Scholarly Works, Biomedical Sciences and Pathobiology

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A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Brucella abortus Mediates Host-Pathogen Interactions
Sheehan, Lauren M.; Caswell, Clayton C. (American Society for Microbiology, 2017-05)
In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. IMPORTANCE Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the Alphaproteobacteria and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In Brucella abortus, AbcR1 and AbcR2 are functionally redundant, as only a double abcR1 abcR2 (abcR1/2) deletion results in attenuation in vitro and in vivo. In the present study, we confirmed that the AbcR sRNAs have redundant regulatory functions and defined two six-nucleotide motifs, M1 and M2, that the AbcR sRNAs utilize to control gene expression. Importantly, only M2 was linked to B. abortus virulence. Further investigation of M2-regulated targets identified BAB2_0612 as critical for colonization of B. abortus in mice, highlighting the significance of AbcR M2-regulated transcripts for Brucella infection. Overall, our findings define the molecular mechanism of the virulence-associated AbcR system in the pathogenic bacterium B. abortus.
Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model
Hay, Alayna N.; Imran, Khan Mohammad; Hendricks-Wenger, Alissa; Gannon, Jessica M.; Sereno, Jacqueline; Simon, Alex; Lopez, Victor A.; Coutermarsh-Ott, Sheryl; Vlaisavljevich, Eli; Allen, Irving C.; Tuohy, Joanne L. (MDPI, 2023-10-09)
Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.
Abrogation of atypical neurogenesis and vascular-derived EphA4 prevents repeated mild TBI-induced learning and memory impairments
Greer, Kisha; Gudenschwager-Basso, Erwin K.; Kelly, Colin; Cash, Alison; Kowalski, Elizabeth A.; Cerna, Steven; Ocampo, Collin Tanchanco; Wang, Xia; Theus, Michelle H. (2020-09-21)
Brain injury resulting from repeated mild traumatic insult is associated with cognitive dysfunction and other chronic co-morbidities. The current study tested the effects of aberrant neurogenesis in a mouse model of repeated mild traumatic brain injury (rmTBI). Using Barnes Maze analysis, we found a significant reduction in spatial learning and memory at 24 days post-rmTBI compared to repeated sham (rSham) injury. Cell fate analysis showed a greater number of BrdU-labeled cells which co-expressed Prox-1 in the DG of rmTBI-injured mice which coincided with enhanced cFos expression for neuronal activity. We then selectively ablated dividing neural progenitor cells using a 7-day continuous infusion of Ara-C prior to rSham or rmTBI. This resulted in attenuation of cFos and BrdU-labeled cell changes and prevented associated learning and memory deficits. We further showed this phenotype was ameliorated in EphA4f.(/f)/Tie2-Cre knockout compared to EphA4f.(/f) wild type mice, which coincided with altered mRNA transcript levels of MCP-1, Cx43 and TGF beta. These findings demonstrate that cognitive decline is associated with an increased presence of immature neurons and gene expression changes in the DG following rmTBI. Our data also suggests that vascular EphA4-mediated neurogenic remodeling adversely affects learning and memory behavior in response to repeated insult.
The activation of the IFN beta induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable
Overend, Christopher C.; Cui, Junru; Grubman, Marvin J.; Garmendia, Antonio E. (2017-03)
Background It has been recognized that the expression of type I interferon (IFN alpha/beta) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. Objective Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFN beta) and downstream signaling events. Methods In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFN beta protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. Results These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFN beta protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFN beta signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFN beta production. Conclusion The results of this study demonstrate that the induction of IFN beta and signaling in PAMs after PRRSV infection is variable.
Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis
Rai, Pallavi; Chuong, Christina; LeRoith, Tanya; Smyth, James W.; Panov, Julia; Levi, Moshe; Kehn-Hall, Kylene; Duggal, Nisha K.; Weger-Lucarelli, James (Elsevier, 2021-11-01)
The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.
