Michael B. Hildreth

Structure-Skin Permeability Relationship of Dendrimers

ABSTRACTPurposeTo investigate skin penetration of poly (amidoamine) (PAMAM) dendrimers as a function of surface charge and molecular weight in presence and absence of iontophoresis.MethodsDendrimers were labeled with fluoroisothiocynate (FITC); skin penetration of dendrimers was studied using excised porcine skin in-vitro. Skin penetration of FITC-labeled dendrimers was quantified using confocal laser scanning microscope (CLSM). G2-G6 NH2, G3.5-COOH and G4-OH dendrimers were used.ResultsCationic dendrimers showed higher skin penetration than neutral and anionic dendrimers. Skin penetration of cationic dendrimer increased linearly with increase in treatment time. Iontophoresis enhanced skin penetration of cationic and neutral dendrimers. Increase in current strength and current duration increased skin transport of dendrimers. Passive and iontophoretic skin penetration of cationic dendrimers was inversely related to their molecular weight. Dendrimer penetrated the skin through intercellular lipids and hair follicles. With iontophoresis, dendrimer was also found in localized skin regions.ConclusionsThe study demonstrates that the physicochemical properties of dendrimers influence their skin transport. Findings can be used to design dendrimer-based nanocarriers for drug delivery to skin.

Weather and Land Cover Influences on Mosquito Populations in Sioux Falls, South Dakota

ABSTRACT This study compared the spatial and temporal patterns of Culex tarsalis Coquillett and Aedes vexans Meigen populations and examined their relationships with land cover types and climatic variability in Sioux Falls, SD. Between 24 and 30 CDC CO2-baited light traps were set annually in Sioux Falls from May to September 2005–2008. Land cover data were acquired from the 2001 National Land Cover Dataset and the percentages of selected land cover types were calculated within a 600-m buffer zone around each trap. Meteorological information was summarized from local weather stations. Cx. tarsalis exhibited stronger spatial autocorrelation than Ae. vexans. Land cover analysis indicated that Cx. tarsalis was positively correlated with grass/hay, and Ae. vexans was positively correlated with wetlands. No associations were identified between irrigation and the host-seeking population of each species. Higher temperature in the current week and 2 wk prior and higher precipitation 3–4 wk before collection of host-seeking adult mosquitoes had positive influences on Cx. tarsalis abundance. Temperature in the current week and rainfall 2–3 wk before sampling had positive influences on Ae. vexans abundance. This study revealed the different influences of weather and land cover on important mosquito species in the Northern Great Plains region, which can be used to improve local vector control strategies and West Nile virus prevention efforts.

Ecological Niche of the 2003 West Nile Virus Epidemic in the Northern Great Plains of the United States

Background The incidence of West Nile virus (WNv) has remained high in the northern Great Plains compared to the rest of the United States. However, the reasons for the sustained high risk of WNv transmission in this region have not been determined. To assess the environmental drivers of WNv in the northern Great Plains, we analyzed the county-level spatial pattern of human cases during the 2003 epidemic across a seven-state region. Methodology/Principal Findings County-level data on WNv cases were examined using spatial cluster analysis, and were used to fit statistical models with weather, climate, and land use variables as predictors. In 2003 there was a single large cluster of elevated WNv risk encompassing North Dakota, South Dakota, and Nebraska along with portions of eastern Montana and Wyoming. The relative risk of WNv remained high within the boundaries of this cluster from 2004–2007. WNv incidence during the 2003 epidemic was found to have a stronger relationship with long-term climate patterns than with annual weather in either 2002 or 2003. WNv incidence increased with mean May–July temperature and had a unimodal relationship with total May–July precipitation. WNv incidence also increased with the percentage of irrigated cropland and with the percentage of the human population living in rural areas. Conclusions/Significance The spatial pattern of WNv cases during the 2003 epidemic in the northern Great Plains was associated with both climatic gradients and land use patterns. These results were interpreted as evidence that environmental conditions across much of the northern Great Plains create a favorable ecological niche for Culex tarsalis, a particularly efficient vector of WNv. Further research is needed to determine the proximal causes of sustained WNv transmission and to enhance strategies for disease prevention.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

BackgroundMagnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development.MethodsUVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle.ResultsMagnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells.ConclusionsMagnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Effect of mouse strain variations and cortisone treatment on the establishment and growth of primary Echinococcus multilocularis hydatid cysts.

