MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

Anti-glomerular basement membrane glomerulonephritis: Characterization of an epitope, antibody response, and the potential role of IL-4Ralpha in disease progression

Anti-Glomerular Basement Membrane Glomerulonephritis (anti-GBM GM) is one of the earliest described autoimmune disorders. Patients present with proteinuria, anti-GBM antibodies, and renal failure. Studies have implicated a T Helper 1 (TH1) response in disease induction and a T Helper 2 (TH2) response for disease progression. A 13 amino acid long peptide sequence spanning residues 28 through 40 [pCol(28–40)] of the Collagen IV α3 non-collagen domain (Col IV α3 NCD) is immunogenic and induces anti-GBM GN. In order to fully understand disease initiation, this peptide was further characterized. Peptides were created containing one amino acid substitution for the entire length of pCol(28–40) and induction of anti-GBM GN was monitored. When residues 31, 33, or 34 contained the substitution, anti-GBM GN was unable to be induced. Thus, residues 31, 33, and 34 of pCol(28–40) are required for induction of anti-GBM. Glomerular injury is observed as early as 14 days post anti-GBM GN induction. However, the presence of anti-GBM antibodies is not observed until 20 days post immunization. An enlarged lymph node adjacent to the diseased kidney exhibits B cell activation after renal injury and produces antibodies toward GBM. Thus, anti-GBM antibodies are a consequence of the initial renal injury. Differences between disease susceptible and disease resistant rat strains exist in the expression of IL-4Rα, a major player in the TH2 response. IL-4Rα signaling is regulated by soluble IL-4Rα (sIL-4Rα). Low expression levels of sIL-4Rα result in the stabilization of IL-4 binding, while elevated expression sequesters IL-4. Quantitative PCR experiments noted low siL-4Rα expression levels in disease susceptible rats. Induction of an immune response toward sIL-4Rα in this strain was responsible for delayed disease progression in 15 out of the 17 experimental animals. Antibody transfer and in vivo biological activity experiments confirmed that delayed disease development was due to anti-sIL-4Rα antibodies. Together these experiments indicate that a T-cell epitope is required for activation of a TH1 autoimmune response and anti-GBM antibodies are a consequence of renal injury. More importantly, a role for IL-4Rα signaling is implicated in the progression of anti-GBM GN. ^

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

THE ASSOCIATION BETWEEN CANCER-RELATED-FATIGUE, RESPONSE TO FATIGUE TREATMENT, AND CYTOKINES AND THEIR RECEPTORS, IN PATIENTS WITH ADVANCED CANCER

Background: Inflammation is implicated in the development of cancer related fatigue (CRF). However there is limited literature on the mediators of inflammation (namely), cytokines and their receptors, associated with clinically significant fatigue and response to treatment. Methods: We reviewed 37 advanced cancer patients with fatigue (≥4/10), who participated in two Randomized Controlled Trials, of anti-inflammatory agents (Thalidomide and Dexamethasone) for CRF. Responders showed improvement in FACIT-F subscale at the end of study (Day 15). Baseline patient characteristics and symptoms were assessed by FACIT-F, ESAS; serum cytokines [IL-1β and receptor antagonist (IL-1RA), IL-6, IL-6R, TNF-α and sTNF-R1 and R2, IL-8, IL-10, IL-17] levels measured by Luminex. Data were analyzed using principal component analysis (PCA) [reporting cumulative variance (variance) for the first four components] to determine their association with fatigue and response to treatment. Results: Females were 54%. Mean (SD) was as follows for age, 61(14); baseline FACIT (F) scores, 21.4(8.6); ESAS Fatigue item, 6.5(1.9); and FACIT-F change, 6.4(9.7); ESAS (fatigue) change, -2 (2.41). Baseline median in pg/mL for IL-6, TNF-α, IL-1β were 31.9; 18.9; 0.55, respectively. Change in IL-6 negatively correlated with change in FACIT-F scores (p=0.02). Baseline CRF (FACIT-F score) was associated with IL-6, IL-6R and IL-17, Variance = 78% whereas IL-10, IL-1RA, TNF-α and IL-1β were associated with improvement of CRF, Variance=74%. Conversely, IL-6 and IL-8 were associated with no improvement or worsening of CRF, Variance= 93%. Conclusions: Change in IL-6 negatively correlated with change in FACIT-F scores. IL-6, IL-6R and IL-17 are associated with CRF while IL-6 and IL-8 were associated with no improvement of CRF. Further studies are warranted confirm our findings.

