Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity.

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants.

Expression of Hematopoietic Stem and Endothelial Cell Markers in Canine Hemangiosarcoma.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

Tensile strength and early healing of self-locking and surgeon's knots.

OBJECTIVE: To compare the biomechanical properties and healing of ventral midline celiotomies (VMC) closed with a self-locking knot combination and forwarder start and Aberdeen end (F-A) vs a traditional knot combination and surgeon's start and end (S-S). STUDY DESIGN: In vivo, experimental. ANIMALS: Twenty-one horses. METHODS: Fourteen horses underwent VMC, which was closed with either an F-A (n = 7) or an S-S (n = 7) knot combination. Incisions were subjectively graded by masked evaluators for dehiscence, edema, and drainage. Biomechanical testing was performed on three abdominal segments, and histology was performed on one segment from each animal after humane euthanasia 10 days post-VMC. The abdominal wall of control horses (n = 7, no celiotomy) was collected for biomechanical testing. RESULTS: Forwarder start and Aberdeen end and S-S horses had less tensile strength compared with control horses (P ≤ .001). No differences were detected between treatment groups for any variable evaluated, including tensile strength (P = .975), location of failure (P = .240), and histologic healing at the knot (P = .600). CONCLUSION: Closure of VMC with self-locking knots resulted in biomechanical and healing features similar to those with a traditional closure technique, with neither restoring the tensile strength of the linea alba. CLINICAL SIGNIFICANCE: Results of this study provide evidence to support a clinical trial to evaluate long-term performance of the F-A self-locking knot closure in horses.

Housefly (Musca domestica) and Blow Fly (Protophormia terraenovae) as Vectors of Bacteria Carrying Colistin Resistance Genes.

Flies have the capacity to transfer pathogens between different environments, acting as one of the most important vectors of human diseases worldwide. In this study, we trapped flies on a university campus and tested them for mobile resistance genes against colistin, a last-resort antibiotic in human medicine for treating clinical infections caused by multidrug-resistant Gram-negative bacteria. Quantitative PCR assays we developed showed that 34.1% of Musca domestica (86/252) and 51.1% of Protophormia terraenovae (23/45) isolates were positive for the mcr-1 gene, 1.2% of M. domestica (3/252) and 2.2% of P. terraenovae (2.2%, 1/45) isolates were positive for mcr-2, and 5.2% of M. domestica (13/252) and 44.4% of P. terraenovae (20/45) isolates were positive for mcr-3 Overall, 4.8% (9/189) of bacteria isolated from the flies were positive for the mcr-1 gene (Escherichia coli: 8.3%, 4/48; Enterobacter cloacae: 12.5%, 1/8; Providencia alcalifaciens: 11.8%, 2/17; Providencia stuartii: 4.9%, 2/41), while none were positive for mcr-2 and mcr-3 Four mcr-1-positive isolates (two P. stuartii and two P. alcalifaciens) from blow flies trapped near a dumpster had a MIC for colistin above 4 mg/ml. This study reports mcr-1 carriage in Providencia spp. and detection of mcr-2 and mcr-3 after their initial identification in Belgium and China, respectively. This study suggests that flies might contribute significantly to the dissemination of bacteria, carrying these genes into a large variety of ecological niches. Further studies are warranted to explore the roles that flies might play in the spread of colistin resistance genes.IMPORTANCE Antimicrobial resistance is recognized as one of the most serious global threats to human health. An option for treatment of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) bacteria with multiple drug resistance was the reintroduction of the older antibiotic colistin. However, a mobile colistin resistance gene (mcr-1) has recently been found to occur widely; very recently, two other colistin resistance genes (mcr-2 and mcr-3) have been identified in Belgium and China, respectively. In this study, we report the presence of colistin resistance genes in flies. This study also reports the carriage of colistin resistance genes in the genus Providencia and detection of mcr-2 and mcr-3 after their initial identification. This study will stimulate more in-depth studies to fully elucidate the transmission mechanisms of the colistin resistance genes and their interaction.

Adding a Second Quinol to a Redox-Responsive MRI Contrast Agent Improves Its Relaxivity Response to H2O2.

