Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

PLASMACYTOID DENDRITIC CELLS SENSE SKIN INJURY AND PROMOTE WOUND HEALING THROUGH TYPE I INTERFERONS

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells, which selectively express intracellular Toll-like receptors (TLR)-7 and TLR-9 and have the capacity to produce large amounts of type I IFNs (IFN-a/b) in response to viruses or host derived nucleic acid containing complexes. pDCs are normally absent in skin but accumulate in the skin of psoriasis patients where their chronic activation to produce IFN-a/b drives the disease formation. Whether pDCs and their activation to produce IFN-a/b play a functional role in healthy skin is unknown. Here we show that pDCs are rapidly and transiently recruited into healthy human and mouse skin upon epidermal injury. Infiltrating pDCs were found to sense nucleic acids in wounded skin via TLRs, leading to the production of IFN-a/b. The production of IFN-a/b was paralleled by a short lived expression of cathelicidins, which form complexes with extracellular nucleic acids and activated pDCs to produce IFN-a/b in vitro. In vivo, cathelicidins were sufficient but not necessary for the induction of IFN-a/b in wounded skin, suggesting redundancy of this pathway. Depletion of pDCs or inhibition of IFN-a/bR signaling significantly impaired the inflammatory response and delayed re-epithelialization of skin wounds. Thus we uncover a novel role of pDCs in sensing skin injury via TLR mediated recognition of nucleic acids and demonstrate their involvement in the early inflammatory process and wound healing response through the production of IFN-a/b.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Studies of MEKK2 activation mechanisms and its biological role in LPS/TLR4 signaling

MEKK2 is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that controls the MAPK and IKK-NF-κB pathways. The MAPK and IKK pathways are intracellular signaling networks that are crucial for the Toll-like receptor (TLR) mediated innate immunity, cellular stress and many other physiological responses. Members of the MAP3K family are central to the activation of these processes. However, the molecular mechanisms underlying stimuli-mediated MAP3K activation remain largely unknown. In this study, we identified a key phosphoserine residue, Ser-519 in MEKK2, and its equivalent site Ser-526 in MEKK3 within their activation loop whose phosphorylation are essential for their optimal activation. Mutation of this regulatory serine to an alanine severely impaired MEKK2 activation and MEKK2 signaling to its downstream targets. To demonstrate that physiological stimuli induce this serine phosphorylation, we generated an antibody that specifically recognizes the phosphorylated serine residue. We found that many, but not all, of the MAPK agonists, including the TLR ligands, growth factors, cytokines and cellular stresses, induced this regulatory serine phosphorylation in MEKK2, suggesting an involvement of MEKK2 in the activation of the MAPK cascade leading to different cellular responses. We further investigated the specific role of MEKK2 in LPS/TLR4 signaling by using MEKK2−/− mice. We found that MEKK2 was selectively required for LPS-induced ERK1/2 activation, but not JNK, p38 or NF-κB activation. We also found that MEKK2 was involved in TLR4 dependent induction of proinflammatory cytokines and LPS-induced septic shock. In conclusion, we identified a key regulatory serine residue in the activation loop of MEKK2 whose phosphorylation is a key sensor of receptor- and cellular stress-mediated signals. We also demonstrated that MEKK2 is crucial for TLR4-mediated innate immunity. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

The relationship between oral contraceptives and pelvic inflammatory disease: Policy implications and recommendations

One of the most widely accepted noncontraceptive benefits of oral contraceptive use is the reduction in the development of pelvic inflammatory disease (PID) and its sequelae in users. While much of the research over the past forty years has found an association between oral contraceptive use and reduced rates of PID [Senanayake, 1980], more recent studies have qualified and even challenged this widely held belief. [Henry-Suchet, 1997; Ness 1997; Ness, 2001] PID, an infection in the upper genital tract causing infertility and ectopic pregnancy, affects over one million women in the United States each year, exacting an enormous toll on women's reproductive and emotional health, as well as our economy. [CDC Factsheet, 2007] This thesis examines the public health implications of pelvic inflammatory disease and the use of oral contraceptives. Sixteen original studies are reviewed and analyzed, thirteen of which found a protective benefit with oral contraceptive use against PID and three more recent studies which found no protective benefit or association between oral contraceptive use and PID. Analysis of the research findings suggests a need for additional research, provider and patient education, and an increased government role in addressing the ongoing and significant public health concerns raised by current rates of Chlamydia- and gonorrheal-PID. ^

Crack and powder cocaine federal legislation: A sentencing disparity and potential for policy improvement

