Theraputic Effects of Genistein, Minozac, and Fosteum in a Mouse Model of Mucopolysaccaridosis Type IIIB (Sanfilippo Syn...

MISSING IMAGE

Material Information

Title:
Theraputic Effects of Genistein, Minozac, and Fosteum in a Mouse Model of Mucopolysaccaridosis Type IIIB (Sanfilippo Syndrome B)
Physical Description:
1 online resource (77 p.)
Language:
english
Creator:
Gibney, Joseph Michael
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences, Molecular Genetics and Microbiology
Committee Chair:
Heldermon, Coy D
Committee Members:
Rowe, Thomas C
Scott, Edward W
Kraft, John

Subjects

Subjects / Keywords:
mucopolysaccharidosis
Molecular Genetics and Microbiology -- Dissertations, Academic -- UF
Genre:
Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Sanfilipposyndrome, or Mucopolysaccharidosis type IIIB (MPS IIIB), is a genetically inherited lysosomal storage disease caused by the absence of N-acetylglucosaminidase(NAGLU). The absence of NAGLU results in accumulation of the glycosaminoglycan(GAG) substrate, heparan sulphate, in the lysosomes causing them to become distended. This leads to the stimulation of a pro-inflammatory state,increasing the activation of immune cells. The immune response may exacerbate damage resulting from abnormal lysosomal function. Sanfilippo syndrome most severely affects the central nervous system with initially delayed behavioral development progressing to neurocognitive regression. Systemic manifestations include organomegaly, diminished vision and hearing, and altered motor function. No curative treatment currently exists for MPS IIIB. Minozac is a CNS-penetrant small molecule previously shown to suppress proinflammatory cytokine upregulation in mouse models of seizure and traumatic brain injury. Genistein®is a tyrosine kinase inhibitor, which acts on both EGF and IGF receptors. Fosteum®is Genistein® with the addition of cholecalciferol and citrated zinc bisglycinate. Evidence suggests that Genistein® reduces heparan sulfate production causing NAGLU substrate reduction and a decrease in proinflammatory cytokine upregulation, while Minozac inhibits the production of proinflammatory cytokines TNFa and IL-1ß. We report the effect of treatment with these compounds separately, or in combination, for four weeks in MPS IIIB and wild type mice at ages 10 and 28 weeks. The primary outcome measures include the effects of these compounds on pro-inflammatory cytokine levels and immune cell activation in the brain. Additional measures of effects of these agents on vascular growth factors and lysosomal distention are reported. Genistein®shows a possible role in treatment for Sanfilippo Syndrome, while Minozac and Fosteum®did not show any positive alteration on disease progression. Further studies evaluating the effect of these agents on lifespan and functional aspects of hearing, vision, and motor ability are anticipated.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Joseph Michael Gibney.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Heldermon, Coy D.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2012
System ID:
UFE0044677:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 THERAP E UTIC EFFECTS OF GENISTEIN MINOZAC, AND FOSTEUM IN A MOUSE MODEL OF MUCOPOLYSACCARIDOSIS TYPE IIIB (SANFILIPPO SYNDROME B) By JOSEPH GIBNEY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

PAGE 2

2 2012 Joseph Gibney

PAGE 3

3 ACKNOWLEDGMENTS Many thanks to my PI, Dr. Coy Heldermon, my committee members, Dr. Ed Scott and Dr. Tom Rowe, and my fe llow lab students, Amber Himmler, Jae Elkind, and Erin Leclair, without you this thesis would have been impossible Additionally, thanks to the Omerod lab group, especially Aditya Asokan, for their help with the bead assay, to Dr. Mandel for his work with our tissue IHC analysis, and to Dr. Waterson for the drug donation.

PAGE 4

4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ........................ 3 LIST OF TABLES ................................ ................................ ................................ 6 LIST OF FIGURES ................................ ................................ ............................... 7 ABSTRACT ................................ ................................ ................................ ......... 9 CHAPTER 1 INTRODUCTION ................................ ................................ .......................... 11 Mucopolysa ccaridoses and Sanfilippo Syndrome ................................ .............. 11 Physical Disease Manifestations ................................ ................................ ..... 12 Previously Attempted Treatments ................................ ................................ ... 13 NAGLU Mouse Model ................................ ................................ ................... 13 Neuroinflammation ................................ ................................ ........................ 14 Administered Drugs ................................ ................................ ...................... 15 Cytokines Assessed ................................ ................................ ...................... 16 Hypothesis ................................ ................................ ................................ ... 17 Aims ................................ ................................ ................................ ........... 17 2 METHODS ................................ ................................ ................................ ... 19 Animals ................................ ................................ ................................ ....... 19 Preliminary Cytokine Map ................................ ................................ .............. 19 Blood and Tissue Collection and Processing ................................ .............. 19 Cytokine Profile Analysis ................................ ................................ .......... 20 Statistical Analysis ................................ ................................ .................. 21 One Month Treatment Experiment ................................ ................................ .. 21 Ho mogenization ................................ ................................ ........................... 22 Bradford Assay Assessing Protein Concentration ................................ ............. 23 BioRad Custom Array Cytokine Assay ................................ ............................. 24 Plate Wash Method ................................ ................................ ................. 24 Plate Layout ................................ ................................ ........................... 24 Preparation of Standards ................................ ................................ ......... 24 Preparation of Samples and Coupled Beads ................................ .............. 24 Running the Assay ................................ ................................ .................. 25 Reading the Pl ate ................................ ................................ ................... 25 Removal of Outliers ................................ ................................ ................. 26 Statistical Analysis ................................ ................................ .................. 26 Secondary Qu antification of Cytokines ................................ ............................ 27 Secondary Enzyme Assay ................................ ................................ ............. 28 Additional Minozac Study ................................ ................................ ............... 29

PAGE 5

5 FGFb ELISA Procedures ................................ ................................ ......... 29 MCP 1 ELIS A Procedures ................................ ................................ ........ 30 Data analysis for ELISAs ................................ ................................ ......... 31 3 RESULTS ................................ ................................ ................................ .... 32 Preliminary Cytokine Map ................................ ................................ .............. 32 Brain Ho mogenate Analysis ................................ ................................ ..... 32 Blood Plasma Analysis ................................ ................................ ............ 33 Cytokine Levels Measured by Bead Assay ................................ ....................... 33 Basic Fibroblast Growth Factor (FGFb) ................................ ...................... 33 ................................ ........... 34 Other Cytokines Tested ................................ ................................ ........... 34 Secondary Quantification of Cytokines ................................ ............................ 35 Secondary Enzyme Fluorescence Assay ................................ ......................... 36 Additional Minozac Application Study ................................ .............................. 36 ................................ ................. 37 Basic Fibroblast Growth Factor (FGFb) ................................ ...................... 37 4 DISCUSSION ................................ ................................ ............................... 68 5 FUTURE AIMS ................................ ................................ ............................. 72 6 ONGOING EXPERIMENTS ................................ ................................ ........... 73 WORKS CITED ................................ ................................ ................................ .. 74 BIOGRAPHICAL SKETCH ................................ ................................ ................... 77

PAGE 6

6 LIST OF TABLES Table page 3 1 Myriad Rules Based Medicine brain cytokine results reported in g/mL. ........... 38 3 2 Myriad Rules Based Medicine brain cytokine results reported in ng/mL. ........... 39 3 3 Myriad Rules Based Medicine brain cytokine results reported in pg/mL. ........... 40 3 4 Myriad Rules Based Medicine blood cytokine results re ported in g/mL. .......... 42 3 5 Myriad Rules Based Medicine blood cytokine results reported in ng/mL. .......... 43 3 6 Myriad Rules Based Medicine blood cytokine results reported in pg/mL. .......... 44 3 7 Cohort setup. The experimental setup for the brain cytokine analysis across two time points and five treatments in MPSIIIB mice. ................................ ..... 45 3 8 Values used to determine cytokine concentration. ................................ ......... 46 3 9 Membrane array map. provided by Ray Biotech. ................................ ........... 59

PAGE 7

7 LIST OF FIGURES Figure page 3 1 Fold increase of 24 week old diseased mice brain homogenate analytes in comparison to unaffected heterozygote brain homogenate analytes.. .............. 41 3 2 Brain IL 1a concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ........................... 47 3 3 Brain IL 1a concentrations across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ........................... 48 3 4 Brain IL 6 concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ................................ ....... 49 3 5 Brain IL 6 concentrations across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ................................ ....... 50 3 6 Brain MIP 1a concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ........................... 51 3 7 Brain MIP 1a concentrations across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ........................... 52 3 8 Brain TNFa concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ........................... 53 3 9 Brai n TNFa concentrations across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ........................... 54 3 10 Brain FGFb concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ........................... 55 3 11 Brain FGFb concentrations across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ........................... 56 3 12 Brain VEGF concentrations across four treatment types in 10 week old MPSIIIB mice. ................................ ................................ ........................... 57 3 13 Brain VEGF concentratio ns across four treatment types in 28 week old MPSIIIB mice. ................................ ................................ ........................... 58 3 14 X rayed image of membrane assay. ................................ ............................ 59 3 15 Paired dif ferences in cytokine expression ................................ ..................... 61 3 16 Seco ndary enzyme fluorescent assay of 10 week old MPSIIIB mice. ............... 62

PAGE 8

8 3 17 Secondary enzyme fluorescent assay of 28 week old MPSIIIB mice. ............... 63 3 18 Graph of standard curve used to derive the M CP1 cytoki ne levels in Figure 3 19 .. ................................ ................................ ................................ ......... 64 3 19 MCP1 ELISA. This graph represents the amount of the cytokine MCP1 in brains from the Minozac modality study. ................................ ...................... 65 3 20 Graph of standard curve used to derive the FGFb cytokine levels in Figure 3 21 .. ................................ ................................ ................................ ......... 66 3 21 FGFb ELISA. This graph represents the amount of the cytokine FGFb in brains from the Minozac modality study. ................................ ...................... 67

