Browsing by Subject "in vitro"
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Item Development and Characterization of an In-Vitro Tissue Culture Model for Equine Laminitis(2014-07-25) Johnson, MargareteEquine laminitis, a disease affecting the laminar tissue in the hoof, is a common and debilitating disease in horses with a significant impact on the equine industry. Currently nearly all laminitis studies are conducted in live horses, a process that is both expensive and limited in biological replicates. Thus the development of an in vitro model for the disease is an important step in advancing laminitis research. Recent evidence suggests that apolipoprotein A-IV (apoA-IV) may be involved in the chronic form of the disease but little is known about this protein in the horse, and its effects on the laminar tissue are unknown. The primary goal of this project was to produce a model for inducing inflammation in slices of laminar tissue in culture. We tested two inflammatory agents: interleukin 6 (IL-6) and lipopolysaccharide (LPS) and measured their effect on the expression of inflammatory cytokines and seven laminitis-associated genes found to be differentially expressed in horses with induced laminitis. The second goal of the project was to test the effects of apoA-IV on laminar tissue inflammation in our model in the presence and absence of the two inflammatory agents, and to further characterize the protein in horses by determining its sequence and expression pattern in this animal. The laminar tissue remained alive and contamination-free over the course of the experiment, showing the viability of our culture. IL-6 did not induce changes in gene expression consistent with those found in horses with laminitis. However, the addition of LPS led to changes in cytokine expression mimicking those seen in horses with induced laminitis and increased two of the seven laminitis-associated genes. The addition of apoA-IV had no effect on laminar tissue inflammation by itself or in the presence of IL-6 or LPS. We found the highest expression of APOA4 in the liver followed by the small intestine, a pattern unique in its high hepatic contribution. A better understanding of how apoA-IV is produced and functions in horses may shed light on its role in laminitis. In the future our tissue culture model could be used in testing agents suspected of causing laminar tissue inflammation and eventually in the development and testing of potential treatments for laminitis.Item Evaluation of anticancer potential of sorghums with different genetic characteristics and levels of phenolic compounds(2009-05-15) Guajardo Flores, SaraTo evaluate the anticancer potential of sorghum phenolic compounds, different experiments including in vitro and in vivo tests were performed. A set of 25 sorghum samples was evaluated for phenolic (total phenols, flavonoids, anthocyanins and tannins) content, hydrophilic and lipophilic antioxidant capacity using de Oxygen Radical Absorbance Capacity assay (ORAC), and screened for citotoxic properties in mammary, colon and hepatic mammalian cancer cell lines in vitro. Results indicated that there was a wide variability in the phytochemical profile among the different sorghums. Among the 25 samples, sumac sorghum bran had the highest amount of phenolic compounds, flavonoids, tannins and the highest ORAC values. It exerted the highest percent inhibition (near 100%) in mammary, colon and liver cancer cell lines. Sumac sorghum bran was selected for further investigation. Methanolic extracts from sumac whole grain, bran and tannin removed bran were tested in vitro at different concentrations in hormone dependent MCF-7 mammary cancer cells and non hormone dependent Caco2 and HepG2 colon and liver cancer cells. Results indicated that the methanolic extract from sumac bran inhibited 100% of MCF-7 cancer cells at a concentration of 0.5 mg/ml and that the citotoxic effect could be partially due to the tannin content of the extract. Concentrations of 0.5 and 1.5 mg/ml were selected for an in vivo preventive cancer study with 7,12-dymethylbenz(a)-anthracene (DMBA) induced female rats. Bran at low and high concentrations and the correspondent amount of methanol extracts were included in the diet. It was observed that sumac methanol extract at low concentration promoted tumor appearance and development, whereas sumac bran had a preventive effect, however, there were no significant differences in rats treated and un-treated with sumac. Differences between in vitro and in vivo results could be due to