MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have been found in urine and have shown diagnostic potential in human ...nephropathies. Here, we aimed to characterize, for the first time, the feline urinary miRNAome and explore the use of urinary miRNA profiles as non-invasive biomarkers for feline pyelonephritis (PN). Thirty-eight cats were included in a prospective case-control study and classified in five groups: healthy Control cats (n = 11), cats with PN (n = 10), cats with subclinical bacteriuria or cystitis (SB/C, n = 5), cats with ureteral obstruction (n = 7) and cats with chronic kidney disease (n = 5). By small RNA sequencing we identified 212 miRNAs in cat urine, including annotated (n = 137) and putative novel (n = 75) miRNAs. The 15 most highly abundant urinary miRNAs accounted for nearly 71% of all detected miRNAs, most of which were previously identified in feline kidney. Ninety-nine differentially abundant (DA) miRNAs were identified when comparing Control cats to cats with urological conditions and 102 DA miRNAs when comparing PN to other urological conditions. Tissue clustering analysis revealed that the majority of urine samples clustered close to kidney, which confirm the likely cellular origin of the secreted urinary miRNAs. Relevant DA miRNAs were verified by quantitative real-time PCR (qPCR). Eighteen miRNAs discriminated Control cats from cats with a urological condition. Of those, seven miRNAs were DA by both RNAseq and qPCR methods between Control and PN cats (miR-125b-5p, miR-27a-3p, miR-21-5p, miR-27b-3p, miR-125a-5p, miR-17-5p and miR-23a-3p) or DA between Control and SB/C cats (miR-125b-5p). Six additional miRNAs (miR-30b-5p, miR-30c, miR-30e-5p, miR-27a-3p, miR-27b-39 and miR-222) relevant for discriminating PN from other urological conditions were identified by qPCR alone (n = 4) or by both methods (n = 2) (P<0.05). This panel of 13 miRNAs has potential as non-invasive urinary biomarkers for diagnostic of PN and other urological conditions in cats.
Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical ...outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain.
The innate immune system is paramount in the response to and clearance of influenza A virus (IAV) infection in non-immune individuals. Known factors include type I and III interferons and antiviral ...pathogen recognition receptors, and the cascades of antiviral and pro- and anti-inflammatory gene expression they induce. MicroRNAs (miRNAs) are increasingly recognized to participate in post-transcriptional modulation of these responses, but the temporal dynamics of how these players of the antiviral innate immune response collaborate to combat infection remain poorly characterized. We quantified the expression of miRNAs and protein coding genes in the lungs of pigs 1, 3, and 14 days after challenge with swine IAV (H1N2). Through RT-qPCR we observed a 400-fold relative increase in IFN-λ3 gene expression on day 1 after challenge, and a strong interferon-mediated antiviral response was observed on days 1 and 3 accompanied by up-regulation of genes related to the pro-inflammatory response and apoptosis. Using small RNA sequencing and qPCR validation we found 27 miRNAs that were differentially expressed after challenge, with the highest number of regulated miRNAs observed on day 3. In contrast, the number of protein coding genes found to be regulated due to IAV infection peaked on day 1. Pulmonary miRNAs may thus be aimed at fine-tuning the initial rapid inflammatory response after IAV infection. Specifically, we found five miRNAs (ssc-miR-15a, ssc-miR-18a, ssc-miR-21, ssc-miR-29b, and hsa-miR-590-3p)-four known porcine miRNAs and one novel porcine miRNA candidate-to be potential modulators of viral pathogen recognition and apoptosis. A total of 11 miRNAs remained differentially expressed 14 days after challenge, at which point the infection had cleared. In conclusion, the results suggested a role for miRNAs both during acute infection as well as later, with the potential to influence lung homeostasis and susceptibility to secondary infections in the lungs of pigs after IAV infection.
Background
Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs ...(miRNAs) have promise as non‐invasive biomarkers in serum and feces for diagnosis of GIC.
Hypothesis/Objectives
Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats.
Animals
Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client‐owned.
Methods
Cats were recruited for an international multicenter observational prospective case‐control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real‐time PCR (RT‐qPCR).
Results
Serum miR‐223‐3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval CI, 0.760‐1.0), sensitivity of 90% (95% CI, 59.6‐99.5%), and specificity of 77.8% (95% CI, 45.3‐96.1%). Serum miR‐223‐3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE.
Conclusion and Clinical Importance
Serum miR‐223‐3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.
Background
Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as ...diagnostic and prognostic markers of GI disease in humans and dogs.
Hypothesis/Objectives
Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. Aims: (a) identify miRNAs that differentiate GIC from CIE, (b) use high‐throughput reverse transcription quantitative real‐time PCR (RT‐qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs.
Animals
Twenty‐four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client‐owned.
Methods
An international multicenter observational prospective case‐control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT‐qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE.
Results
The best diagnostic performance for distinguishing GIC from CIE was fecal miR‐451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR‐223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR‐27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR‐20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR‐148a‐3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR‐652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR‐451 and miR‐223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%).
Conclusions and Clinical Importance
When used as part of a diagnostic RT‐qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.
is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence ...potential of
cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of
infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.
In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell ...culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.
MicroRNAs (miRNAs) contribute to post-transcriptional modulation of the host response during influenza A virus (IAV) infection and may be involved in shaping disease severity. Differential disease ...severity was achieved in two groups of pigs by immunization of one group with a commercial swine IAV vaccine prior to heterologous IAV (H1N2) challenge of both groups. Lung tissue was harvested 1, 3, and 14 days after challenge and miRNA expression was quantified. Gene Ontology term enrichment analysis was employed to examine the functional relevance of genes potentially regulated by differentially expressed miRNAs in pigs with varying degrees of disease severity following IAV infection. Results suggested that the miRNA response associated with less severe disease may modulate host mechanisms essential for viral life cycle, e.g. transcription, translation, and protein trafficking. During more severe disease, miRNA-mediated regulation may focus on dampening virus-specific processes e.g. virion assembly and viral protein processing, and controlling host metabolism.
•Influenza A viruses (IAV) can induce disease of varying severity in the host.•MicroRNAs are involved in modulating the host response during IAV infection.•Different IAV disease severity levels are associated with distinct miRNA profiles.
Nasal mucosal explant (NEs) cultured at an air–liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom ...microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses.
Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs.
Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2–43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2–16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2–26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.
Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic ...diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.
PDF