PAGE 9

9 Abstract o f Thesis Presented to the Graduate School of t he University o f Florida in Partial Fulfillment of the Requirements for the Degree o f Master of Science THERAPEUTIC EFFECTS OF GENISTEIN MINOZAC, AND FOSTEUM IN A MOUSE MODEL OF MUCOPOLYSACCARIDOSIS TYPE IIIB (SANFILIPPO SYNDROME B) By Joseph Gibney August 2012 Chair: Coy Heldermon Major: Medical Science Sanfilippo syndrome, or Mucopolysaccharidosis type IIIB (MPS IIIB), is a genetically inherited lysosomal storage disease caused by the absence of N acetylglucosaminidase (NAGLU). The absence of NAGLU results in accumulation of the glycosaminoglycan (GAG) substrate, heparan sulphate, in the lysosomes causing them to become distended. This leads to the stimula tion of a pro inflammatory state increasing the activation of immune cells. The immune response may exacerbate damage resulting from abnormal lysosomal function. Sanfilippo syndrome most severely affects the central nervous system with initially delayed b ehavioral development progressing to neurocognitive regression. Systemic manifestations include organomegaly, diminished vision and hearing, and altered motor function. No curative treatment currently exists for MPS IIIB. Minozac is a CNS penetrant small m olecule previously shown to suppress proinflammatory cytokine upregulation in mouse models of seizure and traumatic brain injury. Genistein is a tyrosine kinase inhibitor, which acts on both EGF and IGF receptors. Fosteum is Genistein with the addition of cholecalciferol and citrated zinc bisglycinate Evidence suggests that Genistein

PAGE 10

10 reduces heparan sulfate production causing NAGLU substrate reduction and a decrease in pro inflammatory cytokine upregulation while Minozac inhibits th e We report the effect of treatment with these compounds separately, or in combination, for four weeks in MPS IIIB and wild type mice at ages 10 and 28 weeks. The primary outcome measures include the effects of these compounds on pro inflammatory cytokine levels and immune cell activation in the brain. Additional measures of effects of these agents on vascular growth factors and lysosomal distention are reported. Genistein shows a possible role in tre atment for Sanfilippo Syndrome while Minozac and Fosteum did not show any positive alteration on disease progression Further studies evaluating the effect of these agents on lifespan and functional aspects of hearing, vision, and motor ability are antic ipated.

PAGE 11

11 CHAPTER 1 INTRODUCTION Mucopolysaccaridoses and Sanfilippo Syndrome The collective Mucopolysaccaridoses are a group of disorders resulting from the malfunctioning or absence of lysosomal enzymes used to degrade glycosaminoglycans (GAGs). They are part of a larger group of diseases called Lysosomal Storage Diseases (LSDs), all of which result in lysosomal malfunction in one or more essential pathways. All mucopolysaccaridoses result from the absence of one enzyme in the glycosaminoglycan degradation pathway. The absence of this enzyme leads to the cellular buildup of long ch ain sugar carbohydrates. Mucopolysaccaridosis type IIIB ( Sanfi lippo Syndrome type B) (MPSIIIB), an autosomal recessive disorder, is one of the most common mucopolysaccaridoses, at 1 in 50,000 births (Van de Kamp, 1981) All MPS disorders are all c haracter ized by the accumulation of glycosaminoglycans in the cell lysosome (Heldermon, 2007 ) (Neufeld and Muenzer, 1995). In addition to MPSIIIB, there are three other versions of Sanfilippo Syndrome. MPSIIIA results from a deficiency of sulfamidase, MPSIIIC resu lts from a deficiency in N acetylglucosaminide transferase, and MPSIIID results from a lack of glucosamine 6 sulfatase (Nidiffer FD, Kelley TE, 1983) The four disorders are biochemically distinct stemming from the malfunction of completely different enzy mes yet all of these enzymes act on the HS degradation pathway, and thus result in phenotypic manifestations that are largely clinically indistinguishable (Nidiffer FD, Kelly TE, 1983).

PAGE 12

12 Physical Disease Manifestations Phenotypically, patients affected wi th MPSIII often first present with symptoms stemming from deficiencies in the central nervous system. The first characterized phase of the disease is associated with cognitive and linguistic delay. In the early stages the presentation of this disease is re adily mistaken for a learning impediment and not as a symptom of a systemic disease. Phase one begins between the ages of one and four. The second phase of the disease, often beginning between the ages of three and four, is hallmarked by severe behavioral disturbances including tantrums, marked hyperactivity, decreased attention span, aggression, heightened night time activity, and disturbed sleep patterns Physical growth through phase two is normal, with no readily identifiable physical indicators of dise ase presence (Cleary, 1993) The third stage of the illness usually begins around age ten and is characterized by readily noticeable developmental characteristics These may include poor balance and consequently, a reduction in motility. At this time poin t, a decline in connective tissue quality and joint stiffening is also seen. These factors together usually mark the beginning of the affected individuals becoming wheelchair bound and fully dependent on outside care. In the fourth and final stage of the disease, s wa llowing difficulties are common due to a loss of muscular control. Additionally, frequent episodes of aspiration of food and saliva are also seen in the late stages of progression (Cleary, 1993). In the end, d eath is common in the early to mid teens Death is often due to increased respiratory complications or heart failure U ncontrolled infection has also been noted as a cause of death (Heldermon, 2007).

PAGE 13

13 Previously Attempted Treatments Though many therapies have been proposed no treatment has successfully corrected either the cellular, like the expansion of the lysosome within the cell, or physical manifestations of the MPSIIIB disease, like decreased cognitive ability and motor function loss S upraphysiologic levels of NAGLU enzyme administered intravenously, have demonstrated some degree of attenuat ion of the physical manifestations of the disease, but it has shown little or no significant ability to correct neurologic pathology (Sands et al., 1994). Further, one group hypothesized that a bone marrow transplant (BMT) into diseased MPSIIIB mice or application of intracranial (IC) AAV 2/5 hNAGLU would attenuate disease symptoms. They found that IC AAV NAGLU increased lifespan and improved motor function the best. They also found that c ombination of the two treatments decreased the lysosomal inclusion size and improved hearing. Despite the positive effects of BMT+IC AAV NAGLU, no histological improvements were seen (Heldermon, 2010). NAGLU Mouse Model A mouse model of MPSIIIB was create d by Li et al. in 1999. This was accomplished through the replacement of an 852 bp fragment within exon 6 of the NAGLU gene with a neomycin resistance gene. Also within this vector construct a cassette with the Herpes Simplex virus thymidine kinase gene w as cloned upstream to allow for cellular differentiation (Li, 1999). Several phenotypic traits such as m otor function, Purkinje cell loss, circadian rhythms, auditory response, and retinal function in this model were determined to be

PAGE 14

14 similar to those obse rved in humans This mouse was deemed suitable for the s tudy and develop ment of MPSIIIB therapie s (Heldermon et al., 2007). Neuroinflammation New pathways have been proposed for investigat ion in an attempt to provide a more comprehensive understanding of treatments that may offer aid to affected individuals. Lysosomal distention, and thus cellular distention and death, has been hypothesized to play a role in increasing the progression of disease through the activation of the inflammatory pathway (Heldermon 2010). In normal, non diseased brains, activated glia trigger a neuroinflamatory cascade. This cascade is well studied, and its goal is to protect the brain The neuroinflamatory response is proliferated through the production and secretion of proinflam matory cytokines Chronic neuroinflmmation can lead to cellular damage through neuronal and synaptic dysfunction. proinflammatory cytokines has presented a novel target in a wide array of neur odegenerative disorders like Alzheimers disease or traumatic brain injury (Van Eldik, 2007) The accumulation of HS has s hown the ability to initiate the inflammatory cascade including cytokine and chemokine production activated glia and microglia leukocyte recruitment, and the maturation of inflammatory cells (Taylor and Gallo, 1996) (Chrzaszcz et al., 2010 ). Further, microgliosis and the proliferation of reactive astrocytes have shown a correlation to the glut of undegraded substrate seen in MPSII IB ( Ohmi et al., 2009).

PAGE 15

15 Lending credence to the idea that inflammation plays a major role in MPSIIIB disease progression, an experiment by DiRosario tested if the application of immunosuppressant s in an MPSIIIB mouse model would attenuate effects of the d isease. They showed that prednisone, increase s lifespan and decreases glial fibrillary acidic protein (GFAP) concentration in MPSIIIB mice (DiRosario, 2009). Administered Drugs trihydroxyisoflavone or Genistein is a soy derived isoflavone and tyrosine kinase inhibitor that has shown the ability to decrease the production of HS, via receptor inactivation, in an MPSIIIB mouse model (Jakobkiewicz Banecka et al., 2007) (Ruitjer et al., 2007). Genistein is also known to act as an in hibitor of different growth factor receptors. For its use in MPSIIIB, Genistein the epidermal growth factor receptor (EGFR) This receptor mediates the production of heparan sulfate in the cell. Additionally, a one y ear study of human subjects with MPSIIIA or B administered Genistein at 5 mg/kg/day orally (PO) was performed. They found that Genistein significant ly decrease d GAG urine concentration. Additionally slightly improved cognitive function was observed (Pio trowska, 2008). Genistein has been shown to be safe in humans in large doses, and it is currently available as a nutraceutical, without a prescription (Ulmann, 2005). Minozac is a central nervous system (CNS) penetrant chemically manufactured molecule Minozac has previously demonstrated the ability to decrease neural proinflammatory cytokines such as (Van Eldik and Wainwright, 2003) Used in mouse models of traumatic brain injury and electroconvulsive shock induced seizures Minozac sho wed the ability to lower the impact of the proinflammatory

