siRNA high throughput screening identifies regulators of chloropicrin and hydrogen fluoride injury in human corneal epithelial cell models.

Corneal injuries induced by various toxicants result in similar clinical presentations such as corneal opacity and neovascularization. Many studies suggest that several weeks post-exposure a convergence of the molecular mechanisms drives these progressive pathologies. However, chemical agents vary in toxicological properties, and early molecular responses are anticipated to be somewhat dissimilar for different toxicants. We chose 3120 targets from the Dharmacon Human Druggable genome to screen for chloropicrin (CP) and hydrogen fluoride (HF) corneal injury as we hypothesized that targets identified in vitro may be effective as therapeutic targets in future studies. Human immortalized corneal epithelial cells (SV40-HCEC) were used for screening. Cell viability and IL-8 were analyzed to down-select hits into validation studies, where multiplex cytokine analysis and high content analysis were performed to understand toxicant effect and target function. Some endpoints were also evaluated in a second human immortalized corneal epithelial cell line, TCEpi. Over 20 targets entered validation studies for CP and HF; of these, only three targets were shared: NR3C1, RELA, and KMT5A. These findings suggest that early molecular responses to different toxicants may be somewhat distinctive and present dissimilar targets for possible early intervention.

Development of mouse models for the study of chloropicrin and hydrogen fluoride ocular injury.

The possibility of chemical terrorism within the United States is a rising concern, with the eye being one of the most sensitive tissues to toxicant exposure. We sought to develop mouse models of toxicant-induced ocular injury for the purpose of evaluating potential therapeutics. Chloropicrin (CP) and hydrogen fluoride (HF) were selected for the study owing to their reportedly high potential to induce ocular injury. Eyes of female BALB/c mice were exposed to CP or HF vapor in order to produce a moderate injury, as defined by acute corneal epithelial loss followed by progressive corneal pathology with the absence of injury to deeper eye structures. Clinical injury progression was evaluated up to 12 weeks postexposure, where a significant dose-dependent induction of corneal neovascularization was measured. Histopathology noted epithelial necrosis and stromal edema as early as 24 h after exposure but was resolved by 12 weeks. A significant increase in inflammatory cytokine concentrations was measured in the cornea 24 h after exposure and returned to baseline by day 14. The ocular injury models we developed here for CP and HF exposure should serve as a valuable tool for the future evaluation of novel therapeutics and the molecular mechanisms of injury.

Improving Techniques to Study Equine Cervical Mucociliary Clearance.

Postbreeding bacterial uterine infections inflict major losses on the equine industry. Microcurrents propelled by ciliated cells between the folds of the uterus and cervix have been proposed as a means by which contaminants are expelled. Previous data have shown possible ciliary microcurrents propelling carbon particles, occasionally rotating, through cervical folds. However, adherence to the epithelium may have interfered with movement of carbon in these studies. Therefore, we tested potentially nonadherent substances to reveal ciliary microcurrents on the equine cervix under high magnification videoendoscopy. We hypothesized that polyethylene green microspheres 1-5 and 70 µm in diameter, would be superior to carbon in revealing microcurrents on the cervical epithelium and that 50 µm hemispherically coated bichromal microspheres would display rotation. A suspension containing these microspheres and carbon was deposited onto the cervix of five estrous mares, and movement of each type of particle was recorded under high-magnification videoendoscopy for 10-20 minutes. Particles were subjectively assessed for movement between folds, past stationary points, in opposing directions and at different speeds. Visibility, aggregation, motion, and rotation were scored numerically and analyzed by the Kruskal-Wallis test. Backward rotation of bichromal spheres was interpreted as evidence of ciliary activity. Overall, carbon scored equal to or higher than the microspheres, leading to rejection of the hypothesis. Subjective assessment concluded that cervical movement was closely related to respiratory movements of the mare, and that the constantly moving cervical folds helped clear the deposited particles.

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

GM1-gangliosidosis in American black bears: clinical, pathological, biochemical and molecular genetic characterization.