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

New implications of E1A gene therapy in breast and ovarian cancer

A variety of human cancers overexpress the HER-2/neu proto-oncogene. Among patients with breast and ovarian cancers this HER-2/ neu overexpression indicates an unfavorable prognosis, with a shorter overall survival duration and a lower response rate to chemotherapeutic agents. Downregulation of HER-2/neu gene expression in cancer cells through attenuation of HER-2/neu promoter activity is, therefore, an attractive strategy for reversing the transformation phenotype and thus the chemoresistance induced by HER-2/neu overexpression. ^ A viral transcriptional regulator, the adenovirus type 5 E1A (early region 1A) that can repress the HER-2/neu promoter, had been identified in the laboratory of Dr. Mien-Chie Hung. Following the identification of the E1A gene, a series of studies revealed that repression of HER-2/neu by the E1A gene which can act therapeutically as a tumor suppressor gene for HER-2/ neu-overexpressing cancers. ^ The results of these preclinical studies became the basis for a phase I trial for E1A gene therapy among patients with HER-2/neu-overexpressing breast and ovarian cancer. In this dissertation, three primary questions concerned with new implications of E1A gene therapy are addressed: First, could E1A gene therapy be incorporated with conventional chemotherapy? Second, could the E1A gene be delivered systemically to exert an anti-tumor effect? And third, what is the activity of the E1A gene in low-HER-2/neu-expressing cancer cells? ^ With regard to the first question, the studies reported in this dissertation have shown that the sensitivity of HER-2/neu-overexpressing breast and ovarian cancer to paclitaxel is in fact enhanced by the downregulation of HER-2/neu overexpression by E1A. With regard to the second question, studies have shown that the E1A gene can exert anti-tumor activity by i.v. injection of the E1A gene complexed with the novel cationic liposome/protamine sulfate/DNA type I (LPDI). And with regard to the third question, the studies of low-HER-2/ neu-expressing breast and ovarian cancers reported here have shown that the E1A gene does in fact suppress metastatic capability. It did not, however, suppress the tumorigenicity. ^ Three conclusions can be drawn from the experimental findings reported in this dissertation. Combining paclitaxel with E1A gene therapy may expand the implications of the gene therapy in the future phase II clinical trial. Anti-tumor activity at a distant site may be achieved with the i.v. injection of the E1A gene. Lastly when administered therapeutically the anti-metastatic effect of the E1A gene in low-HER-2/neu-expressing breast cancer cells may prevent metastasis in primary breast cancer. (Abstract shortened by UMI.)^

ANAPHYLACTICALLY - MEDIATED EPITHELIAL CHLORINE ION SECRETORY RESPONSE INISOLATED JEJUNUM OF PARASITIZED GUINEA PIGS (TRICHINELLA SPIRALIS)

Electrophysiological studies were conducted to test the hypothesis that alterations in intestinal epithelial function are associated with immunological responses directed against the enteric parasite, Trichinella spirals. Trichinella antigens were used to challenge sensitized jejunum from infected guinea pigs while monitoring ion transport properties of the tissue in an Ussing-type chamber. The addition of antigen caused increases in transepithelial PD and I(,sc) that were rapidly induced, peaked at 1.5 to 2 min after antigen-challenge, and lasted 10 to 20 min thereafter. The increase in I(,sc) ((DELTA)I(,sc)) varied in a dose-dependent manner until a maximal increase of 40 (mu)A/cm('2) was obtained by the addition of 13 (mu)g of antigenic protein per ml of serosal fluid in the Ussing chamber. Trichinella antigen did not elicit alterations in either PD or I(,sc) of nonimmune tissue. Jejunal tissue from guinea pigs immunized with ovalbumin according to a protocol that stimulated homocytotropic antibody production responded electrically to challenge with ovalbumin but not trichinella antigen. Jejunal tissue which was passively sensitized with immune serum having a passive cutaneous anaphylaxis (PCA) titer of 32 for both IgE and IgG(,1) anti-trichinella anti-bodies responded electrically after exposure to trichinella antigen. Heat treatment of immune serum abolished the anti-trichinella IgE titer as determined by the PCA test but did not decrease either the electrical response of passively sensitized tissue to antigen or the anaphylactically mediated intestinal smooth muscle contractile response to antigen in the classical Schultz-Dale assay. These results strongly support the hypothesis that immunological responses directed against Trichinella Spiralis alter intestinal epithelial function and suggest that immediate hypersensitivity is the immunological basis of the response.^ Additional studies were performed to test the hypothesis that histamine and prostaglandins that are released from mucosal mast cells during IgE or IgG(,1) - antigen stimulated degranulation mediate electrophysiological changes in the intestinal epithelium that are reflective of Cl('-) secretion and mediated intracellularly by cAMP. Pharmacological and biochemical studies were performed to determine the physiological messengers and ionic basis of electrical alterations in small intestinal epithelium of the guinea pig during in vitro anaphylaxis. Results suggest that Cl('-) secretion mediated, in part, by cAMP contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting epithelial responses. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^

11beta-hydroxysteroid dehydrogenases as regulators of pulmonary corticosterone during the granulomatous response to trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis requires development and maintenance of granulomatous lesions, a feature considered to be the pathological hallmark of Tuberculosis (TB) disease. Upon encountering Mtb or mycobacterial antigens, specifically trehalose 6,6'-dimycolate (TDM), a strong local pro-inflammatory response is initiated. Systemic production of anti-inflammatory glucocorticoids (GCs) is also induced. Emergence of these antagonists at the inflammatory foci is counterproductive to development of the granulomatous structure and detrimental to host protection against TB. Therefore, it was hypothesized that local enzymatic regulation of GCs occurs locally at the site of granulomatous inflammation. The experiments described here strongly suggest that 11β-hydroxysteroid dehydrogenases (11βHSDs) shuttle GCs between active and inert forms during the acute granulomatous response, supporting the net reduction of corticosterone. The patterns of GC and 11βHSD regulation were specific to the lung (the site of inflammation) and were not observed in other tissues. Furthermore, 11βHSD2, which decreases corticosterone concentrations, was not expressed in models of dysregulated granulomatous inflammation. These findings suggest that cellular exposure to local active GC concentrations is restricted via 11βHSDs as a mechanism to initiate and maintain granuloma formation. The information derived from the experiments outlined in this dissertation provides a better understanding of the events required for establishment and maintenance of the protective granulomatous response. As a practical consequence, exploiting 11βHSD2 modulation of GCs at the site of Mtb infection may lead to improvement of Tuberculosis treatment strategies.^

Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^