PAGE 16

16 cascade through its effect on the expression of GFAP, as well as the astrocyte marker S100B(Chrzaszcz et al., 2010) Fosteum is a combination of Genistein cholecalciferol (vitamin D3) and citrat ed zinc bisglycinate (Zinc) Zinc supplementation has been shown to decrease levels of phase proteins, as well as erythrocyte sedimentation (Prasad, 2009) while vitamin D3 supplementation has been shown to decrease systemic inflammation by reducing C reactive protein (CRP) levels (Mathias, 2010) Burnett et al. also showed that this FDA approved dietary supplement aids in the management of osteopenia and osteoporosis (Burnett, 2011) Cytokines Assessed The cytokines directly assessed in th is study were chosen based on the results of previous literature and preliminary assays performed by our laboratory. In a preliminary cytokine mapping assay run by Myriad Rules Based Medicine increased concentrations of FGF b MIP 1 were found in whole brain homogenates of MPSIIIB mice against aged matched controls; additionally, IL are known to be attenuated by Minozac (Chrzaszcz et al., 2010). FGF b is not strictly a proinflammatory cytokine, however it does have the ab ility to enhance leukocyte recruitment and CAM expression while in the presence of other proinflammatory cytokines, especially TNF 2006). The gene expression of MIP a cytokine enhancer of macrophage recruitment and macrophages and microglia initiation and creation, has been shown to be increased in the brains of 7 month of MPSIIIB mice (Villani, 2006). VEGF also not strictly a proinflammatory cytokine, is often seen in the cytokine profile of various causes of inflammation because it stimulates the recruitment of monocytes to the site of

PAGE 17

17 inflammation when in the presence of TNF as well as synergizing with FGFb to boost monocyte and polymorphonuclear neutrophils ( PMN ) recruitment (Zitterman and Issekutz, 2006). IL a cy tokine commonly associated with apoptosis, has been shown, in combination with TNF to increase expression of cyclooxygenase 2 (COX 2) and phospholipase A2 (PLA2) COX 2 promotes inflammation by increasing the production of prostaglandin E2 (PGE2) and PLA2 enhances prostaglandin production (Dinarello, 2000) IL 6 has receptors on neurons and glial cells and is also implicated in the PGE2 production. It is an important inhibitor of TNF and IL 1 (Fielding et al 2008) TNF gnificantly upregulated in MPSIIB a ffected mice (Killedar, 2010). Hypothesis We hypothesize that the administration of Genistein Minozac, and Fosteum separately or Genistein and Minozac in combination will decrease proinflammatory cytokine levels in the brains of MPSIIIB mice. We hypothesize that the combination treatment of Genistein and Minozac will be the most effective due to Genistein ability to act on heparin sulfate production proinflammatory cytokine Aims This purpose of this experiment is multi faceted and includes the following aims: To further characterize the amount and types of proinflammatory cytokines upregulated in the mouse model of MPSIIIB To determine if the administratio n of Genistein with its ability to attenuate heparan sulfate production will decrease proinflammatory cytokine levels To determine whether Minozac application will decrease levels of proinflammatory cytokines as shown in a model of traumatic brain inju ry

PAGE 18

18 To determine whether Fosteum a compounding of Genistein with Zinc and Vitamin D3 will both decrease HS production and lessen immune activation To evaluate the potential synergistic effects of Genistein and Minozac, in combination on neuroinflammati on by inhibiting both GAG accumulation and upregulation

PAGE 19

19 CHAPTER 2 METHODS Animals An established colony of MPSIIIB mice was transferred to the University of Florida from Washington University in St. Louis, Missouri in 2009. This colony was mai ntained through strict sibling mating; mutant and heterozygous males were crossed with heterozygous females. To genotype pups born, ACS staff at the University of Florida took small tail tip samples at weaning. These samples were used to genotype th e pups via PCR of the NAGLU gene on exon 6 with the neomycin insertion. Results were then verified using a substrate based fluorescence assay to test for the presence of N acetylglucosaminidase, the enzyme absent in MPSIIIB / mice (Heldermon et al 2007 ). All procedures and handling of mice were conducted in compliance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Florida. Preliminary Cytokine Map Blood and Tissue Collection and Processing Twelve mice were used in this study. Two cohorts (mutant and heterozygous) at two time points each (young and old), were randomly selected. This gave each 1 day) group were aged to 6 months (+/ 15 days). Before brain tissue collection began, blood was collected from mice via cheek puncture. Using an 18 gauge needle, a small hole was opened at the intersection of the mouse retro orbital and submand ibular veins. App roximately 0.5 mL of blood was drained into a 1.5 mL heparinized eppindorf tube. Animals were then sacked primarily by

PAGE 20

20 carbon dioxide narcosis and secondarily by cervical dislocation (according to UF IACUC regulations), and blood was again harvested via a ca rdiac puncture technique. The mouse chest cavity was opened, and an 18 gauge needle was inserted into the left ventricle of the heart. Using a slight vacuum from the attached syringe, blood was collected and then ad ded to the partially filled 1.5 mL heparin ized eppindorf tube containing the previously collected blood The harvesting of brains was conducted swiftly after the completion of blood collection. Brains were excised, placed into a 1.5 mL cryogenic vial, and immediately flash frozen in liquid nitrogen Both blood and brain samples were stored at 80 C. To prepare for analysis, blood samples were thawed and then spun at 13,000xg for two minutes to separate out plasma. Plasma was removed from the original 1.5 mL heparinized eppindo rf tube, placed in a se cond 1.5 mL heparinized eppindorf tube, and refrozen to 80 C prior to cytokine measurements. Brain samples were thawed, w eighed, and transferred to a 15 mL conical filled with 9x volume tissue homogenization buffer, 50mM Tris HCl with 2mM EDTA in ddH 2 O, at pH 7.4. The tissue was then homogenized with an Omni tissue homogenizer while the tube was submerged in an ice bath. Next, the homogenized solut ion was transferred from the 15 mL conical to 4x1.5 mL eppindorf tubes and centrifuged at 13,000xg for 8 minute s. 500 L of supernatant from one eppindorf tube was aspirated into a clean 1.5 mL eppindorf tube and stored at 80 C prior to cytokine measurements. All brain homogenization steps were conducted in a 4 C cold room. Cytokine Profile Analysis Prepared sampl es of blood and brain from each mouse were cold packaged and sent to a collaborator, Myriad Rules Based Medicine (RBM), in Austin, Texas. There,

PAGE 21

21 RBM performed a multi analyte profile combined sandwich immunoassay the samples, testing for the presence and amount of 59 distinct biomarkers and cytokines. Statistical Analysis An F test was run on plasma and brain homogenate concentration levels to determine significance among samples. If any significance was present, as determined by an F test p < 0.05, compar isons between old heterozygous/old mutant, old heterozygous/new mutant, old heterozygous/new heterozygous, old mutant/new mutant, old mutant/new heterozygous, and new mutant/new heterozygous pairs were achieved sample t test to det ermine significance between samples at p < 0.05. One Month Treatment Experiment Two hundred twenty mice, half mutant ( / ) and half heterozygous (+/ ), were sex matched and randomly sorted into five treatment groups: control, Genistein Fosteum Minozac or Genistein + Minozac in combination. These treatments were administered in each treatment cohort, with eleven at each age time point. The treatment time across all mi ce was 28 days. Table 3 7 describes the setup of each cohort. Harlan Teklad aided in the production of all specialty research diets. Mice in the control group were fed chow lacking all soy byproducts; this chow was fortified with corn oil to account for the nutrients lost upon removal of soy. Mice in the Genistein a nd Fosteum groups were fed the same chow as the control animals, except this chow was additionally fortified with either Genistein or Fosteum at .12%. Genistein and Fosteum chow concentrations were based on a mouse average food consumption of 4g/day a nd an average mouse weight of 30g (Bachmanov, 2006) for a total daily

PAGE 22

22 treatment dose of ~160mg/kg/day. Minozac was dissolved in normal saline solution (0.9 %) at a concentration of 0.15mg/100 L and was SQ injected into mice daily. Each mouse received Mino zac at 5mg/kg/day ( Chrzaszcz et al., 2010). The amount of Minozac given was weight adjusted weekly throughout the experiment The amount of chow eaten each day was measured to ensure normal food consumption. After twenty eight days, mice were sacrificed. E ight of eleven in each group were sacrificed primarily by carbon dioxide and secondarily by cervical dislocation (according to UF IACUC regulations). The brains of these mice were excised and flash frozen in liquid nitrogen for later analysis. Three of el even in each group were sacrificed via intraperotineal administration of 200 L Beuthanasia D Special (390mg pentobarbital sodium + 50 mg phenytoin sodium in a 1:20 dilution in 0.9% NaCl) After death, the chest cavity was opened and a 22 gauge needle conn ected to a calibrated peristaltic pump was inserted into the left ventricle of the heart and the right atrium was punctured. Ice cold Tyrode solution was pumped through the mouse until the fluid exiting the right atria was clear. Immediately following th e Tyrode solution, ice cold 4% paraformaldehyde was circulated through the mouse until fixation occurred Brains were then excised and transferred into chilled 4% paraformaldehyde for 24 hours at 4 C with gentle shaking, followed by submersion in a 30% sucrose solution with gentle shaking at 4 C for 24 hours. Brains were stored in this 30% sucrose solution until immunohistochemical analysis was performed. Homogenization Brains previously preserved in liquid nitrogen were removed from the freezer and we ighed prior to thawing. The brains were thawed in 500L of ice cold tissue homogenization buffer. The buffer consisted of 50mM tris HCL, 2mM EDTA, and a

PAGE 23

23 cocktail of protease inhibitors all at 1g/mL (aprotenin, antipain leupeptin, pepstatin A in 2mM P M SF). Brains were homogenized, submerged in an ice bath by hand with a pestle, sonicated, while on ice, and centrifuged at 12,000xg for 10 minutes at 4 C. The supernatant was removed with a pipette and stored in a separate 1.5 mL eppindorf tube, on ice M ore tissue homogenization buffer was added to the tube containing the pellet, raising the volume of pellet + tissue homogenization buffer to 1.5 mL. This process was repeated four times. All supernatant was stored, and the brain pellets were frozen pending successful analysis of the supernatant collected. Bradford Assay Assessing Protein Concentration A Bradford assay was run on a 250L microplate. 1X dye reagent was removed from 4 C storage and allowed to warm to ambient temperature. Serial dilutions of 2 mg/mL bovine serum albumin (BSA) were performed. 5L of each standard and unknown sample and 250L of 1X dye reagent were pipetted into the microplate well; the pipette plunger was depressed repeatedly to mix the solution. The microplate was incubated at r oom temperature for thirty minutes. A spectrophotometer was set to 595nm and zeroed with a blank before the absorbance of the standards and samples was determined. Absorbance of the standards and samples was measured, and built in software was used to plo t unknown values against the standard curve. Absorbance values for each sample were then plotted against this standard curve, and protein concentrations for each sample were derived. This data was used to normalize measured cytokine values by the total pro tein concentration of each sample.

