Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

Systemic Caryospora-like coccidiosis in a clutch of hatchling red-eared slider turtles (Trachemys scripta elegans).

Caryospora-like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia/Caryospora-like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora-like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity.

Weight-independent effects of Roux-en-Y gastric bypass surgery on remission of nonalcoholic fatty liver disease in mice.

OBJECTIVE: Obesity is a driver of non-alcoholic fatty liver disease (NAFLD), and interventions that decrease body weight, such as bariatric surgery and/or calorie restriction (CR), may serve as effective therapies. This study compared the effects of Roux-en-Y gastric bypass surgery (RYGB) and CR on hepatic function in mice with obesity and NAFLD. METHODS: C57BL/6J mice were fed a high-fat diet to promote obesity. At 16 weeks of age, mice were randomized to sham surgery (sham), RYGB, or CR weight matched to RYGB (WM). Body weight/composition, food intake, and energy expenditure (EE) were measured throughout treatment. Liver histopathology was evaluated from H&E-stained sections. Hepatic enzymes and glycogen content were determined by ELISA. Transcriptional signatures were revealed via RNA sequencing. RESULTS: RYGB reduced hepatic lipid content and adiposity while increasing EE and lean body mass relative to WM. Hepatic glycogen and bile acid content were increased after RYGB relative to sham and WM. RYGB activated enterohepatic signaling and genes regulating hepatic lipid homeostasis. CONCLUSIONS: RYGB improved whole-body composition and hepatic lipid homeostasis to a greater extent than CR in mice. RYGB was associated with discrete remodeling of the hepatic transcriptome, suggesting that surgery may be mechanistically additive to CR.

The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer.

Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.

Relationship between Uveal Inflammation and Viral Detection in 30 Cats with Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) virus is the most common infectious cause of uveitis in cats. Confirmatory diagnosis is usually only reached at postmortem examination. The relationship between the histologic inflammatory pattern, which depends on the stage of the disease, and the likelihood of detection of the viral antigen and/or RNA has not been investigated. We hypothesized that viral detection rate by either immunohistochemistry, in situ hybridization or RT-qPCR is dependent upon the predominant type of uveal inflammatory response (i.e., pyogranulomatous vs. plasmacytic). Thus, the aims of this study were to evaluate cases of FIP-induced uveitis, localize the viral antigen and RNA, and assess the relationship between the inflammatory pattern (macrophage- vs. plasma cell-rich) and the likelihood of detecting the FIP antigen and/or RNA. We evaluated 30 cats with FIP-induced uveitis. The viral antigen and/or RNA were detected within uveal macrophages in 11/30 cases, of which 8 tested positive by RT-qPCR. Correlation analysis determined a weak to moderate but significant negative correlation between the degree of plasmacytic uveal inflammation and the likelihood of detecting the FIP antigen and RNA. This study suggests that predominance of plasmacytic inflammation in cases of FIP uveitis reduces the odds of a confirmatory diagnosis through the viral detection methods available.

Horizontal and Vertical Transmission of Powassan Virus by the Invasive Asian Longhorned Tick, Haemaphysalis longicornis, Under Laboratory Conditions.

The Asian longhorned tick, Haemaphysalis longicornis, is an ixodid tick native to East Asia that was first detected in North America outside a port of entry in 2017. This invasive species has since been detected in 17 states. As the invasive range of the tick continues to expand, the vector competence of H. longicornis for pathogens native to North America must be assessed. Here, we evaluate the vector competence of H. longicornis for Powassan virus (POWV) under laboratory conditions. POWV is a North American tick-borne flavivirus that is typically transmitted through the bite of Ixodes species ticks. The invasive range of H. longicornis is expected to overlap heavily with the geographic range of Ixodes scapularis and POWV cases, highlighting the potential for this invasive tick species to amplify POWV transmission in natural foci should the native tick vectors and H. longicornis share similar hosts. In these studies, adult female H. longicornis ticks were infected with POWV via anal pore microinjection. Viral RNA and infectious virions were detected in tick tissues via q-RT-PCR and focus-forming assay, respectively. POWV-injected female ticks were infested on mice, and virus was transmitted to mice during tick feeding, as shown by clinical signs of disease and seroconversion in the tick-exposed mice, as well as the detection of viral RNA in various mouse tissues. A POWV-injected female tick transmitted virus to her larval progeny, indicating that H. longicornis can vertically transmit POWV. These naturally-infected larval ticks were also able to transmit POWV to the mouse on which they fed and to the nymphal stage after molting, further demonstrating that H. longicornis can transmit POWV in the horizontal and transstadial modes. Larval and nymphal ticks were also orally infected with POWV while feeding on viremic mice. Additionally, this study provides the first report of POWV neuropathology based on a natural tick transmission model of POWV. Together, our results suggest that the invasive H. longicornis tick is a competent vector of POWV. These findings underline the growing danger this tick may pose to human health in the United States. Additional scholarship on the tick's biology, ecology, and pathogen transmission dynamics in nature will be important towards understanding the full public health impact of this invasive species.