G(M1)-gangliosidosis is a rare progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of lysosomal ß-galactosidase. We have identified seven American black bears (Ursus americanus) found in the Northeast United States suffering from G(M1)-gangliosidosis. This report describes the clinical features, brain MRI, and morphologic, biochemical and molecular genetic findings in the affected bears. Brain lipids were compared with those in the brain of a G(M1)-mouse. The bears presented at ages 10-14 months in poor clinical condition, lethargic, tremulous and ataxic. They continued to decline and were humanely euthanized. The T(2)-weighted MR images of the brain of one bear disclosed white matter hyperintensity. Morphological studies of the brain from five of the bears revealed enlarged neurons with foamy cytoplasm containing granules. Axonal spheroids were present in white matter. Electron microscopic examination revealed lamellated membrane structures within neurons. Cytoplasmic vacuoles were found in the liver, kidneys and chondrocytes and foamy macrophages within the lungs. Acid ß-galactosidase activity in cultured skin fibroblasts was only 1-2% of control values. In the brain, ganglioside-bound sialic acid was increased more than 2-fold with G(M1)-ganglioside predominating. G(A1) content was also increased whereas cerebrosides and sulfatides were markedly decreased. The distribution of gangliosides was similar to that in the G(M1)-mouse brain, but the loss of myelin lipids was greater in the brain of the affected bear than in the brain of the G(M1) mouse. Isolated full-length cDNA of the black bear GLB1 gene revealed 86% homology to its human counterpart in nucleotide sequence and 82% in amino acid sequence. GLB1 cDNA from liver tissue of an affected bear contained a homozygous recessive T(1042) to C transition inducing a Tyr348 to His mutation (Y348H) within a highly conserved region of the GLB1 gene. The coincidence of several black bears with G(M1)-gangliosidosis in the same geographic area suggests increased frequency of a founder mutation in this animal population.

Relationships between intrauterine infusion of N-acetylcysteine, equine endometrial pathology, neutrophil function, post-breeding therapy, and reproductive performance.

Persistent endometritis in the mare is associated with hypersecretion of mucus by endometrial epithelium and migration of neutrophils into the uterine lumen. This study examines the relationships between N-acetylcysteine (NAC), a mucolytic agent with anti-inflammatory properties, and endometrial architecture, serum neutrophil function, post-breeding therapy, and reproductive performance of NAC-treated mares in a clinical setting. In study 1, endometrial biopsies from mares receiving intrauterine saline (fertile-control, n = 6) or 3.3% NAC (fertile-treatment, n = 6; barren-treatment, n = 10) were evaluated by histology and image analysis. In study 2, phagocytic activity of serum-derived neutrophils was measured after adding 0.5% or 3% NAC. In study 3, pregnancy rates of repeat breeders (n = 44) receiving an intrauterine infusion of 3.3% NAC 24-36 hours before mating (group 1) was recorded, as was first cycle of the season pregnancy rates of reproductively normal mares (group 2, n = 85), and mares treated for bacterial endometritis the cycle before mating (group 3, n = 25). Intrauterine NAC did not adversely affect endometrial histology. Extracellular mucus thickness and staining intensity were reduced in fertile-treatment mares (P < 0.03). Neutrophil function was inhibited by 3% NAC solution, but not by 0.5% NAC (P < 0.05). In study 3, for groups 1, 2, and 3, respectively, the first-cycle pregnancy rates were 77%, 74%, and 56%, and early embryonic death rates were 15%, 13%, and 7%. In group 2 mares treated with uterine lavage and oxytocin post-mating, the pregnancy rate was 89% (39/44), whereas in mares treated with uterine lavage and 1 g ceftiofur, it was 60% (24/40). Of the oxytocin-treated mares, 18% (8/44) had ≥ 1 cm of intrauterine fluid or marked uterine edema, whereas 80% (32/40) of the antibiotic-treated mares did. In conclusion, intrauterine infusion of a 3.3% solution of NAC was not irritating and inhibited the oxidative burst of neutrophils. Repeat breeder mares, with evidence of mucus hypersecretion, but no uterine pathogens, when treated with NAC followed by post-mating uterine lavage and oxytocin (and in some cases intrauterine antibiotics), achieved a pregnancy rate of 77%.

Evaluation and computational characterization of the facilitated transport of Glc carbon C-1 oxime reactivators across a blood brain barrier model.

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.

Synthesis of (±)-7-hydroxylycopodine.

A seven-step synthesis of (±)-7-hydroxylycopodine that proceeds in 5% overall yield has been achieved. The key step is a Prins reaction in 60% sulfuric acid that gave the key tricyclic intermediate with complete control of the ring fusion stereochemistry. A one-pot procedure orthogonally protected the primary alcohol as an acetate and the tertiary alcohol as a methylthiomethyl ether. The resulting product was converted to 7-hydroxydehydrolycopodine by heating with KO-t-Bu and benzophenone in benzene followed by acidic workup. During unsuccessful attempts to make optically pure starting material, we observed the selective Pt-catalyzed hydrogenation of the 5-phenyl group of a 4,5-diphenyloxazolidine under acidic conditions and the Pt-catalyzed isomerization of the oxazolidine to an amide under neutral conditions. In attempts to hydroxylate the starting material so that we could adapt this synthesis to the preparation of (±)-7,8-dihydroxylycopodine (sauroine) we observed the novel oxidation of a bicyclic vinylogous amide to a keto pyridine with Mn(OAc)(3) and to an amino phenol with KHMDS and oxygen.

Investigating the mechanism for maintaining eucalcemia despite immobility and anuria in the hibernating American black bear (Ursus americanus).

