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

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

SARS-CoV-2-specific immunoglobulin Y antibodies are protective in infected mice

Safe, passive immunization methods are required against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants. Immunization of chickens with antigen is known to induce specific IgY antibodies concentrated in the egg yolk and has a good safety profile, high yield of IgY per egg, can be topically applied, not requiring parenteral delivery. Our data provide the first evidence of the prophylactic efficacy of Immunoglobulin Y antibodies against SARS-CoV-2 in mice. Lohmann hens were injected with recombinant SARS-CoV-2 RBD protein; IgY-Abs were extracted from the eggs and characterized using SDS-PAGE. Antiviral activity was evaluated using plaque reduction neutralization tests. In additional experiments, IgY-RBD efficacy was examined in mice sensitized to SARS-CoV-2 infection by transduction with Ad5-hACE2 (mild disease) or by using mouse-adapted virus (severe disease). In both cases, prophylactic intranasal administration of IgY-Abs reduced SARS-CoV-2 replication, and reduced morbidity, inflammatory cell infiltration, hemorrhage, and edema in the lungs and increased survival compared to control groups that received non-specific IgY-Abs. These results indicate that further evaluation of IgY-RBD antibodies in humans is warranted

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

<p>Abstract</p> <p>Introduction</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

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Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTRΔF508/ΔF508 Pigs

Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion.Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(ΔF508/ΔF508) with CFTR(-/-) piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands.These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections

Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals

Alum Adjuvant Enhances Protection against Respiratory Syncytial Virus but Exacerbates Pulmonary Inflammation by Modulating Multiple Innate and Adaptive Immune Cells

Respiratory syncytial virus (RSV) is well-known for inducing vaccine-enhanced respiratory disease after vaccination of young children with formalin-inactivated RSV (FI-RSV) in alum formulation. Here, we investigated alum adjuvant effects on protection and disease after FIRSV immunization with or without alum in comparison with live RSV reinfections. Despite viral clearance, live RSV reinfections caused weight loss and substantial pulmonary inflammation probably due to high levels of RSV specific IFN-γ+IL4-, IFN-γ-TNF-α+, IFN-γ+ TNF-α- effector CD4 and CD8 T cells. Alum adjuvant significantly improved protection as evidenced by effective viral clearance compared to unadjuvanted FI-RSV. However, in contrast to unadjuvanted FI-RSV, alum-adjuvanted FI-RSV (FI-RSV-A) induced severe vaccine- enhanced RSV disease including weight loss, eosinophilia, and lung histopathology. Alum adjuvant in the FI-RSV-A was found to be mainly responsible for inducing high levels of RSV-specific IFN-γ-IL4+, IFN-γ-TNF-α+ CD4+ T cells, and proinflammatory cytokines IL-6 and IL-4 as well as B220+ plasmacytoid and CD4+ dendritic cells, and inhibiting the induction of IFN-γ+CD8 T cells. This study suggests that alum adjuvant in FI-RSV vaccines increases immunogenicity and viral clearance but also induces atypical T helper CD4+ T cells and multiple inflammatory dendritic cell subsets responsible for vaccine-enhanced severe RSV disease

Differential Expression of Cytokines in Response to Respiratory Syncytial Virus Infection of Calves with High or Low Circulating 25-Hydroxyvitamin D3

Deficiency of serum levels of 25-hydroxyvitamin D3 has been related to increased risk of lower respiratory tract infections in children. Respiratory syncytial virus (RSV) is a leading cause of low respiratory tract infections in infants and young children. The neonatal calf model of RSV infection shares many features in common with RSV infection in infants and children. In the present study, we hypothesized that calves with low circulating levels of 25-hydroxyvitamin D3 (25(OH)D3) would be more susceptible to RSV infection than calves with high circulating levels of 25(OH)D3. Calves were fed milk replacer diets with different levels of vitamin D for a 10 wk period to establish two treatment groups, one with high (177 ng/ml) and one with low (32.5 ng/ml) circulating 25(OH)D3. Animals were experimentally infected via aerosol challenge with RSV. Data on circulating 25(OH)D3 levels showed that high and low concentrations of 25(OH)D3 were maintained during infection. At necropsy, lung lesions due to RSV were similar in the two vitamin D treatment groups. We show for the first time that RSV infection activates the vitamin D intracrine pathway in the inflamed lung. Importantly, however, we observed that cytokines frequently inhibited by this pathway in vitro are, in fact, either significantly upregulated (IL-12p40) or unaffected (IFN-γ) in the lungs of RSV-infected calves with high circulating levels of 25(OH)D3. Our data indicate that while vitamin D does have an immunomodulatory role during RSV infection, there was no significant impact on pathogenesis during the early phases of RSV infection. Further examination of the potential effects of vitamin D status on RSV disease resolution will require longer-term studies with immunologically sufficient and deficient vitamin D levels

Variation in MSRA Modifies Risk of Neonatal Intestinal Obstruction in Cystic Fibrosis

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10−5 to 1.08×10−6; Bonferroni P = 0.057 to 3.1×10−3). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10−4). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr−/− and Cftr−/−Msra−/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function