The main purpose of this study was to determine the effects of cortisone on the number and size of primary Echinococcus multilocularis cysts developing in a moderately resistant strain of mice, i.e., C3H/HeJ. Computerized image analysis was used to measure the surface area occupied by hydatid cysts 10 wk after inoculation of the mice with E. multilocularis eggs. Our second objective was to compare the infectivity of primary E. multilocularis hydatid cysts in C57BL/6J-Ay/a (lethal yellow) mice with that in C57BL/6J-a/a (non-agouti black) mice. The data obtained show no difference between the C3H/HeJ and C57BL/6J-a/a strains of mice; yet, the image analysis method was able to detect a slight increase in the total cyst size within the Ay/a mutant of the C57BL/6J strain. Treatment of C3H/HeJ mice with cortisone drastically increased both the number of cysts and the average size of each cyst when the treatment occurred early in the infection.

Immunohistochemical characterization of the distribution of galectin-4 in porcine small intestine

Galectins are an evolutionarily conserved family of 15 different lectins found in various combinations in virtually every type of animal cell. One of the primary galectins expressed in intestinal epithelium is galectin-4, a tandem-repeat galectin with two carbohydrate-recognition domains in a single polypeptide chain. In the current study, we produced an anti-galectin-4 monoclonal antibody (MAb) for determining the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its functions in the small intestine. In immunohistochemistry studies, this MAb detected galectin-4 primarily in the cytoplasm of absorptive epithelial cells lining intestinal villi. Mature epithelial cells at the villous tips stained the most intensely with this MAb, with progressively less intense staining observed along the sides of villi and into the crypts. In addition to its cytoplasmic localization, galectin-4 was also associated with nuclei in villous tip cells, indicating that some galectin-4 may migrate to the nucleus during terminal maturation of these cells. In intestinal crypts, a specific subset of cells, which may be enteroendocrine cells, expressed galectin-4 at a relatively high level. Galectin-4 distribution patterns were similar in all three regions (duodenum, jejunum, and ileum) of porcine small intestine.

Chemopreventive Effects of Sarcophine-diol on Ultraviolet B-induced Skin Tumor Development in SKH-1 Hairless Mice

Sarcophine-diol (SD), one of the structural modifications of sarcophine, has shown chemopreventive effects on 12-dimethylbenz(a)anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor development in female CD-1 mice. The objective of this study was to determine the chemopreventive effects of SD on UVB-induced skin tumor development in hairless SKH-1 mice, a model more relevant to human skin cancer, and to determine the possible mechanisms of action. Carcinogenesis was initiated and promoted by UVB radiation. Female hairless SKH-1 mice were divided into two groups having 27 mice in each group: control and SD treatment. The control group was topically treated with 100 μL acetone and SD treatment group was topically treated with SD (30 μg/100 μL in acetone) 1 hour before each UVB radiation for 32 weeks. Tumor counts were recorded on a weekly basis for 30 weeks. Effects of SD on the expression of caspases were investigated to elucidate the possible mechanism of action. The proteins from epidermal homogenates of experimental mice were used for SDS-PAGE and Western blotting using specific antibodies against caspase-3, caspase-8 and caspase-9 respectively. TUNEL assay was used for determining DNA fragmented apoptotic cells in situ. Results showed that at the end of experiment, tumor multiplicity in control and SD treatment groups was 25.8 and 16.5 tumors per mouse respectively. Furthermore, Topical treatment of SD induced DNA fragmented apoptotic cells by upgrading the expressions of cleaved caspase-3 and caspase-8. This study clearly suggested that SD could be an effective chemopreventive agent for UVB-induced skin cancer by inducing caspase dependent apoptosis.

A single pasture limited treatment approach to estimate production loss from gastrointestinal nematodes in grazing stocker cattle

The objective of this study was to evaluate the use of a single pasture, limited treatment methodology to assess the impact of gastrointestinal nematodes on weight gain in grazing cattle. From a group of 450 British crossbred, yearling spayed heifers, 60 animals were randomly selected (prospective randomization) prior to placement on summer pasture. Each of these 60 animals was weighed, a fecal sample obtained for nematode egg count and a uniquely numbered ear tag applied. A randomly assigned group of 30 received treatment with one ivermectin sustained release bolus, while the remaining 30 served as non-treated controls. The treatment and control groups rejoined the remaining non-treated 390 animals, and were grazed as a single group for 165 days. At grazing conclusion, treatment and control cattle were individually weighed, and fecal samples obtained for nematode egg counts. Treatment was associated with a 0.064 kg per grazing day gain increase, or a 10 kg increase over the grazing season (P = 0.02). Nematode egg counts at grazing initiation were not different between treatment and control (P = 0.30), though egg counts in treated cattle at study end were lower than control (P < 0.0001). Results of this study support the use of a single pasture limited treatment approach for measuring the effect that internal nematodes have on weight gain in grazing cattle under commercial range conditions.

