MicroRNA Biomarkers in Canine Diffuse Large B-Cell Lymphoma.

Lymphoma is among the most common cancer in dogs. Diffuse large B-cell lymphoma (DLBCL) is the predominant type, accounting for up to half of all cases. Definitive diagnosis of DLBCL relies on cytologic evaluation with immunophenotyping, or histopathology and immunohistochemistry when needed. A rapid and specific molecular test aiding in the diagnosis could be beneficial. Noncoding microRNAs (miRNAs) are regulators of gene expression involved in a variety of cellular processes, including cell differentiation, cell cycle progression, and apoptosis. Not surprisingly, miRNA expression is aberrant in diseases such as cancers. Their high stability and abundance in tissues make them promising biomarkers for diagnosing and monitoring diseases. This study aimed to identify miRNA signatures of DLBCL to develop ancillary molecular diagnostic tools. miRNA was isolated from formalin-fixed, paraffin-embedded lymph node tissue from 22 DLBCL and 14 nonneoplastic controls. Relative gene expression of 8 tumor-regulating miRNAs was achieved by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction). The results showed downregulation of the let-7 family of miRNAs and miR-155, whereas miR-34a was upregulated in DLBCL compared to the controls. We demonstrated that the combination of expression levels of miR-34a and let-7f or of let-7b and let-7f achieved 100% differentiation between DLBCL and controls. Furthermore, let-7f alone discriminated DLBCL from nonneoplastic tissue in 97% of cases. Our results represent one step forward in search of a rapid and accurate ancillary diagnostic test for DLBCL in dogs.

Development of NP-Based Universal Vaccine for Influenza A Viruses.

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.

MicroRNAs implicated in canine diffuse large B-cell lymphoma prognosis.

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin lymphoma (NHL) in domestic dogs, with many similarities to its human counterpart. The progression of the disease is rapid, and treatment must be initiated early to achieve cancer remission and extend life. This study examined the relationship between progression-free survival (PFS) and microRNA (miRNA) expression in dogs with DLBCL. miRNAs are small non-coding RNA molecules that typically regulate gene expression post-transcriptionally. They are involved in several pathophysiological processes, including the growth and progression of cancer. Based on the analysis of small RNA sequencing (sRNA-seq) data, we validated a group of miRNAs in lymph nodes from 44 DLBCL-affected dogs with known outcomes. We used quantitative PCR to quantify their expression and report a specific subset of miRNAs is associated with decreased PFS in dogs with DLBCL. The miR-192-5p and miR-16-5p expression were significantly downregulated in dogs with increased PFS. These results indicate that miRNA profiling may potentially identify dogs with DLBCL that will experience poor outcomes following treatment. Identifying specific miRNAs that correlate with the progression of canine DLBCL could aid the development of individualized treatment regimens for dogs.

Hemotropic Mycoplasmas (Hemoplasmas) in Free-Ranging Azara's Agoutis (Dasyprocta azarae) from an Urban Area of Southern Brazil.

Hemotropic mycoplasmas (hemoplasmas) are opportunistic bacteria that attach to the erythrocyte surface, causing infectious anemia in several mammalian species, including rodents. Studies surveying native Azara's agoutis (Dasyprocta azarae) in Brazil are lacking. Accordingly, the present study aimed to assess hemoplasmas infection in free-ranging agoutis from an urban environmental conservation area in Curitiba, southern Brazil. Overall, 11/35 (31.43%) agoutis were positive to hemoplasmas by quantitative PCR (cycle threshold≤34.4). Sequencing of the 16S ribosomal RNA gene indicated Mycoplasma haemomuris infection, closely related to M. haemomuris subsp. ratti, suggesting hemoplasma transmission from urban rats to agoutis. Because the main route of M. haemomuris transmission has been direct rodent-to-rodent infection, the relatively lower positivity that we detected may be the result of low intraspecies contact due to the smaller social units of agoutis, generally consisting of two to four individuals, and low interspecies contact due to only sporadic agouti-rat interactions in urban settings, compared with other rodent species interactions. Further studies should be conducted to determine whether the hemoplasma infection that we found can cause clinical onset and life-threatening anemia in agoutis.

Enhancement of mucosal innate and adaptive immunity following intranasal immunization of mice with a bovine adenoviral vector.