The overproduction of reactive oxygen species has been linked to a wide array of health disorders. The ability to noninvasively monitor oxidative stress in vivo could provide substantial insight into the progression of these conditions and, in turn, could facilitate the development of better diagnosis and treatment options. A mononuclear Mn(II) complex with the redox-active ligand N,N'-bis(2,5-dihydroxybenzyl)-N,N'-bis(2-pyridinylmethyl)-1,2-ethanediamine (H4qtp2) was made and characterized. A previously prepared Mn(II) complex with a ligand containing a single quinol subunit was found to display a modest T1-derived relaxivity response to H2O2. The introduction of a second redox-active quinol both substantially improves the relaxivity response of the complex to H2O2 and reduces the cytotoxicity of the sensor but renders the complex more susceptible to transmetalation. The addition of H2O2 partially oxidizes the quinol subunits to para-quinones, concomitantly increasing the r1 from 5.46 mM-1 s-1 to 7.17 mM-1 s-1. The oxidation of the ligand enables more water molecules to coordinate to the metal ion, providing an explanation for the enhanced relaxivity. That the diquinol complex is only partially oxidized by H2O2 is attributed to its activity as an antioxidant; the complex can both catalytically degrade superoxide and serve as a hydrogen atom donor.

A comparison of microRNA expression profiles from splenic hemangiosarcoma, splenic nodular hyperplasia, and normal spleens of dogs.

BACKGROUND: Splenic masses are common in older dogs; yet diagnosis preceding splenectomy and histopathology remains elusive. MicroRNAs (miRNAs) are short, non-coding RNAs that play a role in post-transcriptional regulation, and differential expression of miRNAs between normal and tumor tissue has been used to diagnose neoplastic diseases. The objective of this study was to determine differential expression of miRNAs by use of RNA-sequencing in canine spleens that were histologically confirmed as hemangiosarcoma, nodular hyperplasia, or normal. RESULTS: Twenty-two miRNAs were found to be differentially expressed in hemangiosarcoma samples (4 between hemangiosarcoma and both nodular hyperplasia and normal spleen and 18 between hemangiosarcoma and normal spleen only). In particular, mir-26a, mir-126, mir-139, mir-140, mir-150, mir-203, mir-424, mir-503, mir-505, mir-542, mir-30e, mir-33b, mir-365, mir-758, mir-22, and mir-452 are of interest in the pathogenesis of hemangiosarcoma. CONCLUSIONS: Findings of this study confirm the hypothesis that miRNA expression profiles are different between canine splenic hemangiosarcoma, nodular hyperplasia, and normal spleens. A large portion of the differentially expressed miRNAs have roles in angiogenesis, with an additional group of miRNAs being dysregulated in vascular disease processes. Two other miRNAs have been implicated in cancer pathways such as PTEN and cell cycle checkpoints. The finding of multiple miRNAs with roles in angiogenesis and vascular disease is important, as hemangiosarcoma is a tumor of endothelial cells, which are driven by angiogenic stimuli. This study shows that miRNA dysregulation is a potential player in the pathogenesis of canine splenic hemangiosarcoma.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Effect of vascular seal configuration using the LigaSure on arterial challenge pressure, time for seal creation, and histologic features.

OBJECTIVE: To determine the effectiveness of a vascular sealing technology on canine carotid arteries using various seal configurations to achieve maximal vessel security. STUDY DESIGN: Ex-vivo study. ANIMALS: Dogs (n = 20). METHODS: Carotid arteries (n = 40) were removed from the mid-cervical region of recently euthanatized dogs. Harvested vessels were closed with 2 circumferential ligatures (Group 1) or a vascular sealing device using 1 of 4 seal configurations of 1 or 2 seals combined with 1 or 2 machine activations/seal. The artery was instrumented to measure intraluminal pressure to evaluate the security of each seal during saline infusion. Maximum intraluminal pressure was recorded for each group, and time for application of each sealing protocol was compared using 1-way ANOVA and Tukey's test for multiple comparisons. Histologic features of the sealing protocols were evaluated. RESULTS: Arterial closures for each group were effective in preventing leakage up to 300 mmHg. There was no significant difference in maximum intraluminal pressure between any group. A significant difference (P ≤ .001) was observed for time to seal creation between the groups using 1 and 2 seals. Histologic evaluation showed no differences between the different sealing protocols. CONCLUSION: Vessel sealing using a single seal created with a single activation cycle was adequate for sealing canine carotid arteries. Histologic examination did not demonstrate any disadvantages to multiple seals or multiple cycle activations.

Surgical technique and short-term outcome for experimental laparoscopic closure of the epiploic foramen in 6 horses.

