Magnetic Resonance Imaging of Pulmonary Lesions in Guinea Pigs Infected with Mycobacterium tuberculosis

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines

Integrated pathways associated with metastasis and chemoresistance in canine osteosarcoma

2015 Summer.Osteosarcoma (OS) is the most common canine primary bone tumor affecting 10,000 dogs every year. This aggressive cancer is characterized by both a high rate of metastasis and chemotherapeutic resistance. It is estimated up to 80% of patients carry silent metastases at the time of diagnosis, and most will progress despite removal of the primary tumor and chemotherapy. Canine OS is strikingly similar to the disease in humans following a similar clinical course and sharing genetic and molecular aberrations. Thus the canine disease has gained recognition as a relevant spontaneous tumor model for human OS. Unfortunately, survival rates for both species have plateaued with no significant gains made in the last 20-30 years. New treatment strategies are needed and will likely consist of combined therapies including conventional chemotherapy drugs along with targeted and immune modulating agents. The success of clinical trials to evaluate these novel therapies will rely on improved understanding of molecular pathways contributing to progression and chemotherapy resistance of OS. Further, molecular characterization of OS will provide biomarkers essential for prognosis, treatment planning and patient monitoring. Gene expression profiling of canine tumors from poor responders compared to tumors from good responders implicated pathways critical to normal bone development including hedgehog and Wnt/β-catenin. During bone development, there is significant crosstalk between these pathways and the Notch signaling pathway, a third developmental pathway associated with growth and survival in a variety of human cancers. We performed pathway focused gene expression studies using canine and human OS cells, canine OS tumors and normal bone samples to evaluate two Notch receptors and two downstream effectors. We identified expression changes consistent with Notch activation in OS compared to normal bone. We further determined that, while expression of three Notch associated genes remained elevated in tumors from the poor responders, expression of hairy/enhancer of split 1 (HES1) was significantly lower in tumors from poor responders than in tumors from good responders. Survival analyses based on immunoreactivity for HES1 in fixed tissues from an independent tumor set confirmed the association between low HES1 expression and poor outcome. To further explore the Notch pathway in OS and elucidate potential mechanisms underlying the disruption of Notch/HES1 signaling in the most aggressive tumors, we performed miRNA expression profiling of canine tumors. Our goals included identification of miRNA signatures associated with patient outcome in OS as well as integration of miRNA and gene expression data for additional pathway-focused explorations. Further, we endeavored to find miRNA biomarkers in serum of OS patients with prognostic potential. We successfully identified a tumor-based three-miRNA signature and a serum-based two-miRNA signature that separated patients into distinct outcome groups with good accuracy. In silico miRNA-mRNA interaction analyses of dysregulated miRNAs and Notch-associated genes in tumors compared to normal bone revealed nearly 20 interactions, validated experimentally in other systems, potentially associated with OS. Interaction and pathway analysis of aberrant miRNA and gene expression in tumors from poor responders vs. good responders identified insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), an oncogene of interest in OS, as a common target of seven down-regulated miRNAs. Finally, these analyses suggested interactions with the tumor microenvironment are important to the progression of OS. We expanded our miRNA expression profiling to include microarray analysis of 29 canine cancer cell lines. This allowed us to utilize drug sensitivity data from in vitro assays, where cells were treated with either doxorubicin or carboplatin, to identify "drug-resistance-related" miRNAs associated with outcome in canine OS tumors. We identified an additional miRNA from this cell-based approach, which participated in a tumor-based four-miRNA predictive signature. In silico miRNA-gene regulatory pathway analyses of outcome associated miRNAs and dysregulated genes from predictive doxorubicin and carboplatin models, developed using the COXEN algorithm, implicated the Notch pathway as contributing to doxorubicin resistance. Finally, pathway analyses of the top five miRNAs associated with progression of OS and chemoresistance: let-7b, miR-98, miR-130a, miR-181b and miR-223 implicated the PI3K/AKT pathway in progression of OS. Taken together, the studies described herein, provide an integrated picture of Notch signaling in OS elaborating candidate miRNA-gene interactions associated with development and progression of OS and resistance to doxorubicin. Further, these studies have revealed key miRNA-mRNA interactions that implicate other targetable pathways and thus, may serve as biomarkers for patient stratification, enhancing efforts towards integration of individualized targeted therapies in OS. Finally, we have identified miRNA-based prognostic signatures measurable from OS tumors or patient serum, which laid the groundwork for development of a clinically useful prognostic screen for OS

A New Understanding of Ebola Virus Disease: A Review of Post Ebola Syndrome and Viral Persistence

Ebola Virus Disease (EVD), caused by Ebola virus, is highly fatal and is mostly restricted to African countries. EVD was previously described as an acute illness, but chronic illness can result as well, making infection more of a public health concern in recent years. New developments have demonstrated that individuals can have new and/or lingering symptoms post recovery, termed post-Ebola syndrome. Another phenomenon is the persistence of Ebola virus in immune privileged sites in survivors post recovery that can lead to viral transmission and/or relapsing infection. Four cohort studies were reviewed that looked at symptoms in survivors, with different levels of follow-up and criteria. A total of 1,833 survivors were enrolled in these studies, all survivors of the 2014-2016 outbreak. This outbreak was very difficult to control, which led to more cases of disease and more disease survivors. Symptoms reported most frequently in survivors included joint pain, headache, and fatigue. Persistence of Ebola virus post recovery has most often been detected in semen but has also been found in breast milk and ocular fluid. Viral persistence in semen has led to a minimum of 112 cases of EVD. Breast milk was linked to at least one case of EVD (from mother to child), but ocular persistence has not yet been linked to transmission. Ebola virus is a zoonotic pathogen, making eradication an unlikely scenario. There is increased need for prevention efforts to combat the high rates of post-Ebola syndrome and instances of viral persistence in survivors, including increasing vaccination efforts of the population at risk and receipt of EVD specific treatments in infected individuals. Treatments specific for post-Ebola syndrome need to be assessed to help the growing population suffering from chronic symptoms. Despite the invaluable studies reviewed in this analysis, the pathogenesis and emergence of post-Ebola syndrome and EBOV persistence is relatively unknown and requires further study to make a meaningful impact on the 500 million individuals at risk for EVD in African countries

Klossiella equi Infection in an Immunosuppressed Horse: Evidence of Long-Term Infection

A 13-year-old quarter horse gelding presented with a history of hematuria of approximately 1-year duration, anemia, weight loss over the previous six months, and bilateral nasal discharge of 2-week duration. It was determined that hematuria was most likely caused by the coccidian parasite Klossiella equi. Additional case workup suggested a diagnosis of pituitary pars intermedia dysfunction. Confirmatory testing was declined by the owners and the horse was discharged on medical therapy. Despite initial improvement after discharge, the horse developed unresolving sinusitis approximately 1 year later and was euthanized. Necropsy confirmed the presence of an adenoma of the pars intermedia of the pituitary gland, supporting the initial diagnosis. Additional findings included multiple developmental stages of K. equi present in the kidneys. This finding demonstrates infections with K. equi can be chronic in nature and supports the association of increased severity of klossiellosis and impaired immune function

Magnetic Resonance Imaging of Pulmonary Lesions in Guinea Pigs Infected with Mycobacterium tuberculosis

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines

Chemical Profiling of A‐to‐I RNA Editing Using a Click‐Compatible Phenylacrylamide

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings