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Therapeutic effects of Genistein, Minozac and Fosteum in a mouse model of Mucopolysaccharidosis type IIIb (Sanfilippo sy...

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Title:
Therapeutic effects of Genistein, Minozac and Fosteum in a mouse model of Mucopolysaccharidosis type IIIb (Sanfilippo syndrome B)
Series Title:
Journal of Undergraduate Research
Physical Description:
Serial
Language:
English
Creator:
Himmler, Amber
Gibney, Joseph
Leclair, Erin
Elkind, Jae
Asokan, Aditya
Omerod, Brandi
Mandel, Ronald
Watterson, Martin
Heldermon, Coy
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Genistein
Minozac
Fosteum
Mucopolysaccharidosis type IIIb
Sanfilippo Syndrome B
N-acetylglucosaminidase (NAGLU)
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serial   ( sobekcm )

Notes

Abstract:
In this study, Genistein, Minozac, Fosteum, and a combination treatment of Genistein and Minozac were administered to NAGLU mice, a mouse model for Sanfilippo syndrome type B, to determine if they would attenuate the production of proinflammatory cytokine levels in the brain. The mouse model for this disease is deficient in N-acetyl-glucosaminidase (NAGLU), one of four enzymes responsible for the degradation of heparan sulfate. The cells of NAGLU mice, as well as those of humans affected with Sanfilippo syndrome type B, gradually become distended due to the accumulation of heparan sulfate in the cell’s lysosomes. This retarded degradation is associated with an increase in proinflammatory cytokines in the brain, which is thought to exacerbate the neurodegenerative effects of the disease. Either heterozygous or mutant mice were treated with Genistein, Fosteum, Minozac, or a combination of Genistein and Minozac. Brains were harvested after 28 days of treatments and their homogenates were run on a BioRad custom-array bead assay. Genistein decreased the levels of proinflammatory cytokines in the NAGLU mice. Conversely, Fosteum aggravated neuroinflammation. Minozac was ineffective, and the combination treatment of Genistein and Minozac was more effective than Minozac alone, but not as effective as Genistein alone.

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University of Florida
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University of Florida
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MISSING IMAGE

Material Information

Title:
Therapeutic effects of Genistein, Minozac and Fosteum in a mouse model of Mucopolysaccharidosis type IIIb (Sanfilippo syndrome B)
Series Title:
Journal of Undergraduate Research
Physical Description:
Serial
Language:
English
Creator:
Himmler, Amber
Gibney, Joseph
Leclair, Erin
Elkind, Jae
Asokan, Aditya
Omerod, Brandi
Mandel, Ronald
Watterson, Martin
Heldermon, Coy
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Genistein
Minozac
Fosteum
Mucopolysaccharidosis type IIIb
Sanfilippo Syndrome B
N-acetylglucosaminidase (NAGLU)
Genre:
serial   ( sobekcm )

Notes

Abstract:
In this study, Genistein, Minozac, Fosteum, and a combination treatment of Genistein and Minozac were administered to NAGLU mice, a mouse model for Sanfilippo syndrome type B, to determine if they would attenuate the production of proinflammatory cytokine levels in the brain. The mouse model for this disease is deficient in N-acetyl-glucosaminidase (NAGLU), one of four enzymes responsible for the degradation of heparan sulfate. The cells of NAGLU mice, as well as those of humans affected with Sanfilippo syndrome type B, gradually become distended due to the accumulation of heparan sulfate in the cell’s lysosomes. This retarded degradation is associated with an increase in proinflammatory cytokines in the brain, which is thought to exacerbate the neurodegenerative effects of the disease. Either heterozygous or mutant mice were treated with Genistein, Fosteum, Minozac, or a combination of Genistein and Minozac. Brains were harvested after 28 days of treatments and their homogenates were run on a BioRad custom-array bead assay. Genistein decreased the levels of proinflammatory cytokines in the NAGLU mice. Conversely, Fosteum aggravated neuroinflammation. Minozac was ineffective, and the combination treatment of Genistein and Minozac was more effective than Minozac alone, but not as effective as Genistein alone.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
sobekcm - UF00091523_00602
System ID:
UF00091523:00665