OBJECTIVE: To describe a laparoscopic technique for, and short-term outcome after, closure of the epiploic foramen (EF) in horses. STUDY DESIGN: Descriptive, experimental study. ANIMALS: Healthy, adult horses (n = 6). METHODS: Laparoscopic portals to approach the EF were identified in standing horses. Under laparoscopic observation, the gastropancreatic fold and right lobe of the pancreas were grasped with Babcock forceps and secured to the caudate hepatic lobe using helical titanium coils to obliterate the EF. Surgical procedure time and intra- and postoperative complications were recorded. Serial analysis of select serum enzymes was used as an indication of involvement of the pancreas and liver. Closure was reevaluated at 4 weeks using repeat laparoscopy, and necropsy was performed immediately after. RESULTS: At initial surgery, EF closure was successful in all 6 horses; median surgical time was 40.5 minutes (range, 22-110 minutes). Serum gamma-glutamyl transferase (GGT) and sorbitol dehydrogenase (SDH) were not significantly altered by the surgical procedure; however, aspartate aminotransferase (AST) and amylase (AMY) were transiently increased. At repeat laparoscopic reevaluation, closure was complete in 5 horses, with partial closure of the EF observed in 1 horse. No complications related to the procedure were noted during or after surgery in any horse. CONCLUSIONS: EF closure in the standing horse can be accomplished without complications to the surrounding organs and vessels.

Intrahepatic mucinous cholangiocarcinoma with recurrent colic in a horse case report and literature review of cholangiocarcinoma in horses.

A 17-y-old Arabian mare was presented to the Auburn Large Animal Veterinary Teaching Hospital with a long-term history of intermittent mild recurrent colic that responded to medical treatment. CBC revealed mild lymphopenia; serum biochemistry findings were of increased gamma-glutamyl transferase and creatine kinase activities, hyperferremia, hyperglycemia, hypomagnesemia, and hypokalemia. Abdominocentesis was compatible with low-protein transudate. Due to the progression and duration of clinical signs, the owner elected euthanasia. Postmortem examination and histopathology confirmed a cholangiocarcinoma. The neoplastic cells were arranged in large cysts containing lakes of mucin that comprised 90% of the tumor volume; thus, a mucinous variant was determined. The neoplastic cells had strong cytoplasmic immunolabeling for cytokeratin 19 and lacked immunolabeling for hepatocyte paraffin 1, supporting bile duct origin. Cholangiocarcinomas are infrequent tumors in horses with nonspecific and slow progressive clinical signs, including recurrent colic. Mucinous cholangiocarcinomas are seldom reported in veterinary medicine and, to our knowledge, have not been reported previously in horses.

Nonclassified Lymphoma as a Cause of Radial Nerve Paralysis in a Horse.

A 4-year-old American Quarter Horse gelding was evaluated for acute non-weight-bearing lameness of the right thoracic limb with swelling in the right shoulder region. Physical examination revealed radial nerve paralysis of unknown etiology. The primary differential diagnosis was musculoskeletal trauma. Ultrasonography of the right shoulder region identified a heterogeneous mass that extended from the point of the shoulder to the thoracic inlet. Cytologic analysis of fluid collected by fine needle aspirate of the mass was consistent with large cell lymphoma. Based on the cytological findings, locally invasive neoplasia was diagnosed and considered the likely cause of the radial nerve paralysis. Because of the poor prognosis, the horse was euthanized, and postmortem examination confirmed the diagnosis of a nonclassified large cell lymphoma that extended from the deep tissues of the right pectoral muscle group into the thoracic inlet and pleural cavity, as well as the right brachial plexus. The mass in the region of the brachial plexus encompassed and mechanically compressed all of the nerves within the area, resulting in the clinical sign of radial nerve paralysis. Although neoplasia as a cause of radial nerve paralysis is rare, it should be considered as a differential diagnosis, regardless of age.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Artículo regular­Proliferación de miofibroblastos perisinusoidales hepáticos y respuesta inflamatoria sistémica que precede a la hepatitis por septicemia y toxemia (sep/tox) en pollos de engorde. La septicemia-toxemia (sep/tox) se incluye en la Categoría 1 de seguridad alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA) y es la causa más común y económicamente significativa de decomisos de canales de pollos de engorde. Las lesiones hepáticas se consideran una posible consecuencia de la septicemia y de la contaminación bacteriana asociada con la canal. Por lo tanto, estas lesiones se consideran un indicador de septicemia/toxemia (hepatitis sep/tox). Este estudio se llevó a cabo para analizar las lesiones histológicas que preceden a las lesiones hepáticas muy visibles que conducen a los decomisos debido a septicemia/toxemia en la planta de procesamiento. Se utilizaron hígados de canales de pollos de engorde decomisados por los inspectores del USDA por septicemia/toxemia para establecer criterios microscópicos y generales de hepatitis en etapa terminal de la septicemia/toxemia. Después de la caracterización de la hepatitis por septicemia/toxemia, los pollos de engorde de una granja con un historial de decomisos por septicemia/toxemia se sometieron a examen post mortem e investigación bacteriológica en cuatro intervalos durante los últimos 20 días de producción. Se enviaron cinco pollos sanos y cinco clínicamente enfermos de cuatro casetas a los 18, 25, 32 y 38 días de producción (160 en total). Las lesiones microscópicas que representan la proliferación de miofibroblastos perisinusoidales hepáticos (HPMP), la granulopoyesis extramedular periportal (PEMG), la histocitosis folicular esplénica y la celularidad de la médula ósea (BMC) se clasificaron subjetivamente para cada ave, y la clasificación subjetiva se evaluó con técnicas cuantitativas digitales. La morfología de las células estrelladas hepáticas perisinusoidales y la transformación progresiva de estas células en miofibroblastos se confirmó mediante inmunohistoquímica para actina y desmina del músculo liso. Los cultivos aeróbicos de hígados y vesícula biliar de aves con septicemia/toxemia no produjeron crecimiento de bacterias asociadas con la septicemia. Se observó proliferación de miofibroblastos perisinusoidales hepáticos de leve a severa en todos los grupos de edad, lo que representa el 28% de las aves examinadas. Los aumentos en las células inflamatorias observados por granulopoyesis extramedular periportal y celularidad de la médula ósea se correlacionaron positivamente con proliferación progresiva de miofibroblastos perisinusoidales hepáticos y con hepatitis por septicemia/toxemia en etapa terminal en pollos de engorde.