Adrenocortical Challenge Response and Genomic Analyses in Scottish Terriers With Increased Alkaline Phosphate Activity
Zimmerman, Kurt L.; Panciera, David L.; Hoeschele, Ina; Monroe, William E.; Todd, S. Michelle; Werre, Stephen R.; LeRoith, Tanya; Fecteau, Kellie; Lake, Bathilda B. (Frontiers, 2018-10-09)
Scottish terriers (ST) frequently have increased serum alkaline phosphatase (ALP) of the steroid isoform. Many of these also have high serum concentrations of adrenal sex steroids. The study’s objective was to determine the cause of increased sex steroids in ST with increased ALP. Adrenal gland suppression and stimulation were compared by low dose dexamethasone (LDDS), human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) response tests. Resting plasma pituitary hormones were measured. Steroidogenesis-related mRNA expression was evaluated in six ST with increased ALP, eight dogs of other breeds with pituitary-dependent hyperadrenocorticism (HAC), and seven normal dogs. The genome-wide association of single nucleotide polymorphisms (SNP) with ALP activity was evaluated in 168 ST. ALP (reference interval 8–70 U/L) was high in all ST (1,054 U/L) and HAC (985 U/L) dogs. All HAC dogs and 2/8 ST had increased cortisol post-ACTH administration. All ST and 2/7 Normal dogs had increased sex steroids post-ACTH. ST and Normal dogs had similar post-challenge adrenal steroid profiles following LDDS and HCG. Surprisingly, mRNA of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2) was lower in ST and Normal dogs than HAC. HSD17B2 facilities metabolism of sex steroids. A SNP region was identified on chromosome 5 in proximity to HSD17B2 that correlated with increased serum ALP. ST in this study with increased ALP had a normal pituitary-adrenal axis in relationship to glucocorticoids and luteinizing hormone.We speculate the identified SNP and HSD17B2 gene may have a role in the pathogenesis of elevated sex steroids and ALP in ST.
Advanced sequencing approaches detected insertions of viral and human origin in the viral genome of chronic hepatitis E virus patients
Papp, C-Patrick; Biedermann, Paula; Harms, Dominik; Wang, Bo; Kebelmann, Marianne; Choi, Mira; Helmuth, Johannes; Corman, Victor M.; Thuermer, Andrea; Altmann, Britta; Klink, Patrycja; Hofmann, Joerg; Bock, C-Thomas (Nature Portfolio, 2022-02-02)
The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.
Adventitious viruses persistently infect three commonly used mosquito cell lines
Weger-Lucarelli, James; Rückert, Claudia; Grubaugh, Nathan D.; Misencik, Michael J.; Armstrong, Philip M.; Stenglein, Mark D.; Ebel, Gregory D.; Brackney, Doug E. (2018-08)
Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.
Alantolactone Suppresses Proliferation and the Inflammatory Response in Human HaCaT Keratinocytes and Ameliorates Imiquimod-Induced Skin Lesions in a Psoriasis-Like Mouse Model
Chuo, Wen-Ho; Tung, Yu-Tang; Wu, Chao-Liang; Bracci, Nicole R.; Chang, Yu-Kang; Huang, Hung-Yi; Lin, Chi-Chien (MDPI, 2021-06-25)
Psoriasis is an immune-mediated inflammatory disease that affects 2% to 3% of the world population. Alantolactone, a sesquiterpene lactone, was isolated from Inula helenium and Radix inulae and has several biological effects, including antifungal, anthelmintic, antimicrobial, anti-inflammatory, antitrypanosomal, and anticancer properties. This study aimed to evaluate the antipsoriatic potential of alantolactone in vitro and in vivo and to explore its underlying mechanisms. These results showed that alantolactone significantly attenuated IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) cytokine-induced hyperproliferation in HaCaT keratinocytes. Moreover, M5 cytokines significantly upregulated the mRNA levels of TNF-α, IL-6, IL-1β, and IL-8. However, alantolactone attenuated the upregulation of these inflammatory cytokines. In addition, alantolactone was found to inhibit STAT3 phosphorylation and NF-κB p65 nuclear translocation in HaCaT keratinocytes. Furthermore, alantolactone treatment in mice significantly alleviated the severity of skin lesions (erythema, scaling and epidermal thickness, and inflammatory cell infiltration) and decreased the mRNA expression of inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β, IL-8, IL-17A, and IL-23) in an IMQ-induced-like mouse model. Therefore, our new findings revealed that alantolactone alleviates psoriatic skin lesions by inhibiting inflammation, making it an attractive candidate for future development as an antipsoriatic agent.
