Class of 2012 Abstract / Specific Aims: The incidence of non-response, recurrence, relapse, and rate of complications of Clostridium difficile infections treated with combination of metronidazole and vancomycin versus vancomycin or metronidazole alone over a one-year period by treatment and strain type (i.e. NAP1/BI/027) were evaluated. The incidence of mortality in patients with moderate to severe Clostridium difficile associated diarrhea prescribed metronidazole, vancomycin, or combination metronidazole plus vancomycin as initial therapy was also determined. Additionally, significant factors associated with the use of combination vancomycin-metronidazole as initial therapy for moderate to severe CDAD were characterized. Methods: T This retrospective medical record review has been approved by the Institutional Review Board. Adult patients with stool specimens tested for detection of Clostridium difficile toxin B by PCR between April 2010 and March 2011 at a tertiary care, academic medical center were evaluated. Patients were included in the study if diagnosed with moderate to severe disease and received either monotherapy with metronidazole, monotherapy with oral vancomycin, or combination therapy with metronidazole and oral vancomycin for at least 80% of the first 10 days of treatment. Patients who are discharged alive within 72 hours of admission or who received therapy for less than 48 hours were excluded. Main Results: All patients (N=411) with laboratory evidence of Clostridium difficile during the study time period were evaluated. A total of 26 subjects who received oral vancomycin monotherapy and 56 subjects who received oral vancomycin along with metronidazole for at least 80% of the first 10 days of treatment were identified. Of the subjects who received oral vancomycin monotherapy during the first ten days of therapy, 5 (19%) were classified has a treatment failure or died within the first 21 days of therapy and 5 (19%) had either a recurrence or reappearance of Clostridium difficile associated diarrhea between 22 and 65 days post start of therapy. Of the subjects who received a combination of oral vancomycin and metronidazole during the first 10 days of therapy, 14 (25%) were classified has a treatment failure or died within the first 21 days of therapy and 22 (39%) had either a recurrence or reappearance of Clostridium difficile associated diarrhea between 22 and 65 days post start of therapy. In the combination therapy group, 5 (9%) were reported to have an ileus, toxic megacolon, or necrotic bowel during the first 10 days of therapy. Conclusions: In this study, the subjects who received a combination of oral vancomycin and metronidazole had higher rates of clinical failure, death, and recurrence than subjects who received monotherapy. Current guideline statements recommend combination therapy only in patients with an ileus with Clostridium difficile-associated diarrhea.

Indiana University-Purdue University Indianapolis (IUPUI) / We have previously demonstrated that disabling the transcription factor Nuclear Matrix Protein 4 (NMP4) improved parathyroid hormone (PTH)-induced trabecular bone gain in ovariectomized (OVX) and healthy mice. Here we evaluated whether loss of Nmp4 enhanced bone restoration in OVX mice under concurrent PTH combination therapies and anti-catabolic mono-therapies. Wild type (WT) and Nmp4-/- mice were OVX at 12wks of age followed by therapy regimens, administered from 16wks-24wks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Generally the PTH+RAL and PTH+ZOL therapies were more effective in restoring bone than the PTH mono-therapy. Loss of Nmp4 further improved the restoration of femoral trabecular bone under these treatments. RAL and ZOL mono-therapies moderately increased bone volume but unexpectedly the Nmp4-/- mice showed an enhanced RAL-induced increase in femoral trabecular bone. Immunohistochemical and flow cytometry analyses of the bone marrow and serum profiling for markers of bone formation and resorption indicated that the heightened osteoanabolism of the Nmp4-/- mice under these diverse osteoporosis treatments was partially attributed to an expansion of the osteoprogenitor pool. To address whether the enhanced bone formation observed in Nmp4-/- mice produced structurally sound tissue, mechanical testing was conducted on the femurs of healthy mice treated with intermittent PTH, RAL mono-therapy, or PTH+RAL. Nmp4-/- femurs showed modestly improved mechanical and material properties. At the cellular level, loss of Nmp4 accelerated mineralization in differentiating mesenchymal stem/progenitor cells (MSPCs). Transcriptomic and biochemical analyses indicated that loss of Nmp4 elevated ribosome biogenesis and expanded the capacity of the endoplasmic reticulum for processing protein. Preliminary data showed that disabling Nmp4 increased both aerobic glycolysis and oxidative phosphorylation in osteoprogenitors, which is an emerging hallmark of anabolic osteogenic cells. Transcriptomic analysis also suggested NMP4 targeted pathways driving bone formation. These included but not limited to BMP, IGF1, TGFβ and Wnt signaling pathways. Finally, transcriptomic profiling revealed that Nmp4-/- MSPCs showed a significant perturbation in numerous immunomodulatory pathways, particularly in the interleukin system. The heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, providing a promising target pathway for identifying barriers to pharmacologically-induced bone formation.