Phage constructs targeting gonadotropin-releasing hormone for fertility control: evaluation in cats.

OBJECTIVES: Phage-gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats. METHODS: Sexually mature male cats were injected with a phage-GnRH vaccine using the following treatment groups: (1) single phage-GnRH vaccine with adjuvant; (2) phage-GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage-GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7-9 months. RESULTS: All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24-42% and in group 3 by 15-36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions. CONCLUSIONS AND RELEVANCE: Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

Comparison of microbiota, antimicrobial resistance genes and mobile genetic elements in flies and the feces of sympatric animals.

Flies are well-known vectors of bacterial pathogens, but there are little data on their role in spreading microbial community and antimicrobial resistance. In this study, we compared the bacterial community, antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in flies with those in the feces of sympatric animals. A 16S rRNA-based microbial analysis identified 23 bacterial phyla in fecal samples and 25 phyla in flies; all the phyla identified in the fecal samples were also found in the flies. Bray-Curtis dissimilarity analysis showed that the microbiota of the flies were more similar to the microbiota of the feces of their sympatric animals than those of the feces from the three other animal species studied. The qPCR array amplified 276 ARGs/MGEs in fecal samples, and 216 ARGs/MGEs in the flies, while 198 of these genes were identified in both flies and feces. Long-term studies with larger sample numbers from more geospatially distinct populations and infection trials are indicated to further evaluate the possibility of flies as sentinels for antimicrobial resistance.

Osteoprotegerin inhibits vascular calcification without affecting atherosclerosis in ldlr(-/-) mice.

BACKGROUND: The role of osteoprotegerin in vascular disease is unclear. Recent observational studies show that serum osteoprotegerin levels are associated with the severity and progression of coronary artery disease, atherosclerosis, and vascular calcification in patients. However, genetic and treatment studies in mice suggest that osteoprotegerin may protect against vascular calcification. METHODS AND RESULTS: To test whether osteoprotegerin induces or prevents vascular disease, we treated atherogenic diet-fed ldlr(-/-) mice with recombinant osteoprotegerin (Fc-OPG) or vehicle for 5 months. Vehicle-treated mice developed significant, progressive atherosclerosis with increased plasma osteoprotegerin levels, consistent with observational studies, and approximately 15% of these atherosclerotic lesions developed calcified cartilage-like metaplasia. Treatment with Fc-OPG significantly reduced the calcified lesion area without affecting atherosclerotic lesion size or number, vascular cytokines, or plasma cholesterol levels. Treatment also significantly reduced tissue levels of aortic osteocalcin, a marker of mineralization. CONCLUSIONS: These data support a role for osteoprotegerin in the vasculature as an inhibitor of calcification and a marker, rather than a mediator, of atherosclerosis.