PAGE 24

24 BioRad Custom Array Cytokine Assay Plate Wash Method Plates were washed via magnetic separation. The magnetic wash plate carrier on the BioPlex wash station was installed and primed. The 96 well plate was placed on the m agnetic plate carrier and the MagX2 preloaded program was used to wash the wells 3 times Plate Layout The plate layout was then determined. Standards were assigned to columns one and two, and run in duplicate with the highest concentration in row A and the lowest concentration in row H. Wells A3 and A4 served as blanks and were filled with diluent. All remaining wells were available for sample analysis. Preparation of Standards BioRad provided eight standards for the custom array. Standards were recons tituted and diluted in BioPlex Standard diluent at a 1:4 dilution. The standards were mixed appropriately, a serial dilution was performed, and the dilutions were plated accordingly. Preparation of Samples and Coupled Beads Because the assay was analyzing mouse cytokines from tissue lysates, sample dilutions were pipetted at a ratio of 1:2, lysate s to sample recommendations. This achieved a protein concentration of 200 900g/mL in each well. The amount of sampl e and diluent used was adjusted per sample based on the previously conducted Bradford protein concentration assay. Upon completion, each prepared sample totaled 200L, and this was pipette onto the 96 well plate based on

PAGE 25

25 the previously determined sample la yout. Samples were kept on ice throughout this process. Coupled beads were prepared by adding 72L of 10X beads for each cytokine type (8 cytokine bead reagents x 72 L each= 576 L ) to 175L of assay buffer. The bead mixture was covered in order to minimiz e light exposure. Running the Assay The diluted coupled beads were vortexed at medium speed for 30 seconds and 50L of the bead mixture was added to each well, including those of the standards. The wells were then washed twice using magnetic separation. D iluted standards, blanks and samples were gently vortexed for 1 3 seconds. 50L of each standard, blank and sample were pipetted to previously assigned wells. The plate was incubated on a shaker at room temperature for 45 minutes. During incubation, 38L of detection antibody for each cytokine was pipetted into a tube and diluted with 2700L of detection antibody diluent. The mixture was vortexed and spun, and 25L of the mixture was added to each well. The plate was again incubated at room temperature for thirty minutes. The plate was washed three times via magnetic separation. 50L of diluted Streptavidin PE (60L of streptavidin PE and 5,940L of assay buffer) was added to each well and incubated at room temperature for ten minutes. The plate was again washed three times by magnetic separation. 125L of assay buffer was added to each well before all wells were covered by parafilm and shaken at 1100rpm for 10 minutes. Reading the Plate An optimized software protocol for the Luminex 2 machine was prepared via the BioPlex Pro Assay guideline. The plate was formatted according to the arrangement of

PAGE 26

26 the standards, blanks and samples. The protocol was run and initial cytokine concentrations were determined. Concentrations were determined for each sample an d these were then protein balanced and fit to the standard curve (Table 3 8 ) to derive the final cytokine concentration values This assay was run in duplicate to minimize error Removal of Outliers The mean concentration of each treatment group (by age a nd genotype) for each cytokine was determined from the duplicate runs. If the coefficient of variance for at least one of the two duplicates was greater than 15% from the mean of all values in a treatment group an algorithm was applied to determine which of the two values (from the duplicate wells) should be retained for final data analysis and which value should be discarded as an outlier The algorithm consisted of a formula that considered the two values from the duplicate runs and subtracted these fro excluding both outlying data points ). The duplicate value with the smallest absolute difference from the adjusted mean was selected, and thus included in subsequent analysis. Statistical Analysis Significance by cytokine, was determined first by using a 1 way ANOVA to analyze only untreated mice across a single age cohort (10 weeks or 28 weeks). Significance was noted with yellow stars. Then a 2 way ANOVA was run to compare all treatments ( Genistein Fosteum Minozac, G+M), ages (10 and 28 week old), and genotypes (Het or Mut) against the whole untreated control group. Significance is reported with white stars. Finally, if any T value in the 2 way ANOVA was near significance, a 1 way ANOVA was run for only that cohort against its matched control.

PAGE 27

27 Significance is reported with red stars (this case happened only once: in the FGFb analysis for the 28 week age group treated with Genistein ). Secondary Quantification of Cytokines After acquisition of primary data, the level s of various cytokines for four randomly membrane assay from Ray BioTech [Cat#: AAM ANG 1] for comparison against the BioRad bead assay. To do this, 8 detection membranes, see Table 3 9 were placed in the provided 8 well tray with 2mL blocking buffer. This was incubated at room temperature for 30 minutes. Before adding samples to each well, all samples were protein balanced to 500 g protein/mL (protein values from experiment # 2 were used) via dilution in ddH 2 O. Next, the blocking buffer was decanted and 1mL of adjusted sample was added to each well overnight at 4 C The next day, samples were decanted from each well and the membranes were washed with Wash Buffer One three time s at room temperature with shaking for 5 minutes, followed by the same protocol for Wash Buffer Two, twice. Biotin conjugated anti cytokines were reconstituted with blocking buffer, and 1m L of diluted antibody mixture was added to each well for 2 hrs at ro om temperature. After incubation, 1mL of diluted antibody mixture was decanted, and 2mL of 1000 fold diluted HRP conjugated streptavidin mixture (2 L of HRP conjugated streptavidin + 1998 L of blocking buffer) was added to each well for 2 hours with shakin g. After incubation, wash steps were repeated and the membranes were moved to a dark room. In the dark room, 250 L of detection buffer C and detection buffer D were added, in tandem, to each membrane, after the membrane had been drip dried and placed on a

PAGE 28

28 plastic sheet. Each membrane was incubated for 2 minutes and then was exposed to x ray film for 4 seconds. Membranes were refrozen at 80 C upon successful x ray exposure. See Figure 3 14 The x ray film was scanned into TIFF format using a 1200 dpi scann er, and the image was prepared using freeware ImageJ software. With ImageJ a negative of the Each membrane allowed for duplicates to be run for all cytokines, increasi ng significance. These values were normalized across positive controls (found on each membrane), separated by genotype, and graphed. A one way ANOVA was run across all cytokines by genotype, and then a t test tested between all cytokines. Secondary Enzyme Assay After the acquisition of primary data, the level of 4 methylumbelliferyl D glucuronide, a secondary enzyme in the heparan sulfate pathway, was measured via fluorescence assay. To do this, 50 L of brain homogenate from all samples tested in experime nt #2 and 50L of 4 methylumbelliferyl D glucuronide were added to an eppindorf tube and incub ated for thirty minutes at 37.5 C After 30 minutes, 250L of stop solution was added. 200L of this solution mixture was transferred to a 250L 96 well black bottom plate and read by a Tecan fluorometer (excitation at 364nm and emission 444nm) Fluorescence values were normalized by the protein concentrations derived from the previous Bradford assay and graphed. Significance was determined via one way ANOVA, which analyzed the effects of genotype within a treatment group, and two way ANOVA, which analyzed the effects of age and genotype within a treatment group, with multiple comparison corrections.

PAGE 29

29 Additional Minozac Study The results obtained for Minozac fro m the one month treatment experiment led to the conduction of an additional study to assess the efficacies of different modes of administration when this drug is administered acutely. Thirty 6 month old mice were randomly selected for this study. Six mice, three heterozygous and three mutants, were in each group. Each mouse was weighed and the appropriate dose of Minozac was determined as previously described (5mg/kg). Mice were randomly sorted into five groups, differing by Minozac treatment modality: Cont rol, Subcutaneous (SQ), Intravenous (IV), Intraperitoneal (IP), or Gavage (PO). Twenty four hours post administration, the mice were sacked first by carbon dioxide asphyxiation and then by cervical dislocation. B rains were excised and flash frozen in liq uid nitrogen. In preparation for the ELISA assay, brain s were thawed and moved to a 15 mL conical with 4.5mL of ice cold tissue homogenization buffer, consisting of 50mM tris HCl and 2mM EDTA. They were then mechanically homogenized using an Omni tissue hom ogenizer while submerged in an ice bath The homogenized sample was transferred to eppindorf tubes and spun at 13,000 xg for 5 minutes at 4 C All steps were completed in a 4 C cold room Sampl es were then refrozen at 20 C. FGF b ELISA Procedures FGFb cytokine quantification was performed on the mouse brain supernatant according to the RayBio Mouse FGFb ELISA Kit (Cat#: ELM bFGF 001). A standard curve was created from a 50ng/mL stock provided, via 1:2 fold serial dilutions in tissue homogenization buff er The layout of the 250 L 96 well plate was preplanned; samples were run in duplicate. 100L of each standard and supernatant from sample was added

