Live Vaccination Generates Both Disease Tolerance and Host Resistance During Chronic Pulmonary Infection With Highly Virulent Francisella tularensis SchuS4.

Disease tolerance can preserve host homeostasis and limit the negative impact of infections. We report that vaccinated mice survived pulmonary challenge with the extremely virulent SchuS4 strain of Francisella tularensis for at least 100 days, despite the persistence of large numbers (~104) of organisms. Transfer of 100 of these resident bacteria to naive animals caused 100% lethality, demonstrating that virulence was maintained. Tissue damage in the lung was limited over the course of infection and was associated with increased levels of amphiregulin. Mice depleted of CD4+ cells had reduced amphiregulin and succumbed to infection. In addition, neutralization of interferon-γ or depletion of CD8+ cells resulted in increased pathogen loads, bacteremia, and death of the host. Conversely, depletion of Ly6G+ neutrophils had no effect on survival and actually resulted in reduced bacterial levels. Understanding the interplay between host resistance and disease tolerance will provide new insights into the understanding of chronic infectious diseases.

Asthma increases susceptibility to heterologous but not homologous secondary influenza.

UNLABELLED: Asthma was the most common comorbidity observed among patients hospitalized with influenza A virus during the 2009 pandemic. However, little remains known about how the asthmatic phenotype influences protective immune responses against respiratory viral pathogens. Using the ovalbumin-induced allergic lung inflammation model, we found that asthmatic mice, unlike nonasthmatic mice, were highly susceptible to secondary heterologous virus challenge. While primary virus infection generated protective memory immune responses against homologous secondary virus challenge in both asthmatic and nonasthmatic mice, full protection against heterologous A/California/04/2009 (CA04) viral infection was observed only in nonasthmatic mice. Significant reductions in CA04-specific IgA, IgG, and IgM levels and in CA04-neutralizing activity of bronchoalveolar lavage fluid (BALF) was observed following secondary CA04 challenge of PR8-immunized asthmatic mice. Furthermore, transfer of immune BALF obtained from nonasthmatic, but not asthmatic, donors following secondary viral infection generated protection against CA04 in naive recipients. Nonspecific B-cell activation by CpG inoculation restored protection in PR8-immunized, CA04-challenged asthmatic mice. These results demonstrate a causal link between defective mucosal antibody responses and the heightened susceptibility of asthmatic mice to influenza infection and provide a mechanistic explanation for the observation that asthma was a major risk factor during the 2009 influenza pandemic. IMPORTANCE: The prevalence of asthma worldwide is increasing each year. Unfortunately, there is no cure for asthma. Asthmatic individuals not only suffer from consistent wheezing and coughing but are also believed to be more prone to serious lung infections that result in bronchitis and pneumonia. However, little is known about the influence of asthma on host mucosal immunity. Here we show that antibody responses during secondary heterologous influenza infections are suboptimal and that this is responsible for the increased mortality in asthmatic mice from viral infections. Understanding the mechanism of increased susceptibility will aid in developing new antiviral therapies for asthmatic patients.

Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin.

MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.

Validity of messenger RNA expression analyses of human saliva.

PURPOSE: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. EXPERIMENTAL DESIGN: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase ("no-RT") to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA. RESULTS: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated "RNA" extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, beta-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non-RNA-specific primers on saliva samples yielded PCR products for 36B4, beta-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the "no-RT" and "+RT" conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva. CONCLUSIONS: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.

Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

Phase I and II carcinogen metabolism gene expression in human lung tissue and tumors.

PURPOSE: The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined. EXPERIMENTAL DESIGN: Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting. Tobacco exposure measured by plasma biomarkers nicotine and cotinine, plasma estradiol levels, alpha and beta estrogen receptor (ER) expression in the lung, gender, age, and histological diagnosis were then analyzed using multivariate regression models. RESULTS: In nontumor lung tissue, multivariate models identified several correlates of mRNA expression: (a) CYP1B1 in females (positively: smoke status, P=0.024; ER-beta expression, P=0.024); (b) GSTT1 in females (positively: cotinine, P=0.007; negatively: age, P=0.001; ER-beta expression, P=0.005) and in males (positively: plasma estradiol, P=0.015; ER-beta expression, P=0.025); and (c) NQO1 in females (positively: smoke status, P=0.002) and in males (positively: ER-beta expression, P=0.001). CYP1A1 (mRNA, 9.1%) and GSTM1 (mRNA, 17.5%) are uncommonly expressed in human lung. Confirmation by Western immunoassayed protein is described. The results in nontumor tissue differed from that in tumor tissue. CONCLUSIONS: Regulation of carcinogen metabolism genes expressed in human lung seems impacted by hormonal and gender factors, as well as ongoing tobacco exposure. Expression differences between tumor and nontumor tissue in this pathway have both susceptibility and therapeutic implications.