In Vitro Screening of Forty Medicinal Plant Extracts from the United States Northern Great Plains for Anthelmintic Activity Against Haemonchus Contortus

An egg hatch assay (EHA) and a larval migration assay (LMA) involving Haemonchus contortus was used to evaluate the anthelmintic activity of methanol extracts from 40 plants that are native or naturalized within the U.S.A. Northern Great Plains. Only one of these 40 plants (i.e. Lotus corniculatus) had been previously evaluated for activity against any gastrointestinal nematode. The various extracts were initially screened at 50mg/ml diluted either in 0.5% dimethyl sulphoxide (DMSO) or 3-(N-morpholino) propanesulfonic acid (MOPS buffer), and plants showing 100% inhibition at 50mg/ml, were further evaluated at 8 other concentrations (25-0.19 mg/ml). Extracts with 100% activity with the EHA were again screened with the LMA (50mg/ml). Two extracts with the highest LMA inhibition were also evaluated at lower concentrations (25-3.1mg/ml). Of the 40 methanolic extracts screened, 7 (Chrysothamnus viscidiflorus, Ericameria nauseosa, Liatris punctata, Melilotus alba, Melilotus officinalis, Perideridia gairdneri, and Sanguinaria canadensis) showed significant egg-hatch inhibition in DMSO and MOPS buffer. Three extracts (Geranium viscosissimum, L. corniculatus, and Rhus aromatica) only showed significant inhibition in DMSO. The 8 extracts showing 100% efficacy at 50mg/ml exhibited dose-dependent effects at the 8 lower concentrations, and R. aromatica and E. nauseosa extracts had the lowest ED50 values. Similarly, when these 8 plant extracts were further evaluated with the LMA, the extracts of E. nauseosa and R. aromatica again exhibited the highest activity (p<0.001), with ED50 values of 4.0mg/ml and 10.43 mg/ml respectively. Three other extracts (C. viscidiflorus, M. alba and M. officinalis) also showed inhibitory activity in the LMA. These results support the need for additional evaluations of the nematocidal properties for at least these 5 plants.

Overwintering strategies of a population of anthelmintic-resistant Haemonchus contortus within a sheep flock from the United States Northern Great Plains.

Haemonchus contortus infections have been increasingly reported in ungulates from cold climates even though past studies have shown that the free-living juveniles from this species survive poorly under freezing conditions. Overwintering strategies of H. contortus have not been documented in the Unites States Northern Great Plains. A PCR survey identified H. contortus as vastly predominant trichostrongyle species present (in addition to occasional detections of Teladorsagia sp.) in a closed farm flock of sheep from Brookings County, SD. Benzimidazole (BZ) and avermectin (AV) anthelmintics had been used intensely for many years on this flock. During the autumn season, three fecal egg count reduction tests (doramectin, albendazole, and moxidectin) were performed over a 4 year span to assess drug effectiveness within the flock. Significant drug resistance was found in Haemonchus adults with doramectin (69% efficacy), marginal resistance was found with albendazole (90% efficacy) and no resistance was found in moxidectin (100% efficacy). The following spring, pre-lambing and post-lambing fecals were obtained from albendazole and moxidectin treatment years to assess the resistance of the tissue-dwelling fourth-stage juveniles (J4s) at those times. Albendazole treated pre-lambing fecals averaged only 4 EPG and treated post-lambing fecals increased to 454 EPG, indicating that many of the J4s were not killed during the autumn treatment. Moxidectin pre-lambing fecals averaged only 1 EPG, and post-lambing fecals only increased to 6 EPG in the treated moxidectin population and 1422 EPG in the untreated moxidectin population. In addition to evaluating the ability of H. contortus to overwinter as drug resistant tissue-dwelling J4s, this study also evaluated the overwintering ability of pasture-dwelling, free-living third-stage juveniles at this farm. In the summers of 2010 and 2011, naïve tracer lambs were placed on a H. contortus contaminated pasture for 3 weeks to assess J3 winter survival. In 2010, tracer lambs only averaged 7 EPG whereas drylotted control lambs averaged 2 EPG; in 2011, tracer lambs averaged 2 EPG while the control lambs averaged 1 EPG. These results suggest that at this northern plains farm, yearly transmission of H. contortus is predominately through drug-resistant J4s. This is consistent with other cold-climate, overwintering studies involving H. contortus from Europe.