PAGE 30

30 to each well and incubated at room temperature with gentle shaking for 2.5 hours. The plate was washed fo ur times with 300L wash solution. 100L of prepared biotinylated anti mouse FGFb antibody was added to each well and incubated for one hour at room temperature with gentle shaking followed by the previously described wash step. 100L of prepared HRP Strep tavadin was added to each well and incubated for 45 minutes at room temperature with gentle shaking followed by a wash step. Then, 100L of TMB One Step substrate reagent was added to each well and incubated for half an hour at room temperature in the dark with gentle shaking. 50L of 0.2M sulfuric acid stop solution was added to each well. The plate was immediately read at 450nm. A line of best fit was drawn to the standard curve by integrated software and cytokine concentration was calculated from this line. MCP 1 ELISA Procedures MCP 1 cytokine quantification was performed on the mouse brain supernatant according to the RayBio Mouse MCP 1 ELISA Kit (Cat#: ELM MCP1 001 C ). A standard curve was created from a 50ng/mL stock provided, v ia 1:3 fold serial dilutions in tissue homogenization buffer The layout of the 250 L 96 well plate was preplanned; samples were run in duplicate. 100L of each standard and supernatant from sample was added to each well and incubated at room temperature with gentle shaking for 2.5 hours. The plate was washed four times with 300L wash solution. 100L of prepared biotinylated anti mouse MCP 1 antibody was added to each well and incubated for o ne hour at room temperature with gentle shaking followed by the previously described wash step. 100L of prepared HRP Streptavadin was added to each well and incubated for 45 minutes at room temperature with gentle shaking followed by a wash step. Then,

PAGE 31

31 10 0L of TMB One Step substrate reagent was added to each well and incubated for half an hour at room temperature in the dark with gentle shaking. 50L of 0.2M sulfuric acid stop solution was added to each well. The plate was immediately read at 450nm. A li ne of best fit was drawn to the standard curve by integrated software and cytokine concentration was calculated from this line. Data analysis for ELISAs Data from both FGFb and MCP 1 ELISAs were analyzed using a freeware version of MasterPlex software. V to derive a standard curve with a best fit line. It then determined the adjusted protein concentration values, a nd averaged all duplicate samples, for all unknowns, based on the standard curve. Protein levels for each treatment type were graphed. T tests were run to determine difference between Het and Mut controls. Once significance was verified among controls, one way ANOVAs were run to compare treatment modalities by genotype.

PAGE 32

32 CHAPTER 3 RESULTS Preliminary Cytokine Map Analytes in both brain and blood were measured in four different cohorts: 6 week mice and 24 week quantities below the limit of detection (mean + 3 standard deviations of 20 blank readings) were discarded. Statistical significance for each analyte tested w as determined by T test (*) denotes significance when values were compared within age groups, while () denotes significance when values were compared over time. P values were 0. 0 5 or 0.01, and the number of (*) or () denote the level of significance ach ieved Brain Homogenate Analysis Of the 59 analytes surveyed in the combined sandwich immunoassay, 44 were detected at quantifiable levels. Many cytokines involved in inflammatory response, such 4, IL 10, etc., were elevated as MPSIII B mice age. No significant differences in analyte concentrations exist between mutant and heterozygous mice of the 6 week age group, but a large number of biomarkers exhibit statistically significant differences as mouse age approaches 24 weeks (Tables 3 1 to 3 3 ). Twenty two of the measured analyte concentrations in 24 week old mutant MPSIIIB brain homogenates are statistically significantly greater than those in normal heterozygous mice (of the same age group), 6 week old mutant mice, or both. The fold ch ange of 21 statistically significantly different analyte concentrations in 24 week old mutant MPSIIIB mice against matched controls is graphically displayed in Figure 3 1. O nc ostatin M was left off of the graph due to a lack of confidence in the reported v alues.

PAGE 33

33 Blood Plasma Analysis As seen in T ables 3 4 to 3 6 the circulating cytokines and biomarkers analyzed are similar to those observed in the brain; however, their concentrations are only slightly elevated in most cases. Notably different though, is th e lack of significant differences between the cohorts in terms of concentrations of analytes. MIP show the highest level of significance between old mutants and controls. Cytokine Levels Measured by Bead Assay Brain homogenate levels in MPSII IB mutant (diseased) and heterozygous (unaffected) mice, across four 1 month treatments ( Genistein Fosteum Minozac, and combination G+M) and two time points, were measured, via a custom sandwich immune assay, for IL 1a, IL 6, MIP EGF. Figures 3 2 to 3 13 display the results of this assay. Graphs are ordered by analyte and age. A 2 way ANOVA was run on the data from every analyte measured, to determine significance. (*) denotes significance, with p<0.05, ** p<0.01, *** p<0.001, ** ** p<0.0001. Basic Fibroblast Growth Factor (FGFb) Figures 3 10 and 3 11 show that basic fibroblast growth factor (FGF 2 or FGFb) was significantly upregulated in diseased 10 week old and 28 week old mice compared to their unaffected counterparts. Concentr ations in 10 week old hetero zygous mice averaged 761.2pg/mL and those in 28 week old h eterozygous mice averaged 752.9 pg/m L In contrast, the average concentrations found in the 10 week and 28 week o ld mutant mice were 2030.4pg/mL and 2345.5pg/mL respectively. These differences were significant, via 1 way ANOVA at p<0.0001. Assessed by two way ANOVA, Fosteum significantly exacerbated the upregulation of FGF 2 in 10 week old

PAGE 34

34 heterozygous mice (p<0.01), 28 week old mutant mice (p<0.0001) and 28 we ek old heterozygous mice (p<0.0001). When assessed by 1 way ANOVA, Genistein was effective in significantly decreasing the average concentration of FGFb to 1656.2pg/m L in 28 week old diseased mice (p<0.05). Overall, the combination treatment was less effe ctive than the administration of Genistein alone, but more effective than the administration of Minozac alone. Fosteum significantly increased FGFb concentrations across all mice ages and genotypes. Figures 3 5 and 3 6 show that MIP MPSIIIB mice compared to their heterozygous counterparts (p<0.05). Though none of the drugs tested significantly decreased the concentration of MIP were observable. 1 0 week old diseased mice expressed an aver age concentration of 437.8pg/mL and 28 week old diseased mice expressed an average concentration of 503.2pg/m L Genistein decreased concentrations of MIP old diseased mice to 382.90pg/m L and 427.3pg/mL respectively. The combination treatment of Genistein and Minozac shows a similar decrease to that described in FGF 2. These attenuations are not statistically significant. 2 way ANOVA demonstrated that Fosteum increased the concen tration of this proinflammatory cytokine significantly in 10 week old heterozygous mice (p<0.0001), 10 week old mutant mice (p<0.01) and 28 week old heterozygous mice (p<0.05). Other Cytokines Tested The other four cytokines that were tested, IL 6, 3 2, 3 3, 3 4, 3 5, 3 8, 3 9, 3 12, and 3 13 ) did not yield significant results with respect

PAGE 35

35 to the attenuation of proinflammatory cytokines in the brain homogenates of diseased mice, across treatment groups. In fact, all of these c ytokines, showed (in either the 10 week old group or the 28 week old group) a higher cytokine baseline in the unaffected controls over the diseased MPSIIIB mouse. However, weak trends can be observed. Genistein consistently decreased cytokine concentrations across most cohorts. Fosteum intensified the expression of all six of the surveyed cytokines. Minozac did not seem to alter cytokine expression in any discernible pattern. And, the combination treatment of G enistein and Minozac yielded cytokine levels that were either similar to the expression seen with the Genistein treatment alone, or worse. Secondary Quantification of Cytokines Brain cytokine levels in four randomly selected 28 week old unaffected and f our randomly selected 28 week old MPSIIIB diseased mice from the previous experiment were measur ed via a membrane array (Table 3 9 ), in an effort to confirm trends seen with the bead assay The fluorescent membrane array was imaged on x ray film (Figure 3 14 ), and the relative amount of each cytokine (grayness) was measured via densitometry software. Values were normalized by positive control for each membrane, and background was removed. Figure 3 15 shows the graphed values of relative grayness. Significan ce was determined by T test Three of the twenty four cytokines tested reached significance (p<0.05): GCSF, GM CSF, and IL 12 p70. Several other cytokines nearly achieved 12 p40/p70, IL 9, MCSF, Thro mbopoeitin, and TIMP 2.

PAGE 36

36 Secondary Enzyme Fluorescence Assay All brain homogenate samples from the primary experiment were measured for beta glucuronidase upregulation. beta glucuronidase is an enzyme that has been previously described as upregulated in MSP IIIB mice. To do this a fluorescent substrate cleavage assay was performed and a spectrophotometer was used to measure fluorescence. Figures 3 16 and 3 17 show the average beta glucuronidase levels in diseased and unaffected MPSIIIB mice at 10 week and 28 week old mice, respectively, across four treatment types. Untreated diseased mice from the 10 week cohort showed a significant difference in beta glucuronidase level as compared to its matched control (p<0.05). Further, untreated diseased mi ce from the 28 week cohort showed a significant difference in beta glucuronidase level as compared to its matched control (p<0.0001). Across all treatments in both the 10 week and 28 week old MPSIIIB diseased mice, no statistically significant difference in beta glucuronidase levels was seen. However, of the four treatments, Minozac seems to show a trend toward the reduction of secondary enzyme upregulation. Additional Minozac Application Study Due to the unexpected results from the 1 month primary experim ent, an acute study with Minozac was undertaken to determine if the original application mode (SQ injection 28 days of treatment) was the cause of incongruous results. To do this, unaffected and diseased MPSIIIB mice were treated acutely via several drug application modalities: gavage (PO), subcutaneous injection (SQ), intravenous injection (IV), or

PAGE 37

37 intraperitoneal (IP). Brain FGFb and MCP 1 levels were measured via ELISA. ELISA values were fit to their respective standard curves (Figures 3 18 and 3 20 ). Monocyte Chemotactic C P1) Figure 3 19 shows the adjusted MCP1 values across four treatment modalities. Significance was measured via 1 way ANOVA. Surprisingly, controls of MPSIIIB diseased mice showed significantly (p<0.01) less MCP1 than the ir unaffected counterparts. For the diseased animals, no treatment mode showed a significant difference from another, however, the SQ treatment seems to trend more downward than the control. Additionally, IV treated unaffected mice did reach significance ( p<0.05) against their matched control. Basic Fibroblast Growth Factor (FGFb) Figure 3 21 shows the adjusted FGFb values across four treatment modalities. Significance was measured via 1 way ANOVA. Significance was reached between the MPSIIIB diseased and u naffected controls (p<0.001), but it was not reached among any of the Minozac treatment modalities. However, like MCP1, there seems to be a downward trend in the MPSIIIB diseased mice that were treated SQ, against their matched control.

