Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.

No description available.

Many drugs used in the treatment of various cancers are derived from or influenced by compounds from nature. The soil bacterium Rhodococcus is of interest because of its identified secondary metabolic pathways and the production of novel natural antibiotics from several strains. In this study, a solid agar extraction method was used to collect compounds from strains of Rhodococcus. These bacterial compound extracts were then tested using a MTT assay in order to evaluate their effectiveness in augmenting MCF7 breast cancer cell death. The results of two way ANOVA analyses revealed 18 compound extracts from 15 strains of Rhodococcus that showed significant p-values when assayed with MCF7 breast cancer cells but nonsignificant interaction p-values when assayed with the healthy cell control. These results prompt further identification of specific compounds present in the bacterial extract that caused cell death as well as a mechanism of interaction with the breast cancer cells.

Ethanol usage as a transportation fuel is rapidly increasing in the United States. Production of ethanol from cellulose feedstocks via gasification followed by syngas fermentation offers an environmentally friendly approach that mitigates many of the adverse effects associated with production from corn. In the syngas fermentation process, the hydrogenase enzyme of the fermentation bacterium, Clostridium P11 for this work, supplies electrons to the metabolic pathway, facilitating ethanol production. In this thesis, an assay for P11 hydrogenase activity was developed. It was determined that 1) less than 4 minutes of sparging with 50 sccm H2 is needed to reduce O2 levels to below 1 ppm in a 3 mL aqueous solution, while less than 1 minute of purging at the same rate is needed to fill an air-filled 3.5 mL cuvette to 99.9999% H2, 2) 12.5 mM DTT included in the reaction mixture at pH 6 helps scavenge O2, 3) H2 diffusion is slow compared to enzymatic reaction rates, 4) CO2 lowers media pH, 5) 0.084 atm CO causes 90% inhibition of P11 hydrogenase, 6) prolonged Triton X-100 exposure diminishes hydrogenase activity, and 7) variations in H2 pressure and electron acceptor identity and concentration affect measured hydrogenase activities. The assay developed for P11 hydrogenase activity was used to perform kinetic studies. The Okura rapid-equilibrium rate law best described this activity. A constant that regulates the effect of H2 pressure on hydrogenase activity, KH2, was determined to be independent of electron acceptor and to have a value of 0.31 atm, implying that H2 must be supplied to the syngas fermentation at ~3 atm to maximize hydrogenase activity. KBV and KMV, constants that regulate the effect of benzyl viologen and methyl viologen on hydrogenase activity, were determined to be 1.7-2.4 mM and 10.6 mM, respectively. Additionally, hydrogenase activity was temporally correlated with ethanol production in batch cultures of P11 and strongly dependent on pH. The intracellular pH of P11 was determined to be approximately 5.5.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, characterized by platelet destruction and/or underproduction. The pathophysiology is heterogeneous and can be mediated by autoantibodies and cytotoxic T lymphocytes (CTLs). While platelet destruction in ITP is well documented, there is little support for platelet underproduction due to the inhibition of megakaryocyte growth and considerably less support for CTL-mediated platelet underproduction. Our objective was to develop an assay that could test for CTL-mediated inhibition of megakaryocyte growth (megakaryopoiesis) in ITP, using healthy controls. Peripheral blood from healthy donors was used to prepare hematopoietic stem and progenitor cells (HSPCs). These cells were expanded with StemSpan to culture a large number of megakaryocytes for the CTL assay. Our studies show that CTLs can be stimulated in-vitro using anti-CD3 antibodies and that they can be used after freezing and thawing. We also assessed CTL stimulation via peptide presentation, using viral peptides whom almost 100% of the general population have memory CTL specificity to, in order to activate a lower frequency of CTLs and to model levels of CTL activation in autoimmune disease. Both stimulants were found to stimulate CTLs in healthy donors with donor variability in the IFN-γ ELISpot. The CTL assay was developed by co-culturing thrombopoietin (TPO) stimulated HSPCs with autologous CTLs for 7 days to observe inhibition of megakaryocyte growth. To induce CTL stimulation, CTLs were either incubated with anti-CD3 or HSPCs were incubated with viral peptides before co-culturing with CTLs. Results showed that while viral peptides can be used as an internal control for the CTL assay, it could not serve as a positive control as inhibition was donor dependent. Inhibition of megakaryocyte growth in the presence of anti-CD3 stimulated CTLs was observed in all donors, validating its use as an appropriate positive control to study CD8+ T cell pathophysiology in ITP. / Thesis / Master of Science (MSc)