Alleviation of Collagen-Induced Arthritis by Crotonoside through Modulation of Dendritic Cell Differentiation and Activation
Lin, Shih-Chao; Lin, Chi-Chien; Li, Shiming; Lin, Wan-Yi; Lehman, Caitlin W.; Bracci, Nicole R.; Tsai, Sen-Wei (MDPI, 2020-11-10)
Crotonoside, a guanosine analog originally isolated from Croton tiglium, is reported to be a potent tyrosine kinase inhibitor with immunosuppressive effects on immune cells. Due to its potential immunotherapeutic effects, we aimed to evaluate the anti-arthritic activity of crotonoside and explore its immunomodulatory properties in alleviating the severity of arthritic symptoms. To this end, we implemented the treatment of crotonoside on collagen-induced arthritic (CIA) DBA/1 mice and investigated its underlying mechanisms towards pathogenic dendritic cells (DCs). Our results suggest that crotonoside treatment remarkably improved clinical arthritic symptoms in this CIA mouse model as indicated by decreased pro-inflammatory cytokine production in the serum and suppressed expression of co-stimulatory molecules, CD40, CD80, and MHC class II, on CD11c⁺ DCs from the CIA mouse spleens. Additionally, crotonoside treatment significantly reduced the infiltration of CD11c⁺ DCs into the synovial tissues. Our in vitro study further demonstrated that bone marrow-derived DCs (BMDCs) exhibited lower yield in numbers and expressed lower levels of CD40, CD80, and MHC-II when incubated with crotonoside. Furthermore, LPS-stimulated mature DCs exhibited limited capability to prime antigen-specific CD4⁺ and T-cell proliferation, cytokine secretions, and co-stimulatory molecule expressions when treated with crotonoside. Our pioneer study highlights the immunotherapeutic role of crotonoside in the alleviation of the CIA via modulation of pathogenic DCs, thus creating possible applications of crotonoside as an immunosuppressive agent that could be utilized and further explored in treating autoimmune disorders in the future.
Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2–Priming Protease TMPRSS2
Azouz, N. P.; Klingler, A. M.; Callahan, Victoria; Akhrymuk, Ivan V.; Elez, K.; Raich, L.; Henry, B. M.; Benoit, J. L.; Benoit, S. W.; Noé, F.; Kehn-Hall, Kylene; Rothenberg, M. E. (2021-01-01)
Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 anti-trypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.
Altered Germinal-Center Metabolism in B Cells in Autoimmunity
Shiraz, Ashton K.; Panther, Eric J.; Reilly, Christopher M. (MDPI, 2022-01-05)
B lymphocytes play an important role in the pathophysiology of many autoimmune disorders by producing autoantibodies, secreting cytokines, and presenting antigens. B cells undergo extreme physiological changes as they develop and differentiate. Aberrant function in tolerogenic checkpoints and the metabolic state of B cells might be the contributing factors to the dysfunctionality of autoimmune B cells. Understanding B-cell metabolism in autoimmunity is important as it can give rise to new treatments. Recent investigations have revealed that alterations in metabolism occur in the activation of B cells. Several reports have suggested that germinal center (GC) B cells of individuals with systemic lupus erythematosus (SLE) have altered metabolic function. GCs are unique microenvironments in which the delicate and complex process of B-cell affinity maturation occurs through somatic hypermutation (SHM) and class switching recombination (CSR) and where Bcl6 tightly regulates B-cell differentiation into memory B-cells or plasma cells. GC B cells rely heavily on glucose, fatty acids, and oxidative phosphorylation (OXPHOS) for their energy requirements. However, the complicated association between GC B cells and their metabolism is still not clearly understood. Here, we review several studies of B-cell metabolism, highlighting the significant transformations that occur in GC progression, and suggest possible approaches that may be investigated to more precisely target aberrant B-cell metabolism in SLE.
Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure
Hrubec, Terry C.; Seguin, Ryan P.; Xu, L.; Cortopassi, G. A.; Datta, S.; Hanlon, Alexandra L.; Lozano, A. J.; McDonald, V. A.; Healy, C. A.; Anderson, T. C.; Musse, N. A.; Williams, R. T. (Elsevier, 2021-01-01)
Humans are frequently exposed to Quaternary Ammonium Compounds (QACs). QACs are ubiquitously used in medical settings, restaurants, and homes as cleaners and disinfectants. Despite their prevalence, nothing is known about the health effects associated with chronic low-level exposure. Chronic QAC toxicity, only recently identified in mice, resulted in developmental, reproductive, and immune dysfunction. Cell based studies indicate increased inflammation, decreased mitochondrial function, and disruption of cholesterol synthesis. If these findings translate to human toxicity, multiple physiological processes could be affected. This study tested whether QAC concentrations could be detected in the blood of 43 human volunteers, and whether QAC concentrations influenced markers of inflammation, mitochondrial function, and cholesterol synthesis. QAC concentrations were detected in 80 % of study participants. Blood QACs were associated with increase in inflammatory cytokines, decreased mitochondrial function, and disruption of cholesterol homeostasis in a dose dependent manner. This is the first study to measure QACs in human blood, and also the first to demonstrate statistically significant relationships between blood QAC and meaningful health related biomarkers. Additionally, the results are timely in light of the increased QAC disinfectant exposure occurring due to the SARS-CoV-2 pandemic. Main Findings: This study found that 80 % of study participants contained QACs in their blood; and that markers of inflammation, mitochondrial function, and sterol homeostasis varied with blood QAC concentration.
Alzheimer's Disease-Like Neurodegeneration in Porphyromonas gingivalis Infected Neurons with Persistent Expression of Active Gingipains
Haditsch, Ursula; Roth, Theresa; Rodriguez, Leo; Hancock, Sandy; Cecere, Thomas E.; Nguyen, Mai; Arastu-Kapur, Shirin; Broce, Sean; Raha, Debasish; Lynch, Casey C.; Holsinger, Leslie J.; Dominy, Stephen S.; Ermini, Florian (IOS Press, 2020-01-01)
Background: Porphyromonas gingivalis (P. gingivalis) and its gingipain virulence factors have been identified as pathogenic effectors in Alzheimer's disease (AD). In a recent study we demonstrated the presence of gingipains in over 90% of postmortem AD brains, with gingipains localizing to the cytoplasm of neurons. However, infection of neurons by P. gingivalis has not been previously reported. Objective: To demonstrate intraneuronal P. gingivalis and gingipain expression in vitro after infecting neurons derived from human inducible pluripotent stem cells (iPSC) with P. gingivalis for 24, 48, and 72 h. Methods: Infection was characterized by transmission electron microscopy, confocal microscopy, and bacterial colony forming unit assays. Gingipain expression was monitored by immunofluorescence and RT-qPCR, and protease activity monitored with activity-based probes. Neurodegenerative endpoints were assessed by immunofluorescence, western blot, and ELISA. Results: Neurons survived the initial infection and showed time dependent, infection induced cell death. P. gingivalis was found free in the cytoplasm or in lysosomes. Infected neurons displayed an accumulation of autophagic vacuoles and multivesicular bodies. Tau protein was strongly degraded, and phosphorylation increased at T231. Over time, the density of presynaptic boutons was decreased. Conclusion: P. gingivalis can invade and persist in mature neurons. Infected neurons display signs of AD-like neuropathology including the accumulation of autophagic vacuoles and multivesicular bodies, cytoskeleton disruption, an increase in phospho-tau/tau ratio, and synapse loss. Infection of iPSC-derived mature neurons by P. gingivalis provides a novel model system to study the cellular mechanisms leading to AD and to investigate the potential of new therapeutic approaches.
Ambient and Dosed Exposure to Quaternary Ammonium Disinfectants Causes Neural Tube Defects in Rodents
Hrubec, Terry C.; Melin, Vanessa E.; Shea, Caroline S.; Ferguson, Elizabeth E.; Garofola, Craig; Repine, Claire M.; Chapman, Tyler W.; Patel, Hiral R.; Razvi, Reza M.; Sugrue, Jesse E.; Potineni, Haritha; Magnin, Geraldine; Hunt, Patricia A. (2017-08-15)
Background: Quaternary ammonium compounds are a large class of chemicals used for their antimicrobial and antistatic properties. Two common quaternary ammonium compounds, alkyldimethylbenzyl ammonium chloride (ADBAC) and didecyldimethyl ammonium chloride (DDAC), are combined in common cleaners and disinfectants. Introduction of a cleaner containing ADBAC+DDAC in the vivarium caused neural tube defects (NTDs) in mice and rats. Methods: To further evaluate this finding, male and female mice were dosed in the feed at 60 or 120 mg/kg/day, or by oral gavage at 7.5, 15, or 30 mg/kg ADBAC+DDAC. Mice also received ambient exposure to ADBAC+DDAC from the disinfectant used in the mouse room. Embryos were evaluated on gestational day 10 for NTDs, and fetuses were evaluated on gestational day 18 for gross and skeletal malformations. Results: We found increased NTDs with exposure to ADBAC+DDAC in both rats and mice. The NTDs persisted for two generations after cessation of exposure. Notably, male exposure alone was sufficient to cause NTDs. Equally significant, ambient exposure from disinfectant use in the vivarium, influenced the levels of NTDs to a greater extent than oral dosing. No gross or significant axial skeletal malformations were observed in late gestation fetuses. Placental abnormalities and late gestation fetal deaths were increased at 120 mg/kg/day, which might explain the lack of malformations observed in late gestation fetuses. Conclusion: These results demonstrate that ADBAC+DDAC in combination are teratogenic to rodents. Given the increased use of these disinfectants, further evaluation of their safety in humans and their contribution to health and disease is essential. (C) 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.