PAGE 38

38 Table 3 1. Myriad Rules Based Medicine brain cytokine results reported in g/mL. Average amount of different cytokines found in MPSIIIB mut or het brains in g/mL. A T test was used to determine significance ( *) denotes a statistically significant difference within the ag e group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 New Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) Apo A I 0.0673 0.0661 0.101 0.189 C Reactive Protein 0.0119 0.0153 0.0205 0.025 Fibrinogen 28.9 38.0 37.7 55.1 Haptoglobin 0.388 0.403 0.399 0.613 IgA 0.196 0.229 0.167 0.423 Serum Amyloid P Component 0.0516 0.0564 0.0699 0.120 SGOT 39.6 46.3 35.8 41.7

PAGE 39

39 Table 3 2. Myriad Rules Based Medicine brain cytokine results reported in ng/mL. A verage amount of different cytokines found in MPSIIIB mut or het brains in ng/mL. A T test was used to determine significance. (*) denotes a statistically significant difference within the age group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 New Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) Factor VII 2.25 2.51 1.78 4.89 FGF 9 2.55 2.96 2.55 3.56 FGF basic 22.5 67.2 5.30 215 ** GCP 2 0.0593 0.0710 0.0716 0.132 IL 12p70 0.0167 0.0223 0.0249 0.0338 IL 18 2.35 3.29 1.84 3.98 IL 7 0.0100 0.0113 0.0117 0.0196 M CSF 1 0.216 0.251 0.276 0.414 MIP 0.245 0.214 0.179 0.354 ** MIP 0.263 0.305 0.366 0.520 MMP 9 2.64 3.01 2.22 6.38 MPO 2.14 2.30 1.85 5.90 Myoglobin 13.7 16.1 27.2 16.5 Oncostatin M 0.0245 0.0275 0.0231 0.0553 Tissue Factor 0.427 0.770 0.683 1.58 TIMP 1 0.0337 0.0254 0.0196 0.130 VCAM 1 4.03 4.31 3.67 4.56 vWF 1.23 1.55 1.73 1.68

PAGE 40

40 Table 3 3. Myriad Rules Based Medicine brain cytokine results reported in pg/mL. Average amount of different cytokines found in MPSIIIB mut or het brains in pg/mL. A T test was used to determine significance. (*) denotes a statistically significant difference within the age group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 New Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) CD40 3.97 5.13 5.42 8.12 CD40 Ligand 338 282 361 492 Eotaxin 2.38 3.17 2.83 4.79 ** IFN 2.28 2.51 2.50 3.50 IP 10 8.75 10.7 11.6 34.5 ** IL 49.4 65.0 52.4 120 ** IL 10 42.5 55.1 40.7 78.7 IL 4 7.95 8.52 8.52 13.0 IL 2 3.99 4.23 2.18 6.34 LIF 66.0 63.7 66.0 144 Lymphotactin 6.56 7.83 7.51 15.7 MIP 13.0 16.9 15.8 90.0 ** MIP 2 1.34 1.74 1.67 2.76 ** MDC 22.5 27.6 28.0 53.2 MCP 1 3.96 6.44 3.55 20.3 ** MCP 3 9.86 13.4 10.0 31.0 ** MCP 5 1.90 2.55 2.32 9.58 ** Stem Cell Factor 293 332 258 334 VEGF A 156 224 137 392 **

PAGE 41

41 Figure 3 1. Fold increase of 24 week old diseased mice brain homogenate analytes in comparison to unaffected heterozygote brain homogenate analytes. Averages were taken for 24 week old unaffected and diseased mice analytes. The diseased mouse averages were then divided by the unaffected average in order to calculate the fold increase from normal, and plotted on the above graph. All fold increases were significant (p<0.05). Oncostatin M was left off of the graph due to a lack of confidence in the reported va lues.

PAGE 42

42 Table 3 4. Myriad Rules Based Medicine blood cytokine results reported in g/mL. Average amount of different cytokines found in MPSIIIB mut or het blood in g/mL. A T test was used to determine significance. (*) denotes a statistically significant difference within the age group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 Young Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) Apo A I 36.8 37.1 37.1 35.5 C Reactive Protein 5.35 5.95 4.73 6.92 Fibrinogen 37400 32700 33400 47000 Haptoglobin 25 27 29 48 IgA 35.6 28.8 45.1 40.3 Serum Amyloid P Component 19.7 19.7 18.1 27.1 SGOT 161 175 141 163

PAGE 43

43 Table 3 5 Myriad Rules Based Medicine blood cytokine results reported in ng/mL. The average amount of different cytokines found in MPSIIIB mut or het blood in ng/mL. A T test was used to determine significance. (*) denotes a statistically significant difference wi thin the age group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 Young Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) Factor VII 34.8 33.5 30.7 36.0 GCP 2 10.1 12.5 12.1 17.0 KC/GRO 0.0223 0.0285 0.0623 0.0558 IL 2.85 2.39 1.31 1.86 IL 18 10.3 11.4 10.9 10.8 M CSF 1 6.43 6.96 6.19 6.86 MIP 5.00 5.02 3.61 5.12 MIP 20.9 20.8 14.5 24.7 MIP 2.31 2.59 2.11 2.57 MMP 9 67.6 70.6 70.9 107 MPO 52.5 54.8 56.9 54.6 Myoglobin 627 3300 3760 1890 TPO 33.1 32.2 24.5 32.0 Tissue Factor 7.28 7.28 7.54 8.77 TIMP 1 0.986 0.993 0.874 1.10 VCAM 1 1290 1290 1080 1820 vWF 100 80.4 95.1 113

PAGE 44

44 Table 3 6. Myriad Rules Based Medicine blood cytokine results reported in pg/mL. The average amount of different cytokines found in MPSIIIB mut or het blood in pg/mL. A T test was used to determine significance. (*) denotes a statistically significant differen ce within the age group (ie. Mut vs. Het), while () denotes a statistically significant difference over time (ie. Old vs. New). p<0.5, ** p<0.01, p<0.5, p<0.01 Young Mice Old Mice Analyte Het (n = 3) Mut (n = 3) Het (n = 3) Mut (n = 3) CD40 55.9 48.9 36.4 53.4 CD40 Ligand 1390 1400 1990 1670 Endothelin 1 27.8 27.1 33.8 28.0 Eotaxin 463 504 776 658 IP 10 60.4 63.5 55.4 83.2 IL 276 230 401 367 LIF 943 1140 1050 1110 Lymphotactin 111 96.8 131 166 MIP 104 140 100 251 MIP 2 8.02 7.97 15.5 9.39 MDC 481 549 574 544 MCP 1 61.8 85.7 51.0 86.4 MCP 3 123 196 78.4 127 MCP 5 19.1 23.5 15.5 23.1 Stem Cell Factor 567 637 634 666 T Cell Specific Protein RANTES 0.0218 0.0219 0.0244 0.0375 VEGF A 554 222 7730 291

PAGE 45

45 Table 3 7. Cohort setup. The experimental setup for the brain cytokine analysis across two time points and five treatments in MPSIIIB mice 28 weeks of age. Each treatment cohort consisted of 22 mice at a given age and Tx type ; 11 mice were mutant and 11 mice were heterozygote in each cohort A total of 220 mice were used. Tx Code "Old" "New" Diseased (Mut) Untreated (MU) 11 11 Control (Het) Untreated (HU) 11 11 Diseased Genistein (MG) 11 11 Control Genistein (HG) 11 11 Diseased Fosteum (MF) 11 11 Control Fosteum (HF) 11 11 Diseased Minozac (MM) 11 11 Control Minozac (HM) 11 11 Diseased Combination G+M (MGM) 11 11 Control Combination G+M (HGM) 11 11

PAGE 46

46 Table 3 8. Values used to determine cytokine concentration. Values fit to a standard curve and used to determine cytokine concentration for bead assay (F igures 3 2 through 3 13) Observed concentrations (pg/mL) were matched to Fluorescent intensity. Expected v alues, given by the assay manufacturer, are noted for comparison. Fluorescent Intensity Exp concentration (pg/mL) Obs Concentration (pg/mL) IL 21511 19306 OOR > 16436.8 4826.5 4611.69 3211.5 1206.63 1247 174.8 301.66 283.29 35 75.41 104 18 18.85 39.14 15.5 4.71 2.8 IL 6 15037 11410 11474.98 6240.5 2852.5 2830.77 1074 713.13 730.48 132 178.28 169.08 43 44.57 53.7 29.3 11.14 18.43 MIP 17775 39916 40556.12 15322.8 9979 9970.06 2689 2494.75 2495.1 126 623.69 622.94 75.5 155.92 237.55 14329.5 47111 47145.99 3081.5 11777.75 11754.67 290.5 2944.44 2954.81 23 736.11 727.29 13 184.03 338.11 FGFb 3899 18585.75 18585.31 245.5 4646.44 4648.12 35 1161.61 1148.35 28.5 290.4 641.43 VEGF 5182.5 29861 30070.64 1703.3 7465.25 7383.6 275.5 1866.31 1883.62 33.5 466.58 457.44 24 116.64 247.03

PAGE 47

47 Figure 3 2. Brain IL 1a concentrations across four treatment types in 10 week old MPSIIIB mice. IL 1a concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) ind icate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 48

48 Figure 3 3. Brain IL 1a concentrations ac ross four treatment types in 28 week old MPSIIIB mice. IL 1a concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 49

49 Figure 3 4. Brain IL 6 concentrations across four treatment types in 10 week old MPS IIIB mice. IL 6 concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 50

50 Figure 3 5. Brain IL 6 concentrations across four treatment types in 28 week old MPSIIIB mice. IL 6 concentrations for het and mu t MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 51

