Mpox Viral Lineage Analysis and Technique Development Using Next-generation Sequencing Approach.

BACKGROUND: In response to Mpox endemic and public health emergency, DCHHS aimed to develop NGS based techniques to streamline Mpox viral clade and lineage analysis. METHODS: The Mpox sequencing workflow started with DNA extraction and adapted Illumina's COVIDSeq assay using hMpox primer pools from Yale School of Public Health. Sequencing steps included cDNA amplification, tagmentation, PCR indexing, pooling libraries, sequencing on MiSeq, data analysis, and report generation. The bioinformatic analysis comprised read assembly and consensus sequence mapping to reference genomes and variant identification, and utilized pipelines including Illumina BaseSpace, NextClade, CLC Workbench, Terra.bio for data quality control (QC) and validation. RESULTS: In total, 171 mpox samples were sequenced using modified COVIDSeq workflow and QC metrics were assessed for read quality, depth, and coverage. Multiple analysis pipelines identified the West African clade IIb as the only clade during peak Mpox infection from July through October 2022. Analyses also indicated lineage B.1.2 as the dominant variant comprising the majority of Mpox viral genomes (77.7%), implying its geographical distribution in the United States. Viral sequences were uploaded to GISAID EpiPox. CONCLUSIONS: We developed NGS workflows to precisely detect and analyze mpox viral clade and lineages aiding in public health genomic surveillance.

MicroRNA-145 Antagonism Reverses TGF-ß Inhibition of F508del CFTR Correction in Airway Epithelia.

RATIONALE: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-ß. OBJECTIVES: To demonstrate that miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. METHODS: Primary human CF (F508del homozygous) and non-CF airway epithelial cells were grown to terminal differentiation at the air-liquid interface on permeable supports. TGF-ß (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 µM) and ivacaftor (10 µM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function was done by standard techniques. MEASUREMENTS AND MAIN RESULTS: miR-145 is increased fourfold in CF BAL fluid compared with non-CF (P < 0.01) and increased 10-fold in CF primary airway epithelial cells (P < 0.01). Exogenous TGF-ß doubles miR-145 expression (P < 0.05), halves wild-type CFTR mRNA and protein levels (P < 0.01), and nullifies lumacaftor/ivacaftor F508del CFTR correction. miR-145 overexpression similarly decreases wild-type CFTR protein synthesis (P < 0.01) and function (P < 0.05), and eliminates F508del corrector benefit. miR-145 antagonism blocks TGF-ß suppression of CFTR and enhances lumacaftor correction of F508del CFTR. CONCLUSIONS: miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. Specific antagonists to miR-145 interrupt TGF-ß signaling to restore F508del CFTR modulation. miR-145 antagonism may offer a novel therapeutic opportunity to enhance therapeutic benefit of F508del CFTR correction in CF epithelia.

Altered microRNA Expression Profiles and Regulation of INK4A/CDKN2A Tumor Suppressor Genes in Canine Breast Cancer Models.

microRNA (miRNA) expression profiling of cancer versus normal cells may reveal the characteristic regulatory features that can be correlated to altered gene expression in both human and animal models of cancers. In this study, the comprehensive expression profiles of the 277 highly characterized miRNAs from the canine genome were evaluated in spontaneous canine mammary tumor (CMT) models harboring defects in a group of cell cycle regulatory and potent tumor suppressor genes of INK4/CDKN2 family including p16/INK4A, p14ARF, and p15/INK4B. A large number of differentially expressed miRNAs were identified in three CMT cell lines to potentially target oncogenes, tumor suppressor genes and cancer biomarkers. A group of the altered miRNAs were identified by miRNA target prediction tools for regulation of the INK4/CDKN2 family tumor suppressor genes. miRNA-141 was experimentally validated for INK4A 3'-UTR target binding in the CMT cell lines providing an essential mechanism for the post-transcriptional regulation of the INK4A tumor suppressor gene in CMT models. A well-recognized group of miRNAs including miR-21, miR-155, miR-9, miR-34a, miR-143/145, and miR-31 were found to be altered in both CMTs and human breast cancer. These altered miRNAs might serve as potential targets for advancing the development of future therapeutic reagents. These findings further strengthen the validity and use of canine breast cancers as appropriate models for the study of human breast cancers.

Novel frameshift mutation in the p16/INK4A tumor suppressor gene in canine breast cancer alters expression from the p16/INK4A/p14ARF locus.

The INK4 family of cyclin-dependent kinase inhibitors (CKI) encode important cell cycle regulators that tightly control cell cycle during G1 to S phase. These related genes are considered tumor suppressors as loss of function contributes to the malignant phenotype. Expression of CKIs p16, p14ARF, or p15 were defective in six different canine mammary tumor (CMT) cell lines compared to normal thoracic canine fibroblasts. This suggests CKI defects are frequently responsible for neoplastic transformation in canine mammary carcinomas. p16 and p14ARF are two alternatively spliced products derived from the canine p16/INK4A/p14ARF gene locus. Despite omissions in the published p16 transcript and canine genome and the presence of GC-rich repeats, we determined the complete coding sequence of canine p16 revealing a deletion and frameshift mutation in p16 exon 1α in CMT28 cells. In addition, we determined canine p14ARF mRNA and protein sequences. Mapping of these mutations uncovered important aspects of p16 and p14ARF expression and defects in CMT28 cells shifting the p16 reading frame into p14ARF making a fusion protein that was predicted to be truncated, unstable and devoid of structural and functional integrity. This data describes an important neoplastic mechanism in the p16/INK4A/p14ARF locus in a spontaneous canine model of breast cancer.

Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma.

The cyclin-dependent kinase inhibitors (CKIs) belong to a group of key cell cycle proteins that regulate important cancer drug targets such as the cyclin/CDK complexes. Gene defects in the INK4A/B CKI tumor suppressor locus are frequently associated with human cancers and we have previously identified similar defects in canine models. Many of the cancer-associated genetic alterations, known to play roles in mammary tumor development and progression, appear similar in humans and dogs. The objectives of this study were to characterize expression defects in the INK4 genes, and the encoded p16 family proteins, in spontaneous canine primary mammary tumors (CMT) as well as in canine malignant melanoma (CML) cell lines to further develop these models of spontaneous cancers. Gene expression profiles and characterization of p16 protein were performed by rtPCR assay and immunoblotting followed by an analysis of relevant sequences with bioinformatics. The INK4 gene family were expressed differentially and the genes encoding the tumor suppressor p16, p14, and p15 proteins were often identified as defective in CMT and CML cell lines. The altered expression profiles for INK4 locus encoded tumor suppressor genes was also confirmed by the identification of similar gene defects in primary canine mammary tumor biopsy specimens which were also comparable to defects found in human breast cancer. These data strongly suggest that defects identified in the INK4 locus in canine cell lines are lesions originating in spontaneous canine cancers and are not the product of selection in culture. These findings further validate canine tumor models for use in developing a clear understanding of the gene defects present and may help identify new therapeutic cancer treatments that restore these tumor suppressor pathways based on precision medicine in canine cancers.

CFTR dysfunction increases endoglin and TGF-ß signaling in airway epithelia.

Endoglin (ENG) regulates signaling by transforming growth factor-ß (TGF-ß), a genetic modifier of cystic fibrosis (CF) lung disease severity. We hypothesized that ENG mediates TGF-ß pathobiology in CF airway epithelia. Comparing CF and non-CF human lungs, we measured ENG by qPCR, immunoblotting and ELISA. In human bronchial epithelial cell lines (16HBE), we used CFTR siRNA knockdown and functional inhibition (CFTRINH -172) to connect loss of CFTR to ENG synthesis. Plasmid overexpression of ENG assessed the direct effect of ENG on TGF-ß transcription and signal amplification in 16HBE cells. We found ENG protein to be increased more than fivefold both in human CF bronchoalveolar fluid (BALF) and human CF lung homogenates. ENG transcripts were increased threefold in CF, with a twofold increase in TGF-ß signaling. CFTR knockdown in 16HBE cells tripled ENG transcription and doubled protein levels with corresponding increases in TGF-ß signaling. Plasmid overexpression of ENG alone nearly doubled TGF-ß1 mRNA and increased TGF-ß signaling in 16HBE cells. These experiments identify that loss of CFTR function increases ENG expression in CF epithelia and amplifies TGF-ß signaling. Targeting ENG may offer a novel therapeutic opportunity to address TGF-ß associated pathobiology in CF.

Autologous hybrid cell fusion vaccine in a spontaneous intermediate model of breast carcinoma.

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer.

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.

Canine Mammary Carcinomas: A Comparative Analysis of Altered Gene Expression.

Breast cancer represents the second most frequent neoplasm in humans and sexually intact female dogs after lung and skin cancers, respectively. Many similar features in human and dog cancers including, spontaneous development, clinical presentation, tumor heterogeneity, disease progression and response to conventional therapies have supported development of this comparative model as an alternative to mice. The highly conserved similarities between canine and human genomes are also key to this comparative analysis, especially when compared to the murine genome. Studies with canine mammary tumor (CMT) models have shown a strong genetic correlation with their human counterparts, particularly in terms of altered expression profiles of cell cycle regulatory genes, tumor suppressor and oncogenes and also a large group of non-coding RNAs or microRNAs (miRNAs). Because CMTs are considered predictive intermediate models for human breast cancer, similarities in genetic alterations and cancer predisposition between humans and dogs have raised further interest. Many cancer-associated genetic defects critical to mammary tumor development and oncogenic determinants of metastasis have been reported and appear to be similar in both species. Comparative analysis of deregulated gene sets or cancer signaling pathways has shown that a significant proportion of orthologous genes are comparably up- or down-regulated in both human and dog breast tumors. Particularly, a group of cell cycle regulators called cyclin-dependent kinase inhibitors (CKIs) acting as potent tumor suppressors are frequently defective in CMTs. Interestingly, comparative analysis of coding sequences has also shown that these genes are highly conserved in mammals in terms of their evolutionary divergence from a common ancestor. Moreover, co-deletion and/or homozygous loss of the INK4A/ARF/INK4B (CDKN2A/B) locus, encoding three members of the CKI tumor suppressor gene families (p16/INK4A, p14ARF and p15/INK4B), in many human and dog cancers including mammary carcinomas, suggested their important conserved genetic order and localization in orthologous chromosomal regions. miRNAs, as powerful post-transcriptional regulators of most of the cancer-associated genes, have not been well evaluated to date in animal cancer models. Comprehensive expression profiles of miRNAs in CMTs have revealed their altered regulation showing a strong correlation with those found in human breast cancers. These genetic correlations between human and dog mammary cancers will greatly advance our understanding of regulatory mechanisms involving many critical cancer-associated genes that promote neoplasia and contribute to the promising development of future therapeutics.

Engraftment of canine peripheral blood lymphocytes into nonobese diabetic-severe combined immune deficient IL-2R common gamma chain null mice.

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain -/- (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 10(7) peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45(+) CD4(+)), cytotoxic lymphocytes (CD45(+) CD8(+)), regulatory T cells (CD45(+) CD4(+) Foxp3(+)), and B cells (CD45(+) Ig(+) CD21lo). Canine CD45(+) lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4(+) T cells, of which Foxp-3(+) CD25hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8(+) CTL's and Ig(+) B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.