Gender-dependent expression of alpha and beta estrogen receptors in human nontumor and tumor lung tissue.

Estrogen receptor (ER) expression in human lung has been understudied, particularly in light of its potential biological importance in the female lung cancer epidemic. Reverse transcription-polymerase chain reaction was used to probe mRNA expression of wild-type ERalpha and ERbeta and their splice variants in human bronchogenic tumor and adjacent nontumor specimens. In tumor tissue from 13 women and 13 men, ERalpha was expressed in 85% of women versus 15% in men [P=0.001]. ERbeta was expressed equally in tumors from women versus men [92% vs. 69%, P=ns]. Both ERalpha and beta forms were expressed simultaneously in the lung tumors of 77% of women versus 15% of men [P=0.005]. Among adjacent nontumor lung specimens, 31% of the women expressed ERalpha mRNA versus 0% of men [P=0.101], and 39% of women expressed ERbeta mRNA versus 31% of men [P=ns]; only one woman and no men expressed both ERalpha and beta in nontumor tissue. Females expressed ERalpha [P=0.017], ERbeta [P=0.013], and ERalpha+beta [P=0.002] more frequently in tumor versus nontumor tissue, whereas in males expression of ERalpha, beta and both alpha+beta was not clearly different for tumor versus nontumor tissue. In specimens expressing ERalpha mRNA, the transcript lacking exon 7 (delta7) was the major splice variant with varying contributions from the transcripts delta4, delta3+4, delta5 and others unidentified. Alternative splicing of ERbeta mRNA was observed, but not to as great an extent as for ERalpha mRNA. ERalpha promoter usage in tumors varied among individuals. When the ER receptors were co-expressed in tumors, ERalpha was quantitatively more abundant in the majority of cases than ERbeta. Within this small group of 26 patients, no correlation was found between age, smoking history, plasma nicotine, cotinine, estradiol concentrations or histopathologic type with tumor or nontumor estrogen receptor status of any type. However, several positive correlations imply that: (1) ERalpha expression occurs more often in the lungs of women than men; (2) ERbeta is expressed with approximately equal frequency in the lungs of both genders; and (3) tumors display a higher frequency of both receptor types than nontumors in women. We hypothesize that these putative gender-dependent differences in ERalpha and ERbeta expression could contribute unique phenotypic characteristics to lung cancer development or progression in women.

The 3 prime paradigm of the miR-200 family and other microRNAs.

The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction.

Antifungal testing and high-throughput screening of compound library against Geomyces destructans, the etiologic agent of geomycosis (WNS) in bats.

Bats in the northeastern U.S. are affected by geomycosis caused by the fungus Geomyces destructans (Gd). This infection is commonly referred to as White Nose Syndrome (WNS). Over a million hibernating bats have died since the fungus was first discovered in 2006 in a cave near Albany, New York. A population viability analysis conducted on little brown bats (Myotis lucifugus), one of six bat species infected with Gd, suggests regional extinction of this species within 20 years. The fungus Gd is a psychrophile ("cold loving"), but nothing is known about how it thrives at low temperatures and what pathogenic attributes allow it to infect bats. This study aimed to determine if currently available antifungal drugs and biocides are effective against Gd. We tested five Gd strains for their susceptibility to antifungal drugs and high-throughput screened (HTS) one representative strain with SpectrumPlus compound library containing 1,920 compounds. The results indicated that Gd is susceptible to a number of antifungal drugs at concentrations similar to the susceptibility range of human pathogenic fungi. Strains of Gd were susceptible to amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. In contrast, very high MICs (minimum inhibitory concentrations) of flucytosine and echinocandins were needed for growth inhibition, which were suggestive of fungal resistance to these drugs. Of the 1,920 compounds in the library, a few caused 50%--to greater than 90% inhibition of Gd growth. A number of azole antifungals, a fungicide, and some biocides caused prominent growth inhibition. Our results could provide a theoretical basis for future strategies aimed at the rehabilitation of most affected bat species and for decontamination of Gd in the cave environment.