51 Figure 3 6. Brain MIP 1a concentrations across four treatment types in 10 week old MPSIIIB mice. MIP 1a concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) i ndicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 52

52 Figure 3 7. Brain MIP 1a concentrations a cross four treatment types in 28 week old MPSIIIB mice. MIP 1a concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 w ay ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 53

53 Figure 3 8. Brain TNF concentrations across four treatment types in 10 week old MPSIIIB mice. concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All c ohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 54

54 Figure 3 9. Brain concentrations across four treatment types in 28 week old MPSIIIB mice. concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mi ce and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 55

55 Figure 3 10. Brain FGFb concentrations across four treatment types in 10 week old MPSIIIB mice. FGFb concentrations for het and mut M PSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were con sidered in the 2 way ANOVA. Yellow indicates significance, as determined by 1 way ANOVA, in Mut v Het controls for the 10 week old time point only. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 56

56 Figure 3 11. Brain FGFb concentrations across four treatment types in 28 week old MPSIIIB mice. FGFb concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls, All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA. Yellow indicates significance, as determined by 1 way ANOVA, in Mut v Het controls for the 28 week old time point only. Red indicates sig nificance as determined by 1 way ANOVA for the Genistein treatment only against matched controls, for only the 28 week old time point. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 57

57 Figure 3 12. Brain VEGF concentrations across four treatment types in 10 week old MPSIIIB mice. VEGF concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matche d controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 58

58 Figure 3 13. Brain VEGF concentrations across four treatment types in 28 week old MPSIIIB mice. VEGF concentrations for het and mut MPSIIIB mice in four different treatment groups: Genistein Fosteum Minozac, and combination Genistein +Minozac. (*) indicate significance as determined by 2 way ANOVA against matched controls. All cohorts (10 week old mice and 28 week old mice) were considered in the 2 way ANOVA. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 59

59 Table 3 9. Membrane array map. Provided by Ray Biotech as a part of the membrane assay kit. The position of each antibody blot on the membrane array is displayed All cytokine blots were run in duplicate. A B C D E F G H 1 Pos Pos Neg Neg Blank Eotaxin Fas Ligand bFGF 2 Pos Pos Neg Neg Blank Eotaxin Fas Ligand bFGF 3 G CSF GM CSF IFNg IGF II IL 1a IL 1b IL 12 p40/p70 IL 12 p70 4 G CSF GM CSF IFNg IGF II IL 1a IL 1b IL 12 p40/p70 IL 12 p70 5 IL 13 IL 6 IL 9 Leptin MCP1 M CSF MIG PF 4 6 IL 13 IL 6 IL 9 Leptin MCP1 M CSF MIG PF 4 7 TIMP 1 TIMP 2 Thrombo VEGF Blank Blank Pos 8 TIMP 1 TIMP 2 Thrombo VEGF Blank Blank Pos

PAGE 60

60 Figure 3 14. X rayed image of membrane assay. 8 total cytokine detection membrane assays were completed. The top row represents four, randomly selected, 28 week old control untreated brain homogenates; the bottom row represents four, randomly selected, diseased untreated brain homogenates. These homogenate samples are from Table 2.

PAGE 61

61 Figure 3 15. Paired differences in cytokine expression. Individual blots from the membranes in Fig. 3a were measured for density. These duplicated mea surements were normalized by positive control relative density. Negative blots were subtracted as background; values below background are negative as shown for Eotaxin, Fas Ligand, etc. The average cytokine expression across the all four membranes in each group (het or mut) was determined. These averages were plotted, and T tests were run on each group (Het v Mut) to determine significance. (*) denotes significance (p<0.05)

PAGE 62

62 Figure 3 16. Secondary enzyme fluorescent assay of 10 week old MPSIIIB mice. Lev els glucuronidase, as determined by a substrate based fluorescence assay, in MPSIIIB 10 week old control and diseased mice across four treatments. Significance was reached using a 1 way ANOVA for controls. No significance was found using a 2 way ANOVA across groups vs. untreated. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 63

63 Figure 3 17. Secondary enzyme fluorescent assay of 28 week old MPSIIIB mice. Levels glucuronidase, as determined by a substrate based fluorescence assay, in MPSIIIB 2 8 week old control and diseased mice across four treatments. Significance was reached using a 1 way ANOVA for controls. No significance was found using a 2 way ANOVA across groups vs. untreated. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 64

64 Figure 3 1 8. Graph of standard curve used to derive the M CP1 cytokine levels in Figure 3 19 The best fit line to the standard dilution that came with the MCP1 ELISA kit was determined using MasterPlex 2010 freeware software. Best fit curve was defined using four p arameter logistics. The range o f detection for MCP1 was 0 2000 pg/mL r 2 = 0.9999254.

PAGE 65

65 Figure 3 19. MCP1 ELISA. This graph represents the amount of the cytokine MCP1 in brains from the Minozac modality study. There were four treatment modalities: gavage (PO), subcutaneous injection (SQ), intravenous injection (IV), and intraperitoneal injection (IP). Significance was determined via 1 way ANOVA vs. matched controls. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 66

66 Figure 3 20 Graph of standar d curve used to derive the F GFb cytokine levels in Figure 3 21 The best fit line to the standard dilution that came with the FGFb ELISA kit was determined using MasterPlex 2010 freeware software. Best fit curve was defined using four parameter logistics. The range o f detection for FGFb was 0 1200 pg/mL r 2 = 0.9999055.

PAGE 67

67 Figure 3 21. FGFb ELISA. This graph represents the amount of the cytokine FGFb in brains from the Minozac modality study. There were four treatment modalities: gavage (PO), subcutaneous injection (SQ), intravenous injection (IV), and intraperitoneal injection (IP). Significance was determined via 1 way ANOVA vs. matched controls. p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

PAGE 68

68 CHAPTER 4 DISCUSSION These experiments offer an important view into the neuroinflammatory processes that occur in lysosomal storage diseases, specifically in MPSIIIB. In the primary brain and blood exploratory map we found that circulating proin flammatory cytokine levels may not accurately represent the degree of proinflammatory cytokine proliferation found in the brain. We also note that many cytokines, some of which are proinflammatory, are indeed upregulated in diseased MPSIIIB brains at a 24 week time point. We confirmed our original assessment that both M CP 1 and FGFb were upregulated in MPSIIIB diseased mice in our 28 day treatment experiment. Untreated d iseased MPSIIIB mice showed a significant upregulation of MIP their ma tched unaffected controls. FGFb is an important cytokine that aids in cell growth and, in the presence of other proinflammatory cytokines (TNF to increase the recruitment of polymorphonuclear (PMN) leukocytes, monocytes and T cells, thereby exacerbating inflammation. Thus, because FGFb participate s in the signal transduction of various inflammatory pathways, we hypothesize that high levels of FGFb may be caused by this inflammatory pathway activation (Quarto, 1994). Further, FGFb forms a complex with its receptor (FGFR 1) that is known to be stabilized by heparan sulfate (Quarto, 1994). We hypothesize that MPSIIIB mouse accumulation of HS brain cells e nhance s the expression of FGF b, via positive feedback or retention/stabiliza tion MIP and has been shown to induce the release of histamine from basophils. Antiserum MIP demonstrated the significant role of this proinflammatory cytokine in models of acute

PAGE 69

69 lun g injury (Cook, 1996). We believe that FGFb and MIP 1 provide likely targets by which future therapies for the treatment of MPSIIIB may be based. Additionally, Genistein ability to reduce GAG production and accumulation in MPSIIIB mice resulted in our using it as a possible reducer of proinflammatory cytokines or proinflammatory related cytokines. Our 28 day treatment experiment showed that Genistein significantly prevented FGFb and nearly significantly prevented MIP upregulation in 28 week old MPSI IIB diseased mice. In all additional cytokines, Genistein seemed to instigate a downward trend in proinflammatory cytokine production that does not quite reach significance However, neither Genistein n or any other drug assessed, was able to decrease se condary enzyme upregulation. Throughout the literature, the effectiveness of Genistein is unclear A newly published double blind, study demonstrated a small, yet statistically significant reduction in GAG concentration in urine and blood plasma followi ng oral administration of Genistein to patients with MPSIIIA or B at 5mg/kg/day for one year However, drug application had no effect on behavior or histology, as measured by hair morphology (de Ruitjer, 2012). The idea of substrate reduction is an intere sting secondary pathway that, when targeted, may offer therapeutic benefit to MPSIIIB patients. However, Genistein may be best used as supplement to other more aggressive treatments (gene therapy, BMT, etc.) by providing the added effect of some substrate reduction, thus positively impacting treatment outcome. Our Minozac results were i n direct contrast to the effects described by Chrzaszcz et al. in a mouse model of traumatic brain injury and electroconvul sive induced seizures We found that Minozac did not mitigate levels of neural cytokines including

PAGE 70

70 the expected However, in our 28 day treatment groups these cytokines were not elevated. Chrzaszcz et al. administered Minozac intraperito neally and acutely (Chrzaszcz et al 2010). Our trial administered the drug subcutaneously and chronically. Due to the unexpected results from Minozac application, a second 24 hour trial was run, comparing the acute application of Minozac to diseased MPS IIIB mice intraperioneally (IP), subcutaneously (SQ), intravenously (IV) or orally (PO) Both the FGFb and MCP 1 ESLISAs run on the brain homogenates of these treated mice did not show any significant differences in cytokine concentration among the various modes of administration, although subcutaneous injection had the highest downward trend in both cytokines measured From this data, we conclude that Minozac does not seem to be a viable drug for decreasing the chronic neuroinflammation seen in MPSIIIB mic e. Fosteum greatly aggravated neuroinflammation as evidenced by the heightened concentrations of neural cytokines measured. We speculate that the cholecalciferol supplement in the Fosteum caused this increase Research has shown that h ouse mice and several species of rats have a cholecalciferol LD50 of ~44 mg/kg (Marshall, 1984) In fact previous research has suggested the addition of cholecalciferol as a secondary lethal agent to rodenticides (Marshall, 1984). H umans have a much higher LD50 to chol ecalciferol than mice. We believe that sub toxic levels of cholecalciferol exacerbated the stress and inflammation seen in the brains of MPSIIIB mice, thus rendering Fosteum an ill advised treatment for mouse MPSIIIB. Interestingly, because cholecalcifero l increases calcium absorption, which can cause hypercalcemia and calcification throughout organ systems, Fosteum may have elucidated a heretofore

PAGE 71

71 unknown interaction between calcium absorption and inflammation in the brains of MPS patients that aggravate s inflammation and escalates disease progression.