the degree of absorption of tannins during the in vivo experiment. To obtain additional data about the effect of sumac extracts on cancer development, a quinone reductase enzyme bioassay was performed. Methanol and hexane extracts from sumac bran induced phase II enzymes in vitro. Phytochemicals of sumac bran sorghum including phenolic compounds and lipid like compounds appeared to have potential for cancer prevention.Item In Vitro Inhibition of Listeria Monocytogenes by Novel Combinations of Food Antimicrobials(2011-02-22) Brandt, Alex LamarListeria monocytogenes is a foodborne pathogenic bacterium responsible for ~500 deaths and a financial burden of ~$2.3 billion each year in the United States. Though a zero tolerance policy is enforced with regard to its detection in cooked ready-to-eat foods, additional preemptive control alternatives are required for certain products. Among these alternatives are strategies permitting the usage of food antimicrobial combinations to control the pathogen. Research on antimicrobial combinations can provide insight into more efficient control of the pathogen, but is currently lacking. The purpose of this study was to evaluate the in vitro inhibition of L. monocytogenes exposed to the antimicrobials e-Poly-L-Lysine (EPL), lauric arginate ester (LAE), and sodium lactate (SL) at pH 7.3, octanoic acid (OCT) at pH 5.0, and nisin (NIS) and acidic calcium sulfate (ACS) at both pH 5.0 and 7.3. A broth dilution assay was used to determine single antimicrobial minimum inhibitory and bactericidal concentrations for L. monocytogenes Scott A, 310, NADC 2783, and NADC 2045. Optical density differences (delta<0.05 at 630 nm) were used to denote inhibition. Concentrations producing population decreases of greater than or equal to 3.0 log10 CFU/ml after incubation were considered bactericidal. Inhibition resulting from combinations of antimicrobials (NIS+ACS, EPL+ACS, SL+ACS, NIS+LAE, OCT+ACS, and OCT+NIS) was assessed using a checkerboard assay, and fractional inhibitory concentrations (FIC) were determined. FIC values were plotted on isobolograms and were used to create FIC indices (FICI). Isobologram curvature was used to classify combinations as synergistic, additive, or antagonistic. From FIC indices, interactions were defined as antagonistic (FICI >1.0), additive (FICI =1.0), or synergistic (FICI <1.0). Strain-dependent factors had a bearing on MIC and MBC values for NIS and EPL. At pH 7.3, NIS+ACS displayed synergistic inhibition, NIS+LAE and EPL+ACS demonstrated additive-type interactions, and the SL+ACS pairing was unable to be defined. At pH 5.0, interpretation of the OCT+NIS interaction also presented challenges, while the OCT+ACS combination resulted in synergistic behavior. Additional studies are needed to validate in vitro findings on surfaces of ready-to-eat meats. Future in vivo studies should investigate the ability of synergistic combinations (NIS+ACS and OCT+ACS) to control the pathogen. Better characterizations of inhibitory mechanisms should also be performed.Item Microplitis croceipes (Hymenoptera: Braconidae): A Life History Study and in vitro Rearing(2012-10-19) McLoud, Laura AnnMicroplitis croceipes (Hymenoptera: Braconidae) is an endoparasitoid and potential biological control agent of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae), an agricultural pest. The first objective of the following research was to amend current larval life history descriptions of M. croceipes. Larval head capsule width measurements were used to distinguish instar, and exuvium in abdominal cavities of post-egression hosts were indicative of a molt during parasitoid egression. Data revealed the larvae of M. croceipes pass through five instars, rather than three, as is indicated in the literature. The second objective was to investigate the suitability of potential artificial diets to be used in in vitro rearing of M. croceipes larvae. Three concentrations each of glucose, trehalose, and protein, as well as a combination diet (derived from initial diet trials) were tested. Growth, molting, and death were noted for each diet, and data indicated that diet had a significant effect for each performance measure (p = 0.0000, p < 0.0001, p < 0.0001, respectively). Data also indicated that trehalose and protein were more vital to larval parasitoid development (growth and molting) than was glucose, but no larvae were reared passed the second instar on an artificial diet. The final goals of the research were to evaluate the plausibility of rearing M. croceipes larvae to