Since its introduction into the United States in the 1980s, crack cocaine has been a harsh epidemic that has taken its toll on a countless number of people. This highly addictive, cheap and readily available drug of abuse has permeated many demographic sectors, mostly in low income, lesser educated, and urban communities. This epidemic of crack cocaine use in inner city areas across the Unites States has been described as an expression of economic marginality and “social suffering” coupled with the local and international forces of drug market economies (Agar 2003). As crack cocaine is a derivative of cocaine, it utilizes the psychoactive component of the drug, but delivers it in a much stronger, quicker, and more addictive fashion. This, coupled with its ready availability and cheap price has allowed for users to not only become very addicted very quickly, but to be subject to the stringent and sometimes unequal or inconsistent punishments for possession and distribution of crack-cocaine. ^ There are many public health and social ramifications from the abuse of crack-cocaine, and these epidemics appear to target low income and minority groups. Public health issues relating to the physical, mental, and economic strain will be addressed, as well as the direct and indirect effects of the punishments that come as a result of the disparity in penalties for cocaine and crack-cocaine possession and distribution. ^ Three new policies have recently been introduced into the United Stated Congress that actively address the disparity in sentencing for drug and criminal activities. They are, (1) Powder-Crack Cocaine Penalty Equalization Act of 2009, (HR 18, 111th Cong. 2009), (2) The Drug Sentencing Reform and Cocaine Kingpin Trafficking Act of 2009, (HR 265, 111th Cong. 2009) and (3) The Justice Integrity Act of 2009, (111th Cong. 2009). ^ Although they have only been initiated, if passed, they have potential to not only eliminate the crack-cocaine disparity, but to enact laws that help those affected by this epidemic. The final and overarching goal of this paper is to analyze and ultimately choose the ideal policy that would not only eliminate the cocaine and crack disparity regardless of current or future state statutes, but will provide the best method of rehabilitation, prevention, and justice. ^

ALLERGEN SENSITIZATION AND IMMUNOMODULATION BY SYNTHETIC LIGANDS, PUL-042, IN AN ALLERGEN-INDUCED ASTHMA MURINE MODEL

Allergen-induced asthma is the leading form of asthma and a chronic condition worldwide. Common allergens are known to contribute to the pathogenesis of this disease. Murine models of allergic asthma have mostly used an intraperitoneal route of sensitization (not airway) to study this disease. Allergic asthma pathophysiology involves the activation of TH2-specific cells, which triggers production of IgE antibodies, the up-regulation of TH2-specific cytokines (i.e. IL-4, IL-5, IL-9 and IL-13), increased airway eosinophilia, and mucin hypersecretion. Although there are several therapeutics currently treating asthmatic patients, some of these treatments can result in drug tolerance and may be linked to increased mortality. CpG oligodeoxynucleotides (ODNs) is a synthetic ligand that targets Toll-like Receptor (TLR) 9. It has been evaluated as a therapeutic agent for the treatment of cancer, infectious diseases, and for treating allergy and asthma. PUL-042 is also a synthetic TLR ligand and is composed of two agonists against TLR2/6 heterodimer and TLR9. Previous studies have evaluated PUL-042 for its ability to confer resistance against bacterial and viral lung infection. These findings, combined with studies performed using CpG ODNs, led to speculation that PUL-042 dampens the immune response in allergen-induced asthma. My thesis research investigated airway route sensitization and airway delivery of PUL-042 to evaluate its effects in reducing an allergen-induced asthma phenotype in a murine model. The results of this study contribute to the foundation for future investigations to evaluate the efficacy of PUL-042 as a novel therapy in allergic-asthma disease.

Venous thromboembolism in patients with acute leukemia: Prevalence, incidence, recurrence of thrombosis, risk factors, and impact on survival

Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the third most preventable cardiovascular disease and a growing public health problem in the United States. The incidence of VTE remains high with an annual estimate of more than 600,000 symptomatic events. DVT affects an estimated 2 million American each year with a death toll of 300,000 persons per year from DVT-related PE. Leukemia patients are at high risk for both hemorrhage and thrombosis; however, little is known about thrombosis among acute leukemia patients. The ultimate goal of this dissertation was to obtain deep understanding of thrombotic issue among acute leukemia patients. The dissertation was presented in a format of three papers. First paper mainly looked at distribution and risk factors associated with development of VTE among patients with acute leukemia prior to leukemia treatment. Second paper looked at incidence, risk factors, and impact of VTE on survival of patients with acute lymphoblastic leukemia during treatment. Third paper looked at recurrence and risk factors for VTE recurrence among acute leukemia patients with an initial episode of VTE. Descriptive statistics, Chi-squared or Fisher's exact test, median test, Mann-Whitney test, logistic regression analysis, Nonparametric Estimation Kaplan-Meier with a log-rank test or Cox model were used when appropriate. Results from analyses indicated that acute leukemia patients had a high prevalence, incidence, and recurrent rate of VTE. Prior history of VTE, obesity, older age, low platelet account, presence of Philadelphia positive ALL, use of oral contraceptives or hormone replacement therapy, presence of malignancies, and co-morbidities may place leukemia patients at an increased risk for VTE development or recurrence. Interestingly, development of VTE was not associated with a higher risk of death among hospitalized acute leukemia patients.^

Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.

