What Is Possible and What Questions Can Be Asked?

In recent years several technologies for the complete analysis of the transcriptome and proteome have reached a technological level which allows their routine application as scientific tools. The principle of these methods is the identification and quantification of up to ten thousands of RNA and proteins species in a tissue, in contrast to the sequential analysis of conventional methods such as PCR and Western blotting. Due to their technical progress transcriptome and proteome analyses are becoming increasingly relevant in all fields of biological research. They are mainly used for the explorative identification of disease associated complex gene expression patterns and thereby set the stage for hypothesis-driven studies. This review gives an overview on the methods currently available for transcriptome analysis, that is, microarrays, Ref-Seq, quantitative PCR arrays and discusses their potentials and limitations. Second, the most powerful current approaches to proteome analysis are introduced, that is, 2D-gel electrophoresis, shotgun proteomics, MudPIT and the diverse technological concepts are reviewed. Finally, experimental strategies for biomarker discovery, experimental settings for the identification of prognostic gene sets and explorative versus hypothesis driven approaches for the elucidation of diseases associated genes and molecular pathways are described and their potential for studies in veterinary research is highlighted

Malignancy Associated MicroRNA Expression Changes in Canine Mammary Cancer of Different Malignancies

MicroRNA has been suspected to be generally involved in carcinogenesis since their first description. A first study supported this assumption for canine mammary tumors when miRNA expression was compared to normal gland. The present study extends these results by comparing the expression of 16 microRNA (miRNA) and 4 small nucleolar RNA (snoRNA) in tumors of different malignancy, for example, adenomas, nonmetastasizing and metastasizing carcinomas as well as lymph node metastases, with each other and with normal mammary gland. All neoplastic tissues differed in their miR-210 expression levels from normal gland. While metastatic cells differed in their expression of mir-29b, miR-101, mir-125a, miR-143, and miR-145 from primary tumors, the comparison of miRNA expression in primary tumors of different malignancy failed to reveal significant differences except for a significant downregulation of mir-125a in metastasizing carcinomas when compared to adenomas

Molecular characterization of the feline T-cell receptor γ alternate reading frame protein (TARP) ortholog

T-cell receptor γ alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor γ sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP

The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern- recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis

Extending the host range of Listeria monocytogenes by rational protein design

SummaryIn causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis

Tailoring the Antibody Response to Aggregated Aß Using Novel Alzheimer-Vaccines

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study

protection by adrenomedullin

Ventilator-induced lung injury (VILI) contributes to morbidity and mortality in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs are susceptible to VILI despite protective ventilation. In a previous study, the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI. We hypothesized that mechanical ventilation (MV) contributes to lung injury and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ failure in ventilated mice with pneumococcal pneumonia. We analyzed in mice the impact of MV in established pneumonia on lung injury, inflammation, bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed the therapeutic potential of AM by starting treatment at intubation. In pneumococcal pneumonia, MV increased lung permeability, and worsened lung mechanics and oxygenation failure. MV dramatically increased lung and blood cytokines but not lung leukocyte counts in pneumonia. MV induced systemic leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung and blood bacterial burden was not affected by MV pneumonia and MV increased lung AM expression, whereas receptor activity modifying protein (RAMP) 1-3 expression was increased in pneumonia and reduced by MV. Infusion of AM protected against MV-induced lung injury (66% reduction of pulmonary permeability p<0.01; prevention of pulmonary restriction) and against VILI- induced liver and gut injury in pneumonia (91% reduction of AST levels p<0.05, 96% reduction of alanine aminotransaminase (ALT) levels p<0.05, abrogation of histopathological changes and parenchymal apoptosis in liver and gut). MV paved the way for the progression of pneumonia towards ARDS and sepsis by aggravating lung injury and systemic hyperinflammation leading to liver, kidney and gut injury. AM may be a promising therapeutic option to protect against development of lung injury, sepsis and extrapulmonary organ injury in mechanically ventilated individuals with severe pneumonia

Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis- inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB- dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury

Protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters

Introduction The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyperinflammatory state generated on infection, reducing lung tissue pathology and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. Methods In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib (STA-9090) in vitro on alveolar epithelial cells and alveolar macrophages to characterise its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection. Results In vitro, STA-9090 reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of proinflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of STA-9090 led to an overall improvement of the clinical condition of infected animals, with reduced oedema formation and lung tissue pathology. Conclusion Altogether, we show that HSP90 inhibition could serve as a potential treatment option for moderate and severe cases of COVID-19

Human alveolar progenitors generate dual lineage bronchioalveolar organoids

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3β inhibitor, CHIR99021. By RNA-seq profiling of GSK-3β knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction