Juan F. Muñoz-Gutiérrez’s research while affiliated with Colorado State University and other places

In this retrospective study, we describe the clinicopathologic and immunohistochemical findings in a series of primary central nervous system (CNS) neoplasms in African hedgehogs (Atelerix albiventris). Twelve CNS neoplasms were found among 762 African hedgehog submissions (1.6%) to a private diagnostic laboratory in an 18-y period. The median age of affected hedgehogs was 3.5 y. No sex predilection was found. Hindlimb paresis, weakness, and ataxia were the most commonly reported clinical signs. Gangliogliomas (n = 6) and astrocytomas (n = 5) were the most commonly observed neoplasms; one oligodendroglioma was found. Gangliogliomas were found in the cerebellar white matter (2 of 6), brainstem (4 of 6), cervical spinal cord (1 of 6), and frontal lobe (1 of 6); one metastasized to the tongue. Gangliogliomas were immunoreactive for neurofilament protein (NFP), glial fibrillary acidic protein (GFAP), S100, and CD34. All astrocytomas were gemistocytic, located in the cerebrum, and none of these neoplasms metastasized. Astrocytomas were positive for GFAP, S100, and CD34, but negative for NFP. The oligodendroglioma was located in the cerebrum, and was positive for S100, but negative for GFAP and NFP.

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrPC) is misfolded into an accumulating, disease-associated isoform (PrPD). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors contributing to cellular permissiveness. We previously isolated five clones from an immortalized subline of ovine microglia, two of which had demonstrated differential permissiveness to a natural isolate of sheep scrapie and distinct transcriptomic profiles. To more robustly identify factors contributing to this activity, relative permissiveness, cell proliferation, selected gene transcript level, and matrix metalloproteinase 2 (MMP2) activity were compared amongst all five clones. Differences in cell proliferation were not detected between clones; however, significant correlations were identified between relative permissiveness and genes associated with cell growth (i.e., RARRES1 and PTN), protein degradation (i.e., CTSB and SQSTM1), and heparin binding (i.e., SEPP1). MMP2 activity varied amongst clones, but did not correlate with permissiveness. These associations support the contribution of cell division and protein degradation on the permissiveness of cultured ovine microglia to PrPD.

Background: Ruminants, including sheep and goats (small ruminants), are key agricultural animals in many parts of the world. Infectious diseases, including many viral diseases, are significant problems to efficient production of ruminants. Unfortunately, reagents tailored to viruses of ruminants, and especially small ruminants, are lacking compared to other animals more typically used for biomedical research. Objective: The purpose of this study was to determine the permissibility of a stably immortalized, sheep microglial cell line to viruses that are reported to infect ruminants: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), small ruminant lentiviruses (SRLV), and bovine respiratory syncytial virus (BRSV). Methods: Sublines A and H of previously isolated, immortalized, and characterized (CD14-positive) ovine microglial cells were used. Bovine turbinate cells and goat synovial membrane cells were used for comparison. Cytopathic changes were used to confirm infection of individual wells, which were then counted and used to calculate the 50% tissue culture infectious dose. Uninoculated cells served as negative controls and confirmed that the cells were not previously infected with these viruses using polymerase chain reaction (PCR). Results: Inoculation of the two microglial cell sublines with laboratory and field isolates of BVDV, BoHV-1, and BRSV resulted in viral infection in a manner similar to bovine turbinate cells. Immortalized microglia cells are also permissive to SRLV, similar to goat synovial membrane cells. Conclusion and clinical relevance: These immortalized sheep microglial cells provide a new tool for the study of ruminant viruses in ruminant microglial cell line.

Chromatophoromas are neoplasms arising from pigment-bearing cells (chromatophores) of the dermis. While isolated cases have been reported in the literature, the prevalence and biological behavior of chromatophoromas in snakes are unknown. Forty-two chromatophoromas were identified among 4663 submissions (0.9%) to a private diagnostic laboratory in a 16-year period. The most commonly affected snakes were colubrids (23 cases, 55%) and vipers (8 cases, 19%). The San Francisco garter snake was the most commonly affected species (6 cases; 14% of all affected snake species and 3.7% of all garter snake submissions). No sex predilection was found. The age of 28 snakes ranged from 5 to 27 years. Single cutaneous chromatophoromas were most commonly observed and presented as pigmented cutaneous masses or plaques along any body segment. Euthanasia or death due to progressive neoplastic disease or metastasis was reported in 8 (19%) and 4 (10%) cases, respectively. The survival time of 4 animals ranged from 4 to 36 months. Microscopically, xanthophoromas, iridophoromas, melanocytic neoplasms, and mixed chromatophoromas were identified, with melanocytic neoplasms being most common. Microscopic examination alone was generally sufficient for the diagnosis of chromatophoroma, but immunohistochemistry for S-100 and PNL-2 may be helpful for diagnosing poorly pigmented cases. Moderate to marked nuclear atypia appears to be consistently present in cutaneous chromatophoromas with a high risk of metastasis, while mitotic count, lymphatic invasion, the level of infiltration, and the degree of pigmentation or ulceration were not reliable predictors of metastasis.

PTA precipitation of PK-resistant PrP from microglia lysates. At passage 3 post-inoculation, cell lysates were collected, treated with PK, and incubated with PTA to increase sensitivity of immunoblotting. PK-resistant PrP was precipitated with PTA only from cells of clone 439. The results of three independent culture replicates inoculated with scrapie-positive brainstem homogenates (PrPSc +, lanes 1–3 and 5–7) and one with scrapie-negative inoculum (PrPSc–, lanes 4 and 8) of each microglia clone are shown, and are representative of three experiments. (TIFF)

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β-induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

Comparative transcriptional profiling of highly permissive versus poorly permissive microglia. (XLSX)

Transcript fold-change in highly permissive microglia compared to poorly permissive microglia under different inoculation conditions. Transcriptional profiles of highly permissive and poorly permissive microglia clones under two different inoculation conditions were compared (i.e., mock VS scrapie and scrapie VS mock). Genes with differential transcription in both comparisons (P < 0.05 [Baggerley’s test and Bonferroni correction]) and known biological function across three culture replicates are shown. Gene IDs are on the x—axis and the fold change in transcription relative to highly permissive microglia is on the y—axis. Positive fold changes indicate up-regulation in highly permissive microglia and negative fold changes indicate up-regulation in poorly permissive microglia. (TIF)

GSEA on transcriptional profiling of highly permissive and poorly permissive ovine microglia. (XLSX)

Assessment of ovine microglia for the presence/absence of amoeboid and ramified microglia. (XLSX)