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University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 1 Therapeutic effects of Genistein, Minozac and Fosteum in a mouse model of Mucopolysacch aridosis type IIIb (Sanfilippo s yndrome B) Amber Himmler Joseph Gibney, Erin Leclair, Jae Elkind Aditya Asokan, Dr. Brandi Omerod, Dr. Ronald Mandel, Dr. Martin Watterson, and Dr. Coy Heldermon College of Medicine, University of Florida In this study, Genistein, Minozac, Fosteum, and a combination treatment of Genistein and Minozac were administered to NAGLU mice, a mouse model for Sanfilippo syndrome type B, to determine if they would attenuate the production of proinflammatory cytokine levels in the brain. The mouse model for this disease is deficient in N acetyl glucosaminidase ( NAGLU), one of four enzymes responsible for the degradation of heparan sulfate. The cells of NAGLU mice, as well as those of humans affected with s lysosomes. This retarded degradation is associated with an increase in proinflammatory cytokines in the brain, which is thought to exacerbate the neurodegenerative effects of the disease. Either heterozygous or mutant mice were treated with Genistein, Fo steum, Minozac, or a combination of Genistein and Minozac. Brains were harvested after 28 days of treatments and their homogenates were run on a BioRad custom array bead assay. Genistein decreased the levels of proinflammatory cytokines in the NAGLU mice. Conversely, Fosteum aggravated neuroinflammation. Minozac was ineffective, and the combination treatment of Genistein and Minozac was mor e effective than Minozac alone, but not as effective as Genistein alone. BACKGROUND Sanf ilippo Syndrome Mucopolysaccharidosis type IIIB ( Sanfilippo syndrome type B, or MPS IIIB) is an autosomal recessive disorder characterized by the lysosomal accumulation of heparan sulfate (HS) (Neufeld and Muenzer, 1989). There are four forms of mucopolysacchari doses (types I IV) typified by a deficiency in one of the four enzymes necessary for the degradation of heparan sulfate. In MPS IIIB, the buildup occurs due to a deficiency in N acetylglucosaminidase (NAGLU) (Heldermon 2007). Patients often present with symptoms in the central nervous system though enzyme deficiencies and lysosomal distensions are also sys temic (Neufeld and Muenzer, 1989 ). In the first phase of the disorder, often occurring between one and four years of age clinical patterns are associa ted with developmental delay, especially in language. The second phase of the illness typically begins between three and four years of age and is characterized by severe behavioral disturbances including increasingly frequent and severe temper tantrums, marked hyperactivity, decreased attention span, aggression, panic attacks and distu rbed sleep patterns Physical g rowth is usually normal. The third phase of the illness occurs a t about ten year s of age and begins with effects in balance and, consequently, motility leaving many affected individuals wheelchair bound by their mid teenage years. Swallowing difficulties are often common as are frequent episodes of aspiration of both food and saliva (Cleary, 1993). Death usually occurs due to respiratory complications, heart failure or infection (Heldermon, 2007). Therap y is limited to supportive care ( Cleary, 1993). NAGLU Mouse Model A mouse model of Sanfillipo s yndrome t ype B was created by repla cing an 852 bp fragment within exon 6, the site within the mouse genome homologous to the human NAGLU gene, with a neo gene (Li, 1999). The mouse model was proved to be representative of the human condition through vigorous histological and behavioral testing (Heldermon, 2007) T reatments To date no treatments have successfully eradicated lysosomal inclusions or corrected the clinical manifestations of MPS IIIB The continuous administration of supraphysiologic levels of enzyme seem to quell the visce ral expressions of the disease, but have little effects on the neurologic symptoms (Sands 1994). Furthermore, a study by Heldermon explored possible treatment modalities in th e form of gene therapy with intracranial (IC) AAV 2/5 hNAG LU and bone marrow tra nsplant (BMT). Treatment with IC AAV was shown to be the most