STIMULATION THROUGH TLR4 INCREASES FVIII INHIBITOR FORMATION IN A MOUSE MODEL OF HEMOPHILIA A

Hemophilia A is a clotting disorder caused by functional factor VIII (FVIII) deficiency. About 25% of patients treated with therapeutic recombinant FVIII develop antibodies (inhibitors) that render subsequent FVIII treatments ineffective. The immune mechanisms of inhibitor formation are not entirely understood, but circumstantial evidence indicates a role for increased inflammatory response, possibly via stimulation of Toll-like receptors (TLRs), at the time of FVIII immunization. I hypothesized that stimulation through TLR4 in conjunction with FVIII treatments would increase the formation of FVIII inhibitors. To test this hypothesis, FVIII K.O. mice were injected with recombinant human FVIII with or without concomitant doses of TLR4 agonist (lipopoysaccharide; LPS). The addition of LPS combined with FVIII significantly increased the rate and the production of anti-FVIII IgG antibodies and neutralizing FVIII inhibitors. In the spleen, repeated in vivo TLR4 stimulation with LPS increased the relative percentage of macrophages and dendritic cells (DCs) over the course of 4 injections. However, repeated in vivo FVIII stimulation significantly increased the density of TLR4 expressed on the surface of all spleen antigen presenting cells (APCs). Culture of splenocytes isolated from mice revealed that the combined stimulation of LPS and FVIII also synergistically increased early secretion of the inflammatory cytokines IL-6, TNF-α, and IL-10, which was not maintained throughout the course of the repeated injections. While cytokine secretion was relatively unchanged in response to FVIII re-stimulation in culture, LPS re-stimulation in culture induced increased and prolonged inflammatory cytokine secretion. Re-stimulation with both LPS and FVIII induced cytokine secretion similar to LPS stimulation alone. Interestingly, long term treatment of mice with LPS alone resulted in splenocytes that showed reduced response to FVIII in culture. Together these results indicated that creating a pro-inflammatory environment through the combined stimulation of chronic, low-dose LPS and FVIII changed not only the populations but also the repertoire of APCs in the spleen, triggering the increased production of FVIII inhibitors. These results suggested an anti-inflammatory regimen should be instituted for all hemophilia A patients to reduce or delay the formation of FVIII inhibitors during replacement therapy.

IL -24; a glycosylated dimeric cytokine affecting monocytes and cytotoxic to melanoma cells

IL-24 is an unusual member of the IL-10 family, which is considered a Th1 cytokine that exhibits tumor cell cytotoxicity. I describe the purification of this novel cytokine from the supernatant of IL-24 gene transfected human embryonic kidney cells and define the biochemical and functional properties of the soluble, human IL-24 protein. ^ I showed IL-24 non-covalently associates with bovine albumin. Immunoaffinity purification followed by cation exchange chromatography resulted in the significant enrichment of N-glycosylated IL-24. This protein elicited dose-dependent secretion of TNF-α and IL-6 from purified human monocytes and TNF-α secretion from PMA differentiated U937 cells. I showed this same protein was cytotoxic to melanoma tumor cells via the induction of IFN-α. ^ I reported IL-24 associates as at least two disulfide linked, N-glycosylated dimers. Enzymatic removal of N-linked-glycosylation from purified IL-24 partially diminished its cytokine and cytotoxic functions. Disruption of IL-24 dimers via reduction and alkylation of intermolecular disulfide bonds nearly abolished IL-24s cytokine function. ^ I elucidated IL-24 induced TNF-α secretion was pSTAT1, pSTAT3 as well as the class II heterodimeric receptors IL-20R1/IL-22R2 independent. I identified a requirement for the heterodimer of Toll-like Receptors 1 and 2 for IL-24s cytokine function and show a physical interaction between IL-24 and the extracellular domain of TLR-1. ^ Thus, I demonstrated that purified N-glycosylated, soluble, dimeric, human IL-24 exhibits both immunomodulatory and anti-cancer activities and these functions remain associated during purification. IL-24 induced TNF-α secretion required an interaction with the heterodimeric receptor TLR-1/2 and IL-24s cytotoxic affect to melanoma tumor cells was in part due to its induction of IFN-β. ^