In utero exposures to mint-flavored JUUL aerosol impair lung development and aggravate house dust mite-induced asthma in adult offspring mice.

There are few reports concerning electronic nicotine delivery system (ENDS) use during pregnancy and no studies on asthma in prenatally JUUL-exposed offspring. Here, we tested the hypothesis that in utero JUUL exposure causes unfavorable birth outcomes and lasting pulmonary health effects in adult offspring. BALB/c dams were exposed to either air or mint-flavored JUUL aerosol, 1-hr/d, 20 consecutive days during gestation. Offspring were sacrificed on post-natal day (PND) 0 or at 11-week of age, following house dust mite (HDM) challenge. Gene expression was assessed in the uterine/placental tissue of the dams and lung responses were assessed in offspring at PND0 and at 11 weeks of age. JUUL-exposed offspring exhibited decreased body weights and lengths at PND0. These birth outcomes were accompanied by dysregulation of 54 genes associated with hypoxia and oxidative stress in the uterine/placental tissues of JUUL-exposed dams, as well as 24 genes in the lungs of the offspring related to Wnt signaling, plus 9 genes related to epigenetics, and 7 genes related to inflammation. At 11 weeks of age, JUUL + HDM exposed mice exhibited pulmonary inflammation when compared to their respective air + HDM controls. Additionally, the JUUL + HDM exposure dysregulated several genes associated with allergies and asthma. Further, the JUUL + HDM females showed decreased methylation of the promoter region of the Il10ra gene. Taken together, our mouse model shows that inhalation of JUUL aerosols during pregnancy affects the intrauterine environment, impairs lung development, and heightens the effects of allergic airway responses later in life.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Exploring the association of intratumoral immune cell infiltrates with histopathologic grade in canine mast cell tumors.

Cutaneous canine mast cell tumors (ccMCTs) vary in their biological behavior, treatment, and prognosis, based on their grade. Immune cell infiltration has been associated with prognosis and response to treatments in some human cancers, and immune-targeting therapeutics are increasingly being explored in veterinary oncology. However, currently little is known about the tumor microenvironment (TME) in ccMCTs. Therefore, the objective of this study was to determine the prevalence of T lymphocytes, T regulatory lymphocytes, PD-1+ cells and macrophages in low- and high-grade ccMCTs. Thirty low-grade and 20 high-grade formalin-fixed paraffin-embedded ccMCT samples were included. Immunohistochemistry (IHC) was performed to detect CD3, FOXP3, Iba1, and PD-1 on sequential sections. Three 400x fields with the highest numbers of CD3+ cells were identified for each tumor. The percentage of CD3+, FOXP3+, and Iba1+ cells, and the number of PD-1+ cells, was quantified in each of these three "hot-spot" fields using ImageJ software. Iba1 expression was significantly greater in high-grade compared to low-grade ccMCTs (mean = 12.5% vs. 9.6%, p = 0.043). PD-1 expression was low overall, but a significantly higher number of PD-1-expressing cells was observed in high-grade ccMCTs (median 1 vs. 0, p = 0.001). No significant difference was noted in CD3 and FOXP3 expression between ccMCT grades. Macrophages and PD-1+ cells were more frequent in high-grade, compared to low-grade ccMCTs. Further studies are needed to define the role of macrophages and rare PD-1+ cells in high-grade ccMCTs.

Comparison of blind and endoscopic-guided orotracheal intubation on laryngeal and tracheal damage in domestic rabbits (Oryctolagus cuniculus).