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 2 efficacious in increasing lifespan (by an average of 112 d ays) and improving motor function as assessed by a r ocking RotaRod BMT was shown to be in effective in improving lifespan or motor function. Combination treatments improved the size s of lysosomal inclusions and hearing as measured by auditory brainstem response (ABR) D espite these results, the improvement of symptoms did not coincide with hist ologic al improvements (Heldermon, 2010) These findings show that replacing NAGLU through ERT, gene therapy with IC AAV or BMT is no t sufficient to correct MPS IIIB Therefore, additional pathways must be investigated in order to provide more comprehensive treatment modalities M arked neuroinflammation in MPS IIIB brains has been thought to play a role in disease progression ( Heldermon, 2010) Glycosaminoglycans (GAGs) such as heparan sulfate have been shown to incite inflammatory events within the cell including cytokine and chemokine production, leukocyte recruitment and the maturation of inflammatory cells (Taylor and Gallo, 1996) Prednisone an immunosuppressant, has been shown to yield the best results in regards to an increase in lifesp an, decrease in GFAP concentration, improvement in motor function and attenuation in the immune response as assessed by T cell activation and leukocyte/lymphocyte concentration (DiRosario, 2009) Administered Drugs Geni stein is an isoflavone and a tyrosine kinase inhibitor. Previous literature has shown Genistein to be effective in decreasing the production of heparan sulfate in MPS IIIB mice ( de Rui j ter 2007). Also known as trihydroxyisoflavone, Genistein acts on several growth fac tor receptors, the most important of which is the epidermal growt h factor receptor (EGFR), which mediate s the production of heparan sulfate in the cell. The level of GAG production has been shown to be attenuated by receptor inactivation via tyrosine kinase inhibition (Jakobkiewicz Banecka, 2007 ). Minozac is a ce ntral nervous system (CNS) penetrant molecule that has demonstrated the ability to decrease neuroinflammation in mouse models of traumatic brain injury and electroconvulsive shock induced seiz ures. response was shown through the decreased expression of glial fibrillary acidic protein (GFAP), as we l l as the astrocyte marker S100B (Chrzaszcz 2010). Fosteum is a combination of Genistein, chole calciferol (vitamin D3) and citrated zinc bisglycinate. It is an FDA approved dietary supplement used to manage osteopenia and osteoporosis (Burnett, 2011). Zinc and cholecalciferol have anti inflammatory implications in other diseases such as rheumatoid arthritis and chronic kidney disease (Prasad, 2009; Mathias, 2010). Hypothesis We hypothesize that the administration of these drugs separately or in tandem, will decrease proinflammatory cytokine levels in the brains of NAGLU mice We hypothesize that the combination treatment of Genistein and Minozac will be the most effective. SUBJECTS AND METHODO LOGY Animals A mouse colony of MPS IIIB mice was transferred to the University of Florida from Washington University in St. Louis, Missouri in 2009. The co lony was maintained through strict sibling mating. Mutant and heterozygous males were crossed with heterozygous females. The genotypes of the pups were determined via PCR of the NAGLU gene on exon 6 with the neomycin insertion. Results were verified using a substrate based fluorescence assay to test for the presence of N acetylglucosaminidase (Heldermon 2007). Experimental Design Two hundred twenty mice half mutant and half heterozygous, were randomly sorted into five treatment groups : control, Genistei n Fosteum, Minozac or Genistein and Minozac in combination. These treatments were administered to mutant ( / ) and heterozygous mice (+/ ) at two time points: six weeks or six months of age. Twenty two mice were in each treatment cohort, with eleven at each age checkpoint. Each study lasted for 28 days. Table 1 describes the treatment setup. Mice in the control group were fed chow that was soy free. Genistein and Fosteum were administered through fortified chow that was also soy free.