Dimethyl fumarate abrogates dust mite-induced allergic asthma by altering dendritic cell function.

INTRODUCTION: Allergic asthma is the most common inflammatory disease of upper airways. Airway dendritic cells (DCs) are key antigen presenting cells that regulate T helper 2 (Th2)-dependent allergic inflammation. Recent studies have shown critical role of airway DCs in the induction of Th2-mediated allergic inflammation and are attractive therapeutic targets in asthma. However, molecular signaling mechanism that regulate DCs function to Th2 immune responses are poorly understood. Here we aim to evaluate the immunomodulatory effect of dimethyl fumarate (DMF), an FDA approved small molecule drug, in the house dust mite (HDM)-induced experimental model of allergic asthma. METHODS: DMF was administered intranasally in the challenge period of HDM-induced murine model of experimental asthma. Airway inflammation, airway hyperreactivity, Th2/Th1 cytokine were assessed. The effect of DMF on DC function was further evaluated by adoptive transfer of HDM-pulsed DMF treated DCs to wild-type naïve mice. RESULTS: DMF treatment significantly reduced HDM-induced airway inflammation, mucous cell metaplasia, and airway hyperactivity to inhaled methacholine. Mechanistically, DMF interferes with the migration of lung DCs to draining mediastinal lymph nodes, thereby attenuates the induction of allergic sensitization and Th2 immune response. Notably, adoptive transfer of DMF treated DCs to naïve mice with HDM challenge similarly reduces the features of allergic asthma. CONCLUSION: This identifies a novel function of DMF on DC-mediated adaptive immune responses in the setting of HDM-induced airway inflammation. Taken together, our results offer a mechanistic rationale for DMF use to target DCs in local lung environment as antiasthmatic therapy.

DNA Vaccine Targeting Gonadotropin-Releasing Hormone Receptor and Its Application in Animal Contraception.

Overpopulation of free-roaming and wildlife animals negatively affects economy and public health in many parts of the world. Contraceptive vaccines are viewed as a valuable option for reducing numbers of unwanted animals. This study develops vaccines for potential use in animal contraception exploiting a DNA platform. Objectives of the study were to generate DNA constructs directed against gonadotropin-releasing hormone receptor (GnRHR), a crucial molecular player in animal reproduction, and characterize them for ability to promote immune responses and suppression of reproductive parameters in vivo. DNA constructs were created to encode for a recombinant protein composed of two domains: GnRHR, the target antigen, and ubiquitin (Ub), a support protein. Ub-GnRHR constructs administered intramuscularly or intradermally or containing different promoters were compared. CMV and EF1α promoters were shown to be superior to CAG. In fertility trials, mice immunized intradermally with Ub-GnRHR construct driven by EF1α had a significantly lower number of fetuses. Importantly, the impaired fertility was achieved with a single DNA immunization and without the use of adjuvants. The study demonstrated for the first time that targeting the GnRH receptor with DNA-based vaccines could be a viable option for animal contraception.

Multidrug-Resistant Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. in Houseflies and Blowflies from Farms and Their Environmental Settings.

Background: Antimicrobial resistance is rising globally at an alarming rate. While multiple active surveillance programs have been established to monitor the antimicrobial resistance, studies on the environmental link to antimicrobial spread are lacking. Methods: A total of 493 flies were trapped from a dairy unit, a dog kennel, a poultry farm, a beef cattle unit, an urban trash facility and an urban downtown area to isolate Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. for antimicrobial susceptibility testing and molecular characterization. Results: E. coli, K. pneumoniae and coagulase-negative Staphylococcus were recovered from 43.9%, 15.5% and 66.2% of the houseflies, and 26.0%, 19.2%, 37.0% of the blowflies, respectively. In total, 35.3% of flies were found to harbor antimicrobial-resistant bacteria and 9.0% contained multidrug-resistant isolates. Three Staphylococcus aureus isolates were recovered from blowflies while three extended spectrum beta lactamase (ESBL)-carrying E. coli and one ESBL-carrying K. pneumoniae were isolated from houseflies. Whole genome sequencing identified the antimicrobial resistance genes blaCMY-2 and blaCTXM-1 as ESBLs. Conclusion: Taken together, our data indicate that flies can be used as indicators for environmental contamination of antimicrobial resistance. More extensive studies are warranted to explore the sentinel role of flies for antimicrobial resistance.

Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors.

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.