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Clostridioides difficile is a Gram positive, spore-producing, anaerobic bacterium. It is considered a nosocomial pathogen due to high incidence rates of C. difficile infections (CDI) in hospitals. However, research reveals an increase in community-associated cases. CDI is most common in the elderly, immunocompromised, or those taking a course of antibiotics. These individuals are more vulnerable to experiencing gut dysbiosis, allowing C. difficile to colonize the colon. CDI are an urgent threat due to their ability to sporulate. Spores are hardy and not eradicated by common disinfectants. They persist on surfaces the patient may have contact with during CDI. Current decontamination methods include the utilization of bleach-based disinfectants followed by ultraviolet (UV) light and adherence to a strict quality control protocol. However, spores may remain in the environment even after this process, thus allowing the pathogen to spread through surface contact. Another problem is the cost incurred by the hospital over time in terms of inpatient and equipment turnover related costs. These reasons make it imperative a better means of disinfection is developed. Silver is a known antimicrobial agent and utilized in clinical settings for burn treatment. Cerium oxide nanoparticles (CNP) possess antioxidant properties and can be used for drug delivery. Preliminary studies show silver-modified CNP (AgCNP) possess antiviral activity against COVID-19 and rhinovirus. To study the antimicrobial effects of the AgCNP against vegetative cells, a three-day time-kill assay was performed. Two strains of C. difficile, R20291 and NAP1, were cultured in BHIS (brain heart infusion, supplemented) and grown overnight. Glass coupons coated with AgCNP1 or AgCNP3, CNP1 or CNP3, or left uncoated were used in this study. They were incubated with cell culture at a variety of time points. The results indicate AgCNP3 may possess bactericidal activity. Further research should be conducted to determine the extent of this activity.

The growing threat of antimicrobial resistance is among the largest concerns in the world today. One method under development to combat this issue is the encapsulation of microbes in microfluidic droplets for single-cell testing. This method may be able to circumvent the need for a traditional positive cell culture which consumes the majority of the testing time using current diagnostic methods. This dissertation presents a method by which to deterministically encapsulate microbes using an artificial intelligence object detection algorithm and a Droplet-On-Demand microfluidic device. To accomplish this, the Droplet-On-Demand microfluidic device was first developed using a unique 3D-printing manufacturing method. An annular Channel-in-Channel droplet generator was developed which produced droplets within the hydrophobic 3D-printed polymeric microfluidic device. Supporting microfluidic unit operations were also developed including pumps, a 3-way flow-thru valve, and a detection window used for visualizing microfluidic particles. Control software was developed using python which controlled pneumatically-actuated membranes within the microfluidic device, the imaging system, and the object detection algorithm. 20-μm and 2-μm test particles were used as non-biological test particles while red blood cells and fluorescent E.coli baceria were used as biological test particles. All test particles were identified and encapsulated and show the flexibility of the system overall and the ability to identify a variety of particles of interest in microfluidic systems. Growth tests were conducted using E.coli bacteria encapsulated within microfluidic droplets with a fluorescent metabolic indicator. The fluorescence of droplets containing actively growing encapsulated bacteria was quantified using a unique first-principles model paired with an image processing protocol to provide relative concentration data to quantify the growth of the E.coli over time. These growth results indicated that bacterial growth in droplets could be detected and quickly quantified in 4 hours and thus provide practical results to clinicians on the susceptibility of bacteria to an antibiotic. This Droplet-On-Demand technology has the capability of providing clinically applicable data from the most basic and fundamental biological source, an individual cell; and that can be done with low concentrations and on any cell that can be visually identified.

Enoxaparin, a low-molecular-weight heparin, is commonly used as an anticoagulant in dogs, and is currently dosed at 0.8mg/kg every 6 hours. With an increase in individual enoxaparin doses, less frequent dosing may be possible, thereby reducing owner inconvenience and expense. The three phases of this study investigated the appropriate dose (Phase one- 0.8mg/kg, SQ once; Phase two- 2mg/kg, SQ once; Phase three- 1.3 mg/kg, SQ q8h for 7 total doses) and dosing interval needed for maximum effectiveness of enoxaparin. A Sonoclot® analyzer and factor Xa activity were used to assess level of anticoagulation in six healthy dogs. Anticoagulation was inconsistent at the 0.8mg/kg dose, while the 2mg/kg dose showed a high level of anticoagulation, and the 1.3mg/kg dose provided more reliable anticoagulation than the other dosages and dosing intervals. Small sample size and the use of same-breed healthy dogs potentially affected the strength of the results.

No description available.