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THERAPEUTIC EFFECTS OF G E NISTEIN MINOZAC AND F OSTEUM University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 3 Table 1. Treatment Setup Minozac was dissolved in normal saline solution (0.9 %) at a concentration of 0.15mg/100l and was injected subcutaneously into the specified mice daily. Each mouse in a cohort receiving Minozac received 5mg of the drug per kilogram of its body weight ( Chrzaszcz 2010 ), which was weight adjusted weekly throughout the experiment The amount of chow eaten each day was measured to ensure normal food consumption After 28 days, the mice were sacrific ed. Eight 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. Three of eleven in each grou p were prepared for subsequent immunohistochemical analysis Assays Cytokine concentrations in the brains of the sacrificed mice were quantified using a BioRad custom array bead assay. Tissues were prepared and the assay was run according to Statistical Analysis For untreated controls, significance was determined using 1 way ANOVA comparing mutants and heterozygote mice. The efficacies of each treatment group wer e analyzed by a 2 way ANOVA, which analyzed the effects of age and gen otype within a treatment group. When t tests indicated that a value was close to significant, an additional 1 way ANOVA, which analyzed the effects of treatment within genotype, was perf ormed RESULTS Basic Fibroblast Growth Factor Fibroblast growth factor 2 (FGF 2 ) was significantly upregulated in mutant new and old mice compared to their heterozygous counterparts as assessed by 1 way ANOVA Concentrations in new heterozygous mice averaged 761.53 pg/ml and those in old heterozygous mice averaged 752.91 pg/ml. In contrast, the average concentrations found in the new and old mutant mice were 2030.38 pg/ml and 2345.46 pg/ ml, respectively (p<0.01 ). See F igure 1. During initial assessment of treatment by 1 way ANOVA Genistein was effective in significantly decreasing the average concentration of FGF 2 to 1656.24 pg/ml in old mutants (p<0.05). As assessed by 2 way ANOVA, comparing the effect of tre atment on each group compared to age matched controls, Minozac did not appear to have any effect on the levels of proinflammatory cytokines. The combination treatment of Genistein and Minozac showed a trend of slightly decreasing FGF 2 concentration, but t his attenuation was not significant. Fosteum was significantly upregulated FGF 2 in new mutant mice (p<0.0001), new heterozygous mice (p<0.0001), old mutant mice (p<0.05), and old heterozygous mice (p<0.001). See F igure 2. Macrophage Inflammatory Protein Analysis by 1 way ANOVA comparing untreated mutant and heterozygote animals revealed that MIP significantly upregulated in old MPS IIIB mice (p<0.05). See F igure 1. Treatment Code 6 week old (new) mice 6 month old (old) mice Total mice Mutant untreated (MU) 11 11 22 Heterozygote untreated (HU) 11 11 22 Mutant Genistein (MG) 11 11 22 Heterozygote Genistein (HG) 11 11 22 Mutant Minozac (MM) 11 11 22 Heterozygote Minozac (HM) 11 11 22 Mutant Fosteum (MF) 11 11 22 Heterozygote Fosteum (HF) 11 11 22 Mutant Genistein+ Minozac (MGM) 11 11 22 Heterozygote Genistein+ Minozac (HGM) 11 11 22

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 4 Though none of the drugs tested significantly decreased the concentration of MIP observable. New mutant mice expressed an average concentration of 437.79 pg/ml and old mutant mice expressed an average concentration of 503.19 pg/ml. Genistein decreased concentrations of MIP old mutant mice to 382.90 pg/ml and 427.27.86 pg/ml, respectively. However, this decrease was not significant. The combination treatment of Genistein and Minozac showed a similar decrease to that described in FGF 2. Neither of these attenuations was statistically significant 2 way ANOVA demonstrated that Fosteum increased the concentration of this proinflammatory cytokine significantly in new mutant mice (p<0.01), new heterozygous mice (p<0.0001), and old heterozygous mice (p<0.05). See Figure 3. Other Proinflammatory Cytok ines The other four cytokines that were tested (TNF VEGF, IL 6 and IL with respect to upregulation in untreated mutants as compared to age matched heterozygotes. See F igure 1. A statistically significant attenuati on of these proinflammatory cytokines of mice in the treatment groups was not observed. However, weak trends were again noted. Genistein consistently decreased levels of TNF 6 and IL significantly in creased the concentrations of IL 6 (p<0.05) and IL F igures 4 and 5. Minozac did not seem to affect levels of TNF The combination treatment of Genistein and Minozac either remained near baseline levels or slightly decreased levels of proinflammatory cytokines. Attenuations seen with the combination treatment did not reach statistical significance. See F igures 4 7. Fosteum intensified the expression of all six of the surveyed cytokines. Fosteum treatment resulted in increased concentrations of TNF new mutants (p<0.001), new heterozygotes (p<0.01), old mutants (p<0.01) and old heterozygotes (p<0.01). See F igure 4. IL (p<0.01) and old mutants after the administration of Fosteum. See F igure 5. Likewise, Fosteum treatment led to an upregulation of IL 6 in new mutants (p<0.05). Minozac exhibited a trend of not altering or worsening the expression of these cytokines. See F igure 6. VEGF was increased in new m utants (p<0.0001), new heterozygotes (p<0.0001), old mutants (p<0.01) and old heterozygotes (p<0.01) following the administration of Fosteum. See F igure 7.