Ursine hibernation uniquely combines prolonged skeletal unloading, anuria, pregnancy, lactation, protein recycling, and lipolysis. This study presents a radiographic and biochemical picture of bone metabolism in free-ranging, female American black bears (Ursus americanus) that were active (spring bears and autumn bears) or hibernating (hibernating bears). Hibernating bears included lactating and non-lactating individuals. We measured serum calcium, albumin, inorganic phosphate, creatinine, bone specific alkaline phosphatase (BSALP), CTX, parathyroid hormone, insulin-like growth factor-I (IGF-l), leptin, 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and sclerostin from 35 to 50 tranquilized hibernating bears and 14 to 35 tranquilized spring bears. We compared metacarpal cortical indices (MCI), measured by digital X-ray radiogrammetry, from 60 hunter-killed autumn bears and 79 tranquilized, hibernating bears. MCI was greater in autumn than winter in younger bears, but showed no seasonal difference in older bears. During hibernation eucalcemia was maintained, BSALP was suppressed, and CTX was in the range expected for anuria. During hibernation 1,25(OH)(2)D was produced despite anuria. 1,25(OH)(2)D and IGF-I were less in hibernating than spring bears. In a quarter of hibernating bears, sclerostin was elevated. Leptin was greater in hibernating than spring bears. In hibernating bears, leptin correlated positively with BSALP in non-lactating bears and with CTX in lactating bears. Taken together the biochemical and radiographic findings indicate that during hibernation, bone turnover was persistent, balanced, and suppressed; bone resorption was lower than expected for an unloaded skeleton; and there was no unloading-induced bone loss. The skeleton appears to perceive that it was loaded when it was actually unloaded during hibernation. However, at the level of sclerostin, the skeleton recognized that it was unloaded. During hibernation leptin appeared anabolic in non-lactating bears and catabolic in lactating bears. We hypothesize that ursine hibernation may represent a natural model in which suppression of the sympathetic nervous system prevents unloading-induced bone loss by influencing leptin's skeletal effects and preventing transmission of loading information.

Immunisation of the equine uterus against Streptococcus equi subspecies zooepidemicus using an intranasal attenuated Salmonella vector.

Attenuated Salmonella enterica serovar Typhimurium MGN707, expressing the SzP protective protein of the MB9 serovar of Streptococcus equi subspecies zooepidemicus (SzP-MB9) was tested for its safety and efficacy as a nebulised intranasal vaccine against streptococcal uterine infections in mares. In a preliminary study, vaccinated mares (n=5) displayed serum, nasal and uterine responses (P<0.05) to S. Typhimurium lipopolysaccharide (St-LPS). Subsequently, vaccinated mares (expressor group, n=7), but not mares vaccinated with the vector only (control group, n=7), displayed significant increases in SzP-MB9 antibodies in serum, nasal and uterine washes (P<0.05). Assuming the uteri of all nine mares were free of streptococci prior to challenge with 6.3 x 10(9) colony forming units of S. e. zooepidemicus MB9, significantly fewer S. e. zooepidemicus were cultured from the uterine flushings of expressor-vaccinated mares (n=4) compared to control-vaccinated mares (n=5) (P<0.001). The only adverse reaction to vaccination was nasal haemorrhage in one mare.

Making sense of equine uterine infections: the many faces of physical clearance.

Equine uterine infections inflict major losses on the equine industry. Persistent inflammation of the oviduct and uterus leads to loss of the conceptus and mares susceptible to infection have weakened uterine defences partly due to retention of inflammatory exudate. Bacteria may trigger inflammation, resist phagocytosis, or adhere to the endometrium and types of infection range from genital commensals in susceptible mares to reproductive pathogens in normal mares. Uterine infections are diagnosed by history, detection of uterine inflammation, and isolation of typical organisms and susceptible mares may be identified by detection of intrauterine fluid during oestrus, or at 6-48 h post-breeding. Therapy includes oxytocin, uterine lavage, antibiotics, and prostaglandin analogues and clinical studies indicate additive benefits of oxytocin and antibiotics. Improved conception rates have been associated with autologous, intrauterine plasma, despite controversy about its bactericidal efficacy. Because of the potential for endometrial damage, intrauterine antiseptics require caution.

The equine immune response to Streptococcus equi subspecies zooepidemicus during uterine infection.

The purpose of this study was to describe strain-specific immune responses to Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) during uterine infection in horses. Five isolates of S. zooepidemicus were differentiated into four strains antigenically by bactericidal testing in blood of 12 horses, and genetically by pulsed-field gel electrophoresis. Eight healthy mares were then divided into two groups, each inoculated with one strain intrauterinely on three successive oestrous cycles followed by a second strain for three successive cycles, first and second strains being reversed for each group. Immune responses to both strains were assessed by bactericidal testing and immunoblotting over eight cycles. Both techniques indicated that immune responses to each strain arose at different times. Immunoblots showed greater binding to the first inoculated strain than to the second (P < 0.05). These data confirm that immune responses to S. zooepidemicus during uterine infection are partly strain-specific.