adulthood in vitro and to investigate post-egression host defensive behavior. Larvae were dissected from their hosts just prior to egression and placed in a cell culture plate in previously collected host hemolymph. Larvae were able to initiate pre-egression behavior in an in vitro environment, and a small percentage (6.67%) exhibited ecdysial splitting of the cuticle, however, no larvae were able to make the final molt in vitro. Post-egression hosts exhibited defensive behavior that may suggest they play a role in protecting pupating parasitoids. When the parasitoid exuvium was pulled from the egression wound in the host, hemolymph loss occurred and duration of the defensive behavior significantly decreased (p < 0.0001), indicating the exuvium acted to plug the egression wound, which prevented the host from bleeding to death and made it possible for the host to exhibit defensive behavior.Item Molecular and in vitro growth comparisons of Encephalitozoon hellem isolates from human and bird hosts(Texas A&M University, 2004-09-30) Waters, Paulette FrancescaMolecular and in vitro comparisons were performed using two isolates of Encephalitozoon hellem, one from an avian host and one from a human host, and one isolate of Encephalitozoon cuniculi from a rabbit. The molecular comparisons were performed by amplifying and sequencing the gene coding for a zinc metallo-aminopeptidase from cDNA and gDNA obtained from each of the isolates. The E. hellem sequences shared >99 % identity between each other and 70% identity with the E. cuniculi sequences. Conserved HEXXH and GXMEN motifs located within the sequences classify the protein as an aminopeptidase of the M1 family, with at least one zinc atom required for catalytic activity. In vitro growth comparisons of the isolates described above were performed under simulated "mammalian and avian conditions". The models utilized mammalian and avian cell lines and sera at incubation temperatures of 37 ?C and 40 ?C, respectively. Three separate experiments were performed. E. cuniculi grew best under the mammalian model and significantly better than both E. hellem isolates under this model. The E. hellem isolates were able to infect and replicate under both the mammalian and avian models, which reflects the zoonotic potential of these isolates.Item Predicting Forage Nutritive Value Using an In Vitro Gas Production Technique and Dry Matter Intake of Grazing Animals Using n-Alkanes(2011-08-08) Aguiar, Andre D.In the first experiment, forage samples (n = 39) were collected during 4 years (2006 ? 2009) from pastures grazed by Santa Gertrudis cattle at the King Ranch, TX. The in vitro gas production technique (IVGP) was performed to understand the pattern of fermentation parameters of the forage and obtain fractional digestion rate (kd) values to predict total digestible nutrients (TDN). The best nonlinear model to describe the IVGP values of the forages was the two-pool logistic equation. The passage rate (kp) of 4%/h was used.. The kp predicted by the Large Nutrient Ruminant System (LNRS) model was 3.66%/h. The average TDN was 55.9% compared to 53.8% using a theoretical equation. In the second experiment, Brahman bulls (n = 16) grazed Coastal bermudagrass pastures [Cynodon dactylon (L.) Pers.] and stocked at a moderate to low grazing pressure. Three periods of fecal collections were made within each period. Bulls were individually fed at 0700 and 1900 h of 400 g of corn gluten pellets containing C32 n-alkanes. Each period was divided in 2 sub periods in which fecal samples were collected 4 times a day (0700, 1100, 1500 and 1900 h). N-alkanes in the forage and feces were determined using gas chromatography. In the third experiment, four methods were used to estimate dry matter intake (DMI): C31 or C33 with or without adjustment for forage C32 (C31_0 and C33_0, respectively). There was a difference between morning (0700 and 1100 h) and afternoon fecal collections (1500 and 1900 h) on the predicted DMI using C31 (P = 0.0010), C33 (P = 0.0001), C31_0 (P = 0.0010), or C33_0 (P < 0.0001). There was no difference in average daily gain (ADG) between low and high residual feed intake (RFI) (P = 0.5709). The nonparametric analysis indicated that preranking animals for efficiency under confinement conditions does not guarantee (P < 0.0001) similar ranking under grazing conditions when using the alkane technique to determine forage DMI. In order to estimate DMI at least 5 d of fecal collection and 2 times a day of collection (0700 and 1500h) are needed to decrease the variability.