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THERAPEUTIC EFFECTS OF G E NISTEIN MINOZAC AND F OSTEUM University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 5 Figure 1. Baseline cytokine concentrations in the brains of new and old MPS IIIB heterozygous and mutant mice based as assessed by the BioRad Custom Cytokine Array Bead Assay. indicates significance as determined by 1 way ANOVA comparing cytokine concentration in age matched mutant and heterozygote brains. **** 0 500 1000 1500 2000 2500 3000 VEGF FGF-2 TNF-a MIP-1a IL-6 IL-1a Concentration (pg/mL) Cyotkine Mu New Hu New Mu Old Hu Old **** ****

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 6 0 500 1000 1500 2000 2500 3000 3500 4000 4500 U G F M G + M Concentration pg/ml Treatments FGF 2 Concentrations in the Brains of New and Old MPS IIIB Mice Mut New Het New Mut Old Het Old Figure 2. Concentration of FGF 2 in 10 week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and combination Genistein+Minozac. indicate significance as determined by 2 way ANOVA against age & genotype matched unt reated controls. Blue indicate significance as determined by a 1 way ANOVA against untreated age matched heterozygote controls. Red indicate significance as determined by a 1 way ANOVA against its corresponding matched, untreated group. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Mut New Het New Mut Old Het Old **** **** *** **** **** **

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THERAPEUTIC EFFECTS OF G E NISTEIN MINOZAC AND F OSTEUM University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 7 Figure 3. Concentration of MIP week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay. Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and combination Genistein+Minozac. indicate significance as determined by 2 way ANOVA against age and genotype matched untreated controls. Blue i ndicate significance as determined by a 1 way ANOVA against untreated heterozygote controls. 0 100 200 300 400 500 600 700 800 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Mut New Het New Mut Old Het Old ** ****

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 8 0 200 400 600 800 1000 1200 1400 1600 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg//ml) Treatments Mut New Het New Mut Old Het Old Figure 4. Concentration of TNF week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay. Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and combination Genistein+Minozac. indicate significance as determined by 2 way ANOVA against age and genotype matched untreated controls. *** ** ** **

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THERAPEUTIC EFFECTS OF G E NISTEIN MINOZAC AND F OSTEUM University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 9 0 10 20 30 40 50 60 70 80 90 U G F M G + M Concentrations pg/ml Treatments Il 1 Concentrations in the Brains of New and Old MPS IIIB Mice Mut New Het New Mut Old Het Old 0 10 20 30 40 50 60 70 80 90 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Concentrations of IL 1 in the Brains of MPS IIIB Mice Across Treatments Mut New Het New Mut Old Het Old Figure 5. Concentration of IL week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay. Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and combination Genistein+M inozac. indicate significance as determined by 2 way ANOVA against age and genotype matched untreated controls. 0 10 20 30 40 50 60 70 80 90 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Mut New Het New Mut Old Het Old **