The rise of antimicrobial resistance (AMR) has culminated in the most pressing problem in modern medicine. The situation is most acute with regards to the management of multi- drug resistant Gram-negative infections (MDRGNB) with common infections increasingly untreatable due to rapidly dwindling therapeutic options. A solution to the problem of AMR is unlikely to be easily found, but revisiting and re-purposing existing antimicrobials is a viable approach in the medium term. This study investigated the use of unorthodox antimicrobial combination therapies for the treatment of MDRGNB, with particular focus on agents of last resort. A systematic review of clinical studies highlighted the potential for polymyxin (colistin) combination therapies (e.g. colistin-rifampicin, colistin-carbapenems), although this could not be supported in a formal meta-analysis. A systematic approach for screening MDRAB for susceptibility to novel colistin combinations using multiple methods was employed and uncovered a number that were more potent than those previously identfied. The most potent combination that was consistently identified was colistin when combined with fusidic acid, despite this drug having no useful activity against MDRGNB on its own. The combination was further evaluated in static time-kill assays against a range of Gram-negative pathogens with defined resistance mechanisms, including to polymyxins and using invertebrate (Galleria mellonella) and murine models of MDRGNB infection. Colistin and fusidic acid combination therapy was subsequently used to successfully treat a case of ventilator-associated pneumonia due to MDR A. baumannii. This work highlights how older drugs can be re-purposed to tackle the problem of AMR using a precision medicine approach. Further studies to elucidate the mechanism of action of the colistin- fusidic acid combination and a formal clinical trial are warranted to investigate the potential utility of this combination in the treatment of MDRGNB with the expressed goal of bridging the current antimicrobial development gap.

Despite recent advances, prognosis in metastatic prostate cancer remains poor. As with other cancers, tumor heterogeneity is an increasingly evident contributor in prostate tumorigenesis and developed resistance. Using in vitro and in vivo model systems, we examined novel diagnostic and therapeutic strategies in prostate cancer. In these studies, combination treatment with amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor, was assessed for its ability to differentially modulate growth signaling in pathway diverse LNCaP (PTEN⁻) and DU-145 (PTEN⁺) human prostate cancer cell and mouse xenograft models. Our results suggest both individual mechanistic signaling activities, as well as benefits of the combination therapy though modulations of MAPK (pERK) and 4EBP1/cyclin D1 in growth signaling divergent PTEN+ and PTEN- prostate cancer cells. Additionally, despite the importance preanalytical tissue preservation on downstream diagnostic assays, exact protocols are not well defined and highly variable clinically and, as such, critical diagnostic information is lost. We show that a novel 2+2 fixation method induces target- and cell-specific alterations in immunostain intensity and efficacy. Importantly, cyclin D1 is increasingly utilized for as a clinical prognostic/diagnostic marker and demonstrated improved immunohistochemical staining efficacy with 2+2 fixation compared with treatment-matched xenograft protein alterations as assessed by western analysis. Finally, pentoxifylline (PTX) is a clinically utilized and well tolerated PDE inhibitor that has shown promise as a radio-/chemo-sensitization and anti-cancer agent against a variety of cancers. In these studies, we demonstrate that PTX induces cell and tumor growth inhibition in LNCaP prostate cancer cells. Mechanistically, PTX induces transient cellular signaling modulations of both the AMPK metabolic and AKT/mTOR growth pathways, while inducing autophagy. Also, PTX sensitizes LNCaP prostate cancer to cytotoxicity induced by first line chemotherapy docetaxel, inducing significant cellular apoptosis and reducing effective docetaxel concentrations by >10 fold for equivalent toxicity in viability assays. These findings nominate PTX as an adjunct therapy for the treatment of prostate cancer. In summary, these studies characterize the targeted signaling modulation by combination erlotinib and amuvatinib therapy, as well as pentoxifylline, for their use as therapies for prostate cancer. A novel fixation protocol was also assessed for improved diagnostic tissue preservation of critical signaling proteins. Further understanding in these areas will aid and expand the development of effective diagnostics, as well as emphasize the benefits of these and similar therapeutics for the treatment of prostate cancer.

Class of 2007 Abstract / Objectives: This 12-month retrospective, naturalistic study determined medication adherence, psychiatric hospitalizations, cost of services, cost of prescriptions, and rates of polypharmacy (less than 4 versus greater than or equal to 4 concomitant psychotropic medications) for patients receiving clozapine versus quetiapine therapy for the treatment of schizophrenia in an outpatient mental health facility. Methods: The clozapine and quetiapine groups were compared for gender, age, medication adherence rates, hospitalizations, cost of care, polypharmacy, and types of concomitant psychotropic medications over 12-months. The polypharmacy groups for clozapine and quetiapine (e.g., greater than or equal to 4 psychotropic medications versus less than 4 psychotropic medications) were compared for medication adherence. Results: A total of 71 patients met the entry criteria (44 = clozapine and 27 = quetiapine). The two groups were similar for age, gender, court order, average daily dose, and hospitalization rates. The clozapine group had a higher medication adherence rate of 0.901 (e.g., 329 days supply) compared to the quetiapine group’s adherence rate of 0.723 (e.g., 264 days supply) (p=0.007). The clozapine group had higher costs for medication, labs, and other services compared to the quetiapine group, as well as total costs of services (p=0.004). The clozapine group was on fewer concomitant psychotropic medications compared to the quetiapine group based on the rates of polypharmacy. Conclusions: Patient on clozapine therapy had improved medication adherence and lower rates of polypharmacy, but higher costs of care compared to quetiapine. The frequent monitoring required with clozapine may result in medication adherence that results in improved efficacy, less polypharmacy, and lower hospitalization rates. Further studies in larger populations are needed to compare different frequency rates of monitoring patients on outcome measures over a longer period of treatment.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