PAGE 72

72 CHAPTER 5 FUTURE AIMS The results of this study are limited by several factors. The definitive role of neuroinflammation and the roles of specific upregulated cytokines, such as FGF 2 and MIP sease pathology of MPSIIIB remain to be definitively determined. Even if neuroinflammation is proven to play a key role in MPSIIIB, f uture studies must evaluate whether cytokines are a viable biomarker for predicting lifespan or symptomatic improvement of Sanfilippo Syndrome. Genistein and Minozac also should be tested to determine their effects on lifespan and potential synergistic effects with other treatment modalities such as gene therapy via intracranial AAV, BMT (Heldermon, 2010) or ERT (Sands, 199 4) The potentially detrimental effects of excess zinc bisglycate and cholecalciferol in the diets of patients with Sanfilippo syndrome elucidated by the Fosteum treatment findings, should also be evaluated.

PAGE 73

73 CHAPTER 6 ONGOING EXPERIMENTS I mmunohistochemical analysis of brains from the primary 28 day treatment experiment is ongoing. Brains from all cohorts are being graded for the presence of: glial fibrillary acidic protein (GFAP), an astrocyte marker; NeuN, a neuronal marker; heparin sulfa te (HS); lysosomal associated membrane protein 1 (LAMP 1); IBA 1, a microglia marker; FGFb, and MCP1. Further analysis is ongoing for the 24 hour Minozac experiment. Brain homogenates have yet to be immunohistochemically analyzed (described above) or analy zed for GFAP and MIP

PAGE 74

74 WORKS CITED Bachmanov A, Reed D, Beauchamp G, Tordoff M. Food Intake, Water Intake, and Drinking Spout Side Preference of 28 Mouse Strains. Behav Genet. 2002: 32(2):435 43 Burnett, B. P., Pillai, L., Bitto, A., Squa drito, F., & Levy, R. M. (2011). Evaluation of CYP450 inhibitory effects and steady state pharmacokinetics of genistein in combination with cholecalciferol and citrated zinc bisglycinate in postmenopausal women. Int J Womens Health, 3 139 150. doi: ijwh 3 139 [pii] 10.2147/IJWH.S19309 Chrzaszcz M, Venkatesan C, Dragisic T, et al. Minozac treatment prevents increased seizure susceptibility in a mouse "two hit" model of closed skull traumatic brain injury and electroconvulsive shock induced seizures. J Neuro trauma. 2010;27(7):1283 95. Cleary MA, Wraith JE. Management of mucopolysaccharidosis type III. Arch Dis Child. 1993;69(3):403 6. Cook DN. The role of MIP 1 alpha in inflammation and hematopoiesis. J Leukoc Biol. 1996;59(1):61 6. Cunningham O, Campion S, Perry VH, et al. Microglia and the urokinase plasminogen activator receptor/uPA system in innate brain inflammation. Glia. 2009;57(16):1802 14. de Ruijter J, Valstar MJ, Narajczyk M, et al. Genistein in Sanfilippo disease: a ran domized controlled crossover trial. Ann Neurol. 2012;71(1):110 20. DiRosario, J, Divers, E, Wang, C, Etter, J, Charrier, A, Jukkola, P et al (2009). Innate and adaptive immune activation in the brain of MPS IIIB mouse model. J Neurosci Res 87 : 978 990. E lkind, J. (2011). Neural cytokine expression profile in the murine model of sanfilippo syndrome type b. University of Florida Journal of Undergraduate Research. 11 (4) Fielding, C. A., McLoughlin, R. M., McLeod, L., Colmont, C. S., Najdovska, M., Grail, D., Jenkins, B. J. (2008). IL 6 regulates neutrophil trafficking during acute inflammation via STAT3. J Immunol, 181 (3), 2189 2195. doi: 181/3/2189 [pii] Futerman, Anthony H., and Gerrit Van Meer. The Cell Biology of Lysosomal Storage Disorders. Nature Revie ws Molecular Cell Biology 5.7 (2004): 554 65. Print. Heldermon CD, Hennig AK, Ohlemiller KK, et al. Development of sensory, motor and behavioral deficits in the murine model of Sanfilippo syndrome type B. PLoS One. 2007;2(8):e772.

PAGE 75

75 Heldermon CD, Ohlemiller KK, Herzog ED, et al. Therapeutic efficacy of bone marrow transplant, intracranial AAV mediated gene therapy, or both in the mouse model of MPS IIIB. Mol Ther. 2010;18(5):873 80. Jakobkiewicz Banecka J, Piotrowska E, Narajczyk M, et al. Genistein media ted inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor dependent pathway. J Biomed Sci 2009;16:26.11. Killedar, S., Dirosario, J., Dive rs, E., Popovich, P. G., McCarty, D. M., & Fu, H. (2010). Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response. J Neuroinflammation, 7 39. doi: 1742 2094 7 39 [pii] 10.1186/1742 2094 7 39 Li HH, Yu WH, Ro zengurt N, et al. Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha N acetylglucosaminidase. Proc Natl Acad Sci U S A. 1999;96(25):14505 10. Marshall, Edward F., "Cholecalciferol: A Unique Toxicant for Rodent Control" (1984). Proceedings of the Eleventh Vertebrate Pest Conference (1984). Paper 22. http://digitalcommons.unl.edu/vpc11/22 Matias, P. J., Jorge, C., Ferreira, C., Borges, M., Aires, I., Amaral, T., Ferreira, A. (2010). Cholecalciferol supplementation in hemodialysis patients: effects on mineral metabolism, inflammation, and cardiac dimension parameters. Clin J Am Soc Nephrol, 5 (5), 905 911. doi: CJN.06510909 [pii] 10.2215/CJN.0651090 9 Neufeld, E. F. and Muenzer, J. (1989). The mucopolysaccharidoses. In: Scriver C. R., Beaudet A. L., Sly W. S. and Valle D. (Eds), The Metabolic Basis of Inherited Disease McGraw Hill: New York. 2465 2494. Nidiffer FD, Kelly TE. Developmental and degener ative patterns associated with cognitive, behavioural and motor difficulties in the Sanfilippo syndrome: an epidemiological study. J Ment Defic Res. 1983;27 (Pt 3):185 203. Ohmi, K., Greenberg, D. S., Rajavel, K. S., Ryazantsev, S., Li, H. H., & Neufeld, E. F. (2003). Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB. Proc Natl Acad Sci U S A, 100 (4), 1902 1907. Piotrowska E, Jakobkiewicz Banecka J, Tylki Szymanska A, et al. Genistin rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: an open label, pilot study in 10 pediatric patients. Curr Ther Res Clin Exp 2008;69:166 179. Prasad, A. S. (2009). Zinc: role in immunity, oxidative stress and chronic inflammation. Curr Opin Clin Nutr Metab Care, 12 (6), 646 652. doi: 10.1097/MCO.0b013e3283312956

PAGE 76

76 Quarto N, Amalric F. Heparan sulfate proteoglycans as transducers of FGF 2 signalling. J Cell Sci. 1994;107 ( Pt 11):3201 12. Rabiau N, Kossai M, Braud M, et al. Genistein and daidzein act on a panel of genes implicated in cell cycle and angiogenesis bypolymerase chain reaction arrays in human prostate cancer cell lines. Cancer Epidemiol 2010;34:200 206. Sands MS, Vogler C, Kyle JW, et al. Enzyme replacement therapy for murine mucopolysaccharidosis type V II. J Clin Invest. 1994;93(6):2324 31. Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans: host associated molecular patterns for initiation and modulation of inflammation Faseb J 2006, 20 : 9 22 Ullmann U, Ullmann U, Metzner J, Frank T, et a l. Safety, tolerability, and pharmacokinetics of single ascending doses of synthetic Genistein (Bonistein) in healthy volunteers. Adv Ther. 2005;22:65 78. Van de Kamp, JJ, Niermeijer, MF, von Figura, K et al. Genetic heterogeneity and clinical variability in the Sanfilippo syndrome (types A, B, and C). Clin Genet 1981;20:152 160. Van Eldik, L., and Wainwright, M.S. (2003). The Janus face of glial derived S100B: beneficial and detrimental functions in the brain. Restorative Neurol. Neurosci. 21, 97 108. Vi llani, G. R., Gargiulo, N., Faraonio, R., Castaldo, S., Gonzalez Y Reyero, E., & Di Natale, P. (2007). Cytokines, neurotrophins, and oxidative stress in brain disease from mucopolysaccharidosis IIIB. J Neurosci Res, 85 (3), 612 622. doi: 10.1002/jnr.21134 Zittermann SI, Issekutz AC. Basic fibroblast growth factor (bFGF, FGF 2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression. Am J Pathol. 2006;168(3):835 46. Zittermann, S. I., & Issekutz, A. C. (2006b ). Endothelial growth factors VEGF and FGFb differentially enhance monocyte and neutrophil recruitment to inflammation. J Leukoc Biol, 80 (2), 247 257. doi: jlb.1205718 [pii] 10.1189/jlb.1205718

PAGE 77

77 BIOGRAPHICAL SKETCH Jo s e ph Gibney graduated from the University of Florida College of Agriculture and Life Sciences in May 2010 with a b iology a nd two minors in environmental s cience and a gricultural and n atural r esource e thics and p olicy. He followed this degree with master s in M edical S cience from the University of Florida College of Medicine in August 2012 He plans to attend medical school in the coming years.