Introduction: Nonhuman adenoviral (AdV) gene delivery platforms have significant value due to their ability to elude preexisting AdV vector immunity in most individuals. Previously, we have demonstrated that intranasal (IN) immunization of mice with BAd-H5HA, a bovine AdV type 3 (BAdV3) vector expressing H5N1 influenza virus hemagglutinin (HA), resulted in enhanced humoral and cell-mediated immune responses. The BAd-H5HA IN immunization resulted in complete protection following the challenge with an antigenically distinct H5N1 virus compared to the mouse group similarly immunized with HAd-H5HA, a human AdV type 5 (HAdV5) vector expressing HA. Methods: Here, we attempted to determine the activation of innate immune responses in the lungs of mice inoculated intranasally with BAd-H5HA compared to the HAd-H5HA-inoculated group. Results: RNA-Seq analyses of the lung tissues revealed differential expression (DE) of genes involved in innate and adaptive immunity in animals immunized with BAd-H5HA. The top ten enhanced genes were verified by RT-PCR. Consistently, there were transient increases in the levels of cytokines (IL-1α, IL-1ß, IL-5, TNF- α, LIF, IL-17, G-CSF, MIP-1ß, MCP-1, MIP-2, and GM-CSF) and toll-like receptors in the lungs of the group inoculated with BAdV vectors compared to that of the HAdV vector group. Conclusion: These results demonstrate that the BAdV vectors induce enhanced innate and adaptive immunity-related factors compared to HAdV vectors in mice. Thus, the BAdV vector platform could be an excellent gene delivery system for recombinant vaccines and cancer immunotherapy.

miRNome expression analysis in canine diffuse large B-cell lymphoma.

Introduction: Lymphoma is a common canine cancer with translational relevance to human disease. Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype, contributing to almost fifty percent of clinically recognized lymphoma cases. Identifying new biomarkers capable of early diagnosis and monitoring DLBCL is crucial for enhancing remission rates. This research seeks to advance our knowledge of the molecular biology of DLBCL by analyzing the expression of microRNAs, which regulate gene expression by negatively impacting gene expression via targeted RNA degradation or translational repression. The stability and accessibility of microRNAs make them appropriate biomarkers for the diagnosis, prognosis, and monitoring of diseases. Methods: We extracted and sequenced microRNAs from ten fresh-frozen lymph node tissue samples (six DLBCL and four non-neoplastic). Results: Small RNA sequencing data analysis revealed 35 differently expressed miRNAs (DEMs) compared to controls. RT-qPCR confirmed that 23/35 DEMs in DLBCL were significantly upregulated (n = 14) or downregulated (n = 9). Statistical significance was determined by comparing each miRNA's average expression fold-change (2-Cq) between the DLCBL and healthy groups by applying the unpaired parametric Welch's 2-sample t-test and false discovery rate (FDR). The predicted target genes of the DEMs were mainly enriched in the PI3K-Akt-MAPK pathway. Discussion: Our data point to the potential value of miRNA signatures as diagnostic biomarkers and serve as a guideline for subsequent experimental studies to determine the targets and functions of these altered miRNAs in canine DLBCL.

Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.

Pythium insidiosum is an infectious oomycete affecting dogs that develop the cutaneous or gastrointestinal form of pythiosis with a poor prognosis. If left untreated, pythiosis may be fatal. This organism is not a true fungus because its cell wall and cell membrane lack chitin and ergosterol, respectively, requiring specific treatment. Identifying the organism is challenging, as a hematoxylin and eosin (H&E) stain poorly stain the P. insidiosum hyphae and cannot be differentiated conclusively from other fungal or fungal-like organisms (such as Lagenidium sp.) morphologically. Our study aimed to develop a nested PCR to detect P. insidiosum and compare it with the traditional histopathologic detection of hyphae. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls from 26 dogs with lesions suggesting the P. insidiosum infection were assessed histologically, and DNA was extracted from the FFPE tissue sections for nested PCR. Agreement between the histologic stains, (H&E), periodic acid-Schiff (PAS), and/or Grocott methenamine silver (GMS) and the nested PCR occurred in 18/26 cases. Hyphae consistent with Pythium sp. were identified via histopathology in 57.7% of the samples, whereas the nested PCR detected P. insidiosum in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Using a combination of histopathology evaluation and nested PCR is recommended to aid in the accurate diagnosis of pythiosis.