Contexte. Le paludisme provoque annuellement le décès d’environ 25 000 enfants de moins de cinq ans au Burkina Faso. Afin d’améliorer un accès rapide à des traitements efficaces, les autorités burkinabées ont introduit en 2010 la prise en charge du paludisme par les agents de santé communautaires (ASC). Alors que son efficacité a été démontrée dans des études contrôlées, très peu d’études ont évalué cette stratégie implantée dans des conditions naturelles et à l’échelle nationale. Objectif. L’objectif central de cette thèse est d’évaluer, dans des conditions réelles d’implantation, les effets du programme burkinabé de prise en charge communautaire du paludisme sur le recours aux soins des enfants fébriles. Les objectifs spécifiques sont : (1) de sonder les perceptions des ASC à l’égard du programme et explorer les facteurs contextuels susceptibles d’affecter leur performance ; (2) d’estimer le recours aux ASC par les enfants fébriles et identifier ses déterminants ; (3) de mesurer, auprès des enfants fébriles, le changement des pratiques de recours aux soins induit par l’introduction d’une intervention concomitante – la gratuité des soins dans les centres de santé. Méthodes. L’étude a été conduite dans deux districts sanitaires similaires, Kaya et Zorgho. Le devis d’évaluation combine des volets qualitatifs et quantitatifs. Des entrevues ont été menées avec tous les ASC de la zone à l’étude (N=27). Des enquêtes ont été répétées annuellement entre 2011 et 2013 auprès de 3002 ménages sélectionnés aléatoirement. Les pratiques de recours aux soins de tous les enfants de moins de cinq ans ayant connu un récent épisode de maladie ont été étudiées (N2011=707 ; N2012=787 ; N2013=831). Résultats. Les résultats montrent que le recours aux ASC est très modeste en comparaison de précédentes études réalisées dans des milieux contrôlés. Des obstacles liés à l’implantation du programme de prise en charge communautaire du paludisme ont été identifiés ainsi qu’un défaut de faisabilité dans les milieux urbains. Enfin, l’efficacité du programme communautaire a été négativement affectée par l’introduction de la gratuité dans les centres de santé. Conclusion. La prise en charge communautaire du paludisme rencontre au Burkina Faso des obstacles importants de faisabilité et d’implantation qui compromettent son efficacité potentielle pour réduire la mortalité infantile. Le manque de coordination entre le programme et des interventions locales concomitantes peut générer des effets néfastes et inattendus. / Context. In Burkina Faso, malaria causes approximately 25,000 deaths every year in children under five. In 2010, national health authorities introduced case management of malaria by community health workers (CHWs) as a way to increase prompt access to effective treatments. While this strategy’s efficacy has been demonstrated in controlled studies, very few studies evaluated its effectiveness under real-world and nation-wide conditions of implementation. Objective. The overarching aim of this thesis is to evaluate the effects of the Burkinabè program on treatment-seeking practices in febrile children. The specific objectives are: (1) to examine CHWs’ perceptions and investigate the contextual factors likely to affect their performance; (2) to estimate the use of CHWs in febrile children and its determinants; (3) to evalauate changes in treatment-seeking practices induced by the introduction of a concomitant intervention – the removal of user fees at health centres. Methods. The study was conducted in two similar health districts, Kaya and Zorgho. The evaluation design integrates quantitative and qualitative components. Interviews were carried out with all CHWs in the study area (N=27). Surveys were repeated every year from 2011 to 2013 in 3002 randomly selected households. Treatment-seeking practices of all children with a recent sickness episode (N2011=707; N2012=787; N2013=831) were examined. Results. Results show that the use of CHWs is really low in comparison to previous controlled studies. Feasibility issues in urban areas and barriers to implementation of the community case management of malaria programme were identified. Moreover, its effectiveness in rural areas was challenged by the removal of user fees at health centres. Conclusion. In Burkina Faso, community case management of malaria faces serious challenges of feasibility and implentation. These challenges compromise the programme’s potential to reduce child morbidity and mortality. The lack of integration between the programme and local concomitant interventions can generate unpredicted adverse effects.