An in vitro evaluation of intravenous lipid emulsion on three common canine toxicants
Jones, Emery; Walton, Stuart A.; Davis, Jennifer; Council-Troche, McAlister (Frontiers, 2024-09-25)
Objective: To determine whether intravenous lipid emulsion (ILE) therapy significantly reduces the concentration of baclofen, ibuprofen, and/or bromethalin in canine whole blood over time. Animals: Seven 500 mL bags of canine DEA 1.1 negative blood were divided into aliquots of 125 mL and randomly assigned to one of three treatment groups (baclofen, ibuprofen, bromethalin) or four control groups (a positive control for each treatment group and a negative control group). Procedures: Injectable ibuprofen (200 mg/kg), baclofen (8 mg/kg), or bromethalin (3 mg/kg) was apportioned into 125 mL aliquots of canine whole blood and incubated for 30 min at 38.5°C. ILE (12.4 mL, Intralipid®) was added to each sample and the solution vortexed [215 rpm for 15 min at 37°C (98.6°F)]. Samples were obtained at designated time points (0, 15, 30, 60, 180, 360 min), centrifuged, and separated into serum and RBC fractions. Serum samples were ultracentrifuged (22,000 g for 10 min at 37°C) to separate lipid rich and poor fractions. Samples were stored at −80°C prior to analysis. Results: A significant decrease in total drug concentration was established for bromethalin and its metabolite desmethylbromethalin compared to positive controls. ILE significantly reduced desmethylbromethalin at the 30-and 360-min time points. The remainder of the desmethylbromethalin time points did not reach significance. Bromethalin concentration was significantly reduced at all time points compared to positive controls. Neither baclofen nor ibuprofen had significant changes in concentration. Conclusion: ILE therapy was effective at reducing the total drug concentration of bromethalin and its metabolite desmethylbromethalin supporting the lipid sink theory. As a single compartment in vitro study, this study does not evaluate other proposed mechanisms of action of ILE therapy. ILE therapy may have other means of significantly decreasing lipophilic drug concentration in cases of toxicosis.
Analysis of lung transcriptome in calves infected with Bovine Respiratory Syncytial Virus and treated with antiviral and/or cyclooxygenase inhibitor
Lebedev, Maxim; McEligot, Heather A.; Mutua, Victoria N.; Walsh, Paul; Carvallo, Francisco R.; Gershwin, Laurel J. (2021-02-18)
Bovine Respiratory Syncytial virus (BRSV) is one of the major infectious agents in the etiology of the bovine respiratory disease complex. BRSV causes a respiratory syndrome in calves, which is associated with severe bronchiolitis. In this study we describe the effect of treatment with antiviral fusion protein inhibitor (FPI) and ibuprofen, on gene expression in lung tissue of calves infected with BRSV. Calves infected with BRSV are an excellent model of human RSV in infants: we hypothesized that FPI in combination with ibuprofen would provide the best therapeutic intervention for both species. The following experimental treatment groups of BRSV infected calves were used: 1) ibuprofen day 3-10, 2) ibuprofen day 5-10, 3) placebo, 4) FPI day 5-10, 5) FPI and ibuprofen day 5-10, 6) FPI and ibuprofen day 3-10. All calves were infected with BRSV on day 0. Daily clinical evaluation with monitoring of virus shedding by qRT-PCR was conducted. On day10 lung tissue with lesions (LL) and non-lesional (LN) was collected at necropsy, total RNA extracted, and RNA sequencing performed. Differential gene expression analysis was conducted with Gene ontology (GO) and KEGG pathway enrichment analysis. The most significant differential gene expression in BRSV infected lung tissues was observed in the comparison of LL with LN; oxidative stress and cell damage was especially noticeable. Innate and adaptive immune functions were reduced in LL. As expected, combined treatment with FPI and Ibuprofen, when started early, made the most difference in gene expression patterns in comparison with placebo, especially in pathways related to the innate and adaptive immune response in both LL and LN. Ibuprofen, when used alone, negatively affected the antiviral response and caused higher virus loads as shown by increased viral shedding. In contrast, when used with FPI Ibuprofen enhanced the specific antiviral effect of FPI, due to its ability to reduce the damaging effect of prostanoids and oxidative stress.