OBJECTIVE: To compare blind and endoscopic-guided techniques for orotracheal intubation in rabbits and the number of intubation attempts with laryngeal/tracheal damage. STUDY DESIGN: Prospective, randomized experimental study. ANIMALS: A total of 24 healthy, intact female New Zealand White rabbits, weighing 2.2 ± 0.2 kg (mean ± standard deviation). METHODS: Rabbits were randomly assigned to blind (group B) or endoscopic-guided (group E) orotracheal intubation with a 2.0 mm internal diameter uncuffed tube. Intramuscular (IM) alfaxalone (7 mg kg-1), hydromorphone (0.1 mg kg-1) and dexmedetomidine (0.005 mg kg-1) were administered, and additional IM alfaxalone (3-5 mg kg-1) and dexmedetomidine (0.025 mg kg-1) were administered to rabbits with strong jaw tone. An intubation attempt was defined as the advancement of the endotracheal tube from the incisors to the laryngeal entrance. Tracheal intubation was confirmed via capnography and anesthesia was maintained with isoflurane for 2 hours. Following euthanasia, laryngeal and tracheal tissues were submitted for histopathology. Quality of anesthesia for orotracheal intubation, intubation procedure and tissue damage were numerically scored. Data were analyzed using Poisson regression, Spearman's correlation, t test, mixed anova, Mann-Whitney U test, Friedman and Chi square tests as appropriate. RESULTS: Median (range) intubation attempts were 2 (1-8) and 1 (1-3) for groups B and E, respectively. More rabbits in group E (91.6%) required additional alfaxalone and dexmedetomidine than in group B (16.7%). Median (range) cumulative histopathology scores were 6 (3-10) and 6 (2-9) for groups B and E, respectively. Scores were highest in the cranial trachea, but there was no difference between groups and no correlation between laryngeal/tracheal damage and the number of intubation attempts. CONCLUSIONS AND CLINICAL RELEVANCE: Both orotracheal intubation techniques were associated with laryngeal/tracheal damage. Although blind orotracheal intubation was associated with a higher number of attempts, the tissue damage was similar between groups.

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10-11, and the PI among siblings (PIsib) was 1.5 x 10-5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

An outbreak of visna-maedi in a flock of sheep in Southern Brazil.

Visna-maedi is a multisystemic and progressive inflammatory disease caused by a non-oncogenic retrovirus (Visna-maedi virus, VMV). An outbreak of visna-maedi occurred in Southern Brazil in sheep with clinical signs of blindness and stumbling gait. At post-mortem examination, all animals had similar lesions, including heavy non-collapsed lungs and multifocal yellow areas in the cerebral white matter, affecting mainly the periventricular region. These lesions corresponded histologically to lymphocytic interstitial pneumonia and histiocytic periventricular encephalitis surrounding areas of necrosis, in addition to significant demyelination in the brain. Serology was performed in all the sheep from the flock and 14% were seropositive for VMV. The presence of VMV was confirmed through PCR and partial sequencing of the 5'LTR. Sequencing demonstrated that the virus had 89.7 to 90.0% of nucleotide identity with VMV strains reported in the USA. This is the first description of clinical disease related to VMV in Brazil leading to economic losses. This study calls for the need to implement control measures to prevent the spread of small ruminant lentiviruses in Brazil.

Canine and feline uveal melanocytic tumours: Histologic and immunohistochemical characteristics of 32 cases.

OBJECTIVE: Gross, histopathological, and immunohistochemical characteristics of uveal melanocytic neoplasms in dogs and cats were investigated. SAMPLES: Thirty-two enucleated globes with uveal melanocytic neoplasms, 27 from dogs and 5 from cats, were examined. PROCEDURES: Morphological characteristics of uveal melanocytic neoplasms in dogs and cats were evaluated with anti-PNL2, anti-Melan-A, anti-Ki-67, anti-caspase-3, and anti-BAP1 immunomarkers. Statistical analysis was performed to compare canine melanocytomas and melanomas. RESULTS: The 32 uveal neoplasms were classified as melanocytomas (19/27 in dogs) or melanomas (8/27 in dogs, 5/5 in cats). Most tumours (84%) were located in the anterior uvea. Neoplastic cells were classified as epithelioid, spindle-shaped, mixed, or special type (balloon and signet ring cells). The percentage of cells with melanin, melanin concentration within cells, anisocytosis and anisokaryosis, mitotic count, lymphocytic inflammation, necrosis, vascular invasion, and glaucoma were also characterized. Anisocytosis, percentage of neoplastic cells with melanin, mitotic count, and indices (proliferation and apoptotic) varied significantly between canine uveal melanomas and melanocytomas; in general, melanomas had greater cell variability, were less pigmented, and had a higher mitotic count. The melanocytic origin of the neoplasms was confirmed by positive anti-PNL2 immunolabelling (29/32) and positive anti-Melan-A immunolabelling (3/32). In canine uveal melanomas, anisocytosis and anisokaryosis correlated with less pigmentation and minimal pigmentation correlated with a high percentage of immunolabelling for caspase-3. CONCLUSIONS: Uveal melanocytomas were more common in dogs, and uveal melanomas were more frequent in cats. Anisocytosis, percentage of neoplastic cells with melanin, and mitotic count are important histologic characteristics of malignancy to evaluate in uveal melanocytic neoplasms. The proliferation and apoptotic indices are relevant when comparing malignant tumours with benign tumours.