Stable expression of miR-200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression.

Regulation of the transcription factor TCF8 (ZEB1) by the microRNA miR-200c and was first identified using a bioinformatic and relative quantitative PCR based approach.(1) Using stable ectopic expression of miR-200c we then demonstrated loss of TCF8 (ZEB1) mRNA and restoration of its primary regulatory target, E-cadherin.(2) Recently, other members of the miR-200 'family' and an additional unrelated microRNA, miR-205, have been reported to be essential for the regulation of TCF8 (ZEB1) and restoration of E-cadherin.(3-5) To investigate, we repeated our initial method(s) and generated individual stable cell-lines, expressing the miR-200 'family' members; miR-200c, miR-200b, miR-141 and the related miR-205. Of these lines, miR-200b produced no mature transcript and miR-205 and miR-141 cells were either morphologically similar to controls or showed some slight changes respectively. However no reduction in TCF8/ZEB1 mRNA or restoration of E-cadherin could be detected in either line. In contrast, cells expressing miR-200c had a significantly altered morphology from mesenchymal to epithelial and restored expression of E-cadherin. These contrasting findings demonstrate that, as transient transfection is in essence an RNAi experiment, results should be treated with caution when investigating microRNAs and that an examination of microRNAs with similar seed regions requires stable ectopic expression to accurately reflect the endogenous mechanism(s).

Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR.

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated approximately 3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against approximately 600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2'-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.

mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts.

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design "RNA-specific" PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these "housekeeper" genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a unique tag sequence not otherwise existing in the human genome. Genomic DNA does not incorporate this RT-inserted unique tag. PCR is then performed using a transcript-specific forward primer and a reverse primer that is identical to the unique tag incorporated at RT. Only cDNA made with this RT primer is compatible with this reverse PCR primer, thus eliminating confounding signal from contaminating gDNA. This method performs RNA-specific qualitative and quantitative evaluation of gene expression, while preserving the sensitivity of standard RT-PCR techniques. Applications to low-copy transcripts in human samples are demonstrated.

Decreased lung tumorigenesis in mice genetically deficient in cytosolic phospholipase A2.

Epidemiological investigations suggest that chronic lung inflammation increases lung cancer risk. Pharmacologic and genetic evidence in mouse models indicates that lipid mediators released during pulmonary inflammation enhance lung tumor formation. Cytosolic phospholipase A2 (cPLA2) catalyzes arachidonic acid (AA) release from membrane phospholipids. AA can then lead to the synthesis of several classes of lipid mediators, including prostaglandin (PG) biosynthesis through the cyclooxygenase (COX) pathway. We investigated a role for cPLA2 in mouse lung tumorigenesis by using mice genetically deficient in cPLA2. After multiple urethane injections into cPLA2 null mice and wild-type littermates, the number of lung tumors was determined. cPLA2 null mice developed 43% fewer tumors (from 16 +/- 2 to 9 +/- 2 tumors/mouse; P < 0.05) than wild-type littermates. cPLA2, COX-1, COX-2 and microsomal prostaglandin E2 synthase (mPGES), examined by immunohistochemistry, are present in alveolar and bronchiolar epithelia and in alveolar macrophages in lungs from naive mice and tumor-bearing mice. Tumors express higher levels of each of these four enzymes than control lungs, as determined by immunoblotting. No differences were detected in the contents of COX-1, COX-2 and mPGES between wild-type and cPLA2 null mice. Although the steady-state levels of prostaglandin E2 and prostaglandin I2 in lung tissue extracts prepared from wild-type or cPLA2 (-/-) mice were not significantly different, both prostaglandins markedly increased in tumors from wild-type mice, an increase that was significantly blunted in tumors from cPLA2 (-/-) mice. These results demonstrate a role for cPLA2 in mouse lung tumorigenesis that may be mediated by decreased prostaglandin synthesis.