Analysis of the Murine Immune Response to Pulmonary Delivery of Precisely Fabricated Nano- and Microscale Particles
Roberts, Reid A.; Shen, Tammy; Allen, Irving C.; Hasan, Warefta; DeSimone, Joseph M.; Ting, Jenny P.-Y. (PLOS, 2013-04-12)
Nanomedicine has the potential to transform clinical care in the 21st century. However, a precise understanding of how nanomaterial design parameters such as size, shape and composition affect the mammalian immune system is a prerequisite for the realization of nanomedicine's translational promise. Herein, we make use of the recently developed Particle Replication in Non-wetting Template (PRINT) fabrication process to precisely fabricate particles across and the nano- and micro-scale with defined shapes and compositions to address the role of particle design parameters on the murine innate immune response in both in vitro and in vivo settings. We find that particles composed of either the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) or the biocompatible polymer polyethylene glycol (PEG) do not cause release of pro-inflammatory cytokines nor inflammasome activation in bone marrow-derived macrophages. When instilled into the lungs of mice, particle composition and size can augment the number and type of innate immune cells recruited to the lungs without triggering inflammatory responses as assayed by cytokine release and histopathology. Smaller particles (80×320 nm) are more readily taken up in vivo by monocytes and macrophages than larger particles (6 µm diameter), yet particles of all tested sizes remained in the lungs for up to 7 days without clearance or triggering of host immunity. These results suggest rational design of nanoparticle physical parameters can be used for sustained and localized delivery of therapeutics to the lungs.
Ancient, globally distributed lineage of Sarcocystis from sporocysts of the Eastern rat snake (Pantherophis alleghaniensis) and its relation to neurological sequalae in intermediate hosts
Verma, Shiv K.; Lindsay, David S.; Rosenthal, Benjamin M.; Dubey, Jitender P. (2016-07)
There is an emerging concern that snakes are definitive hosts of certain species of Sarcocystis that cause muscular sarcocystosis in human and non-human primates. Other species of Sarcocystis are known to cycle among snakes and rodents, but have been poorly characterized in the USA and elsewhere. Although neurological sequalae are known for certain species of Sarcocystis, no such neurological symptoms are known to typify parasites that naturally cycle in rodents. Here, sporocysts of a species of Sarcocystis were found in the intestinal contents of a rat snake (Pantherophis alleghaniensis) from Maryland, USA. The sporocysts were orally infective for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred mice. The KO mice developed neurological signs, and were necropsied between 33 and 52 days post-inoculation. Only schizonts/merozoites were found, and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neuropil. The schizonts and merozoites were located in neuropil, and apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice and CV-1 cell line. DNA extracted from the infected mouse brain, and infected cell cultures revealed the highest identity with Sarcocystis species that employ snakes as definitive hosts. This is the first report of Sarcocystis infection in the endangered rat snake (P. alleghaniensis) and the first report of neurological sarcocystosis in mice induced by feeding sporocysts from a snake. These data underscore the likelihood that parasites in this genus that employ snakes as their definitive hosts constitute an ancient, globally distributed monophyletic group. These data also raise the possibility that neurological sequalae may be more common in intermediate hosts of Sarcocystis spp. than has previously been appreciated.
Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens
Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa FN N.; Sobreira, Tiago JP P.; Hedrick, Victoria E.; Paul, Lake N.; Seleem, Mohamed N. (Nature Publishing Group, 2016-03-03)
Traditional methods employed to discover new antibiotics are both a time-consuming and financially-taxing venture. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). Such an effort led to the discovery of auranofin, a drug initially approved as an anti-rheumatic agent, which also possesses potent antibacterial activity in a clinically achievable range. The present study demonstrates auranofin's antibacterial activity is a complex process that involves inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. We also confirmed that the lack of activity of auranofin observed against Gram-negative bacteria is due to the permeability barrier conferred by the outer membrane. Auranofin's ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins. Additionally, auranofin is capable of eradicating intracellular MRSA present inside infected macrophage cells. Furthermore, auranofin is efficacious in a mouse model of MRSA systemic infection and significantly reduces the bacterial load in murine organs including the spleen and liver. Collectively, this study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antibacterial for treatment of invasive bacterial infections.

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