The BPH/5 Mouse Model of Superimposed Preeclampsia Is Not a Model of HELLP Syndrome.

Preeclampsia (PE) is a multisystemic disease of pregnancy affecting 2-8% of women worldwide. PE-induced liver disease is a rare but important complication of pregnancy. The pathogenesis of liver dysfunction in PE is poorly understood, but is correlated with dysregulated angiogenic, inflammatory, and hypoxic events in the early phase of placental development. Because BPH/5 mice develop the maternal and fetal hallmarks of PE during pregnancy, we hypothesized that they may also share the clinicopathologic findings of the human PE-associated hemolysis elevated liver transaminases low platelets (HELLP) syndrome. Using this model, we determined that microangiopathic hemolysis, thrombocytopenia, and elevated liver enzymes do not occur in mid to late gestation. Pregnant BPH/5 mice do not develop histologic evidence of hepatic inflammation, but they do have increased microsteatosis scores at preconception and in mid to late gestation that progress to macrosteatosis in a subset of mice in late gestation. The transcriptional upregulation of TNF-α, CXCL-10, and TLR-2 occurs in mid gestation prior to the onset of macrosteatosis. The BPH/5 female mouse is not a model of HELLP syndrome, but may be a model of fatty liver disease associated with pregnancy.

Breast cancer growth and proliferation is suppressed by the mitochondrial targeted furazano[3,4-b]pyrazine BAM15.

BACKGROUND: Enhanced metabolic plasticity and diversification of energy production is a hallmark of highly proliferative breast cancers. This contributes to poor pharmacotherapy efficacy, recurrence, and metastases. We have previously identified a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively reduces bioenergetic coupling efficiency and is orally available. Here, we evaluated the antineoplastic properties of uncoupling oxidative phosphorylation from ATP production in breast cancer using BAM15. METHODS: The anticancer effects of BAM15 were evaluated in human triple-negative MDA-MB-231 and murine luminal B, ERα-negative EO771 cells as well as in an orthotopic allograft model of highly proliferative mammary cancer in mice fed a standard or high fat diet (HFD). Untargeted transcriptomic profiling of MDA-MB-231 cells was conducted after 16-h exposure to BAM15. Additionally, oxidative phosphorylation and electron transfer capacity was determined in permeabilized cells and excised tumor homogenates after treatment with BAM15. RESULTS: BAM15 increased proton leak and over time, diminished cell proliferation, migration, and ATP production in both MDA-MB-231 and EO771 cells. Additionally, BAM15 decreased mitochondrial membrane potential, while inducing apoptosis and reactive oxygen species accumulation in MDA-MB-231 and EO771 cells. Untargeted transcriptomic profiling of MDA-MB-231 cells further revealed inhibition of signatures associated with cell survival and energy production by BAM15. In lean mice, BAM15 lowered body weight independent of food intake and slowed tumor progression compared to vehicle-treated controls. In HFD mice, BAM15 reduced tumor growth relative to vehicle and calorie-restricted weight-matched controls mediated in part by impaired cell proliferation, mitochondrial respiratory function, and ATP production. LC-MS/MS profiling of plasma and tissues from BAM15-treated animals revealed distribution of BAM15 in adipose, liver, and tumor tissue with low abundance in skeletal muscle. CONCLUSIONS: Collectively, these data indicate that mitochondrial uncoupling may be an effective strategy to limit proliferation of aggressive forms of breast cancer. More broadly, these findings highlight the metabolic vulnerabilities of highly proliferative breast cancers which may be leveraged in overcoming poor responsiveness to existing therapies.

Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model.

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 103 PFU/mL) versus untreated (8.25 ± 0.25 × 105 PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.