uterotomy. Ethanol was administered five times a week as a 40% solution at 1g/kg of body weight. The mean maternal serum alcohol concentration measured 6h postadministration was 16.3+/-4.36 mg/dl. Tracheas from six full-term lambs were collected to assess ciliary beat frequency (CBF). The lung tissue from all (24) lambs was collected for immunohistochemistry analysis of SP-A and SP-D protein production and fluorogenic real-time quantitative polymerase chain reaction analysis of SP-A and SP-D mRNA levels. Exposure to ethanol during pregnancy significantly blocked stimulated increase in CBF through ethanol-mediated desensitization of cAMP-dependent protein kinase. In addition, preterm born/ethanol-exposed lambs showed significantly decreased SP-A mRNA expression when compared with the preterm born/control group (P=.004); no significant changes were seen with SP-D. The full-term/ethanol-exposed lambs had no significant alterations in mRNA levels, but had significantly less detectable SP-A protein when compared with the full-term/control lambs (P=.02). These findings suggest that chronic maternal ethanol consumption during the third trimester of pregnancy alters innate immune gene expression in fetal lung. These alterations may underlie increased susceptibility of preterm infants, exposed to ethanol in utero, to RSV and other microbial agents.

Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor.

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions.

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time - calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional.

Depletion of alveolar glycogen corresponds with immunohistochemical development of CD208 antigen expression in perinatal lamb lung.

CD208 DC lysosomal-associated protein is a marker of activated human dendritic cells; however, recently it was described as a marker of adult type II pneumocytes in many species including humans and sheep. Our hypothesis was that CD208 is developmentally regulated in lung pneumocytes. Lamb lungs at varying stages of development were stained immunohistochemically for CD208 and with Nile red (a fluorescent stain for lamellar bodies of type II cells) along with pulmonary markers of maturation (glycogen stores and surfactant protein A [SP-A] expression) or proliferation (Ki-67). CD208 staining and Nile red were localized to rare pneumocytes in young fetal lambs (day 115), increasing in frequency and stain intensity with age. Periodic acid-Schiff staining of glycogen granules was most prominent in the young lambs (day 115) with reduced staining through advancing lung development. SP-A was detected in pulmonary epithelia and staining in alveoli increased through gestation with decreased staining at 2 weeks of age. Intranuclear Ki-67 staining decreased through late gestation but was increased in 2-week-old lambs. Ontogeny of CD208 staining and depletion of glycogen were correlated (p<0.0001) and consistent with the premise that CD208 is localized to developing lamellar bodies. The findings suggest that CD208 antigen expression may serve as a marker for pneumocyte maturation in the developing fetal lung.

Expression of select immune genes (surfactant proteins A and D, sheep beta defensin 1, and toll-like receptor 4) by respiratory epithelia is developmentally regulated in the preterm neonatal lamb.

Preterm infants experience enhanced susceptibility and severity to respiratory syncytial virus (RSV) infection. Terminal airway epithelium is an important site of RSV infection and the extent of local innate immune gene expression is poorly understood. In this study, expression of surfactant proteins A and D (SP-AD), sheep beta defensin 1 (SBD1), and toll-like receptor 4 (TLR4) mRNA were determined in whole lung homogenates from lambs. SP-AD and TLR4 mRNA expression increased (p < 0.05) from late gestation to term birth. In addition, gene expression of LCM-retrieved type II pneumocytes (CD208+), adjacent epithelium (CD208-) and bronchial epithelium demonstrated that bronchiole-alveolar junction epithelium (combined CD208 +/-) had significant (p < 0.05) developmental increases in SP-AD, SBD1 and TLR4 mRNA, whereas CD208+ cells had statistically significant increases only with SP-A mRNA. Using immunofluorescence, SP-AD antigen distribution and intensity were also greater with developmental age. These studies show reduced SBD1, SP-AD, and TLR4 expression in the preterm lung and this may underlie enhanced RSV susceptibility.

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.

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.

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants.

RATIONALE: Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions. METHODS: Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 10(7) FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses. RESULTS: Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log10 FFU/mL), which was maintained until day 6 (4.83 Log10 FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log10 M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log10 M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log10 M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants. CONCLUSION: This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants.

Reduced clearance of respiratory syncytial virus infection in a preterm lamb model.

Respiratory syncytial virus (RSV) causes significant respiratory disease in children worldwide. For the study of severe RSV disease seen in preterm infants, a suitable animal model is lacking. The novel hypothesis of this study was that preterm lambs are susceptible to bovine RSV (bRSV) infection, an analogous pneumovirus with ruminant host specificity, and that there would be age-dependent differences in select RSV disease parameters. During RSV infection, preterm lambs had elevated temperatures and respiration rates with mild anorexia and cough compared to controls. Gross lesions included multifocal consolidation and atelectasis with foci of hyperinflation. Microscopic lesions included multifocal alveolar septal thickening and bronchiolitis. Immunohistochemistry localized the RSV antigen to all layers of bronchiolar epithelium from a few basal cells to numerous sloughing epithelia. A few mononuclear cells were also immunoreactive. To assess for age-dependent differences in RSV infection, neonatal lambs were infected similarly to the preterm lambs or with a high-titer viral inoculum. Using morphometry at day 7 of infection, preterm lambs had significantly more cellular immunoreactivity for RSV antigen (P <0.05) and syncytial cell formation (P <0.05) than either group of neonatal lambs. This work suggests that perinatal RSV clearance is age-dependent, which may explain the severity of RSV infection in preterm infants. The preterm lamb model is useful for assessing age-dependent mechanisms of severe RSV infection.