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 10 Figure 6. Figure 6 shows the concentration of IL 6 in 10 week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay. Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and comb ination Genistein+Minozac. indicate significance as determined by 2 way ANOVA against age and genotype matched untreated controls. 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Mut New Het New Mut Old Het Old ****

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THERAPEUTIC EFFECTS OF G E NISTEIN MINOZAC AND F OSTEUM University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 11 Figure 7. Concentration of VEGF in 10 week old (new) and 7 month old (old) mice as assessed by the BioRad Custom Array Bead Assay. Cytokine concentration was determined across four different treatment groups: Genistein, Fosteum, Minozac, and combination Genistein+Mi nozac. indicate significance as determined by 2 way ANOVA against age and genotype matched untreated controls. 0 100 200 300 400 500 600 Untreated Genistein Fosteum Minozac Genistein and Minozac Concentrations (pg/ml) Treatments Mut New Het New Mut Old Het Old **** **** **** **

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A MBER H IMMLER ET AL University of Florida | Journal of Undergraduate Resea rch | Volume 14, Issue 3 | S ummer 2013 12 DISCUSSION Though none of the treatments proved to significantly downregulate proinflammatory cytokines, the up regulations observed in FGF 2 and MIP 1 support the idea that neuroinflammation may indeed be playing a role in the pathology of MPS IIIB FGF 2 is important in cell growth and differentiation and has implications in angiogenesis and tissue healing. FGF 2 binds to the cell surface tyrosine kinase receptor, fibroblast growth factor receptor 1 (FGFR 1). In order to be endocytosed, this complex must be stabilized by heparan sulfate (Quarto, 1994). We speculate that the accumulation of heparan sulfate in the brains of MPS IIIB mice may enhance the expression of FGF 2 via a positive feedback mechanism. Once internalized, FGF 2 participates in the signal transduction of various pathways, many of which are inflammatory (Quarto, 1994). Previous studies have demonstrated that, when expression is juxtaposed alongside other proinflammatory cytokines (especially TNF 2 acts acutely and synergistica lly to augment the recruitment of polymorphonuclear (PMN) leu kocytes, monocytes and T cells ( Zitterman, 2006). Other studies have demonstrated that the internalization of FGF 2 enhances the activity of the plasminogen activator system, as measured by an i ncrease in urokinase plasminogen mRNA. Plasminogen activation plays a significant role in tissue remodeling, cell migration (Quarto, 1994), and inflammation (Cu nningham, 2009). T he activation of the pathway was significantly upregulated in models of chroni c inflammation, induced by inoculation with a murine prion disease (ME7) (Cunningham, 2009). Our study also demonstrated a significant increase in concentration in old mutant mice compared to old heterozygous mi MIP induction of leukocyte chemotaxis, especially in regards to B lymphocytes, CD8+ T cells, NK cells and eosinophils. It has also been shown to induce the releas e of histamine from basophils and induce the expression of CAMs (Cook, 1996). FGF 2 and MIP proinflammatory cytokines, are targets of interest for reducing neuroinflammation in MPS IIIB The efficacies of the drugs tested in this study were assessed to determine whether they could contribute to the alleviation of the sequelae associated with this disorder. Genistein has been used to treat MPS IIIB via substrate reduction. It has been shown to decrease GAG production and lysosomal storage in mouse models of MPS IIIB Our results demonstrated a trend of decreasing neural proinflammatory cytokine levels in mutant mice treated with Genistein. It significantly decreased FGF 2 in old mutant mice compared to untreated old mutant mice. Ge nistein inhibits the autophosphorylation of the EGF tyrosine kinase receptor, thereby initiating a kinase cascade that leads to the downregulation of transcription factors used for HS synthesis ( Jakobkiewicz Banecka 2009 ). The efficacy of this treatment is debated A recent double blind study demonstrated a small yet still statistically significant reduction in GAG concentration in urine and plasma following the oral administration of Genistein to patients with MPS IIIB However, the drug had no effects o n behavior or hair morphology, a common treatment efficacy marker for MPS IIIB (de Rui j t er 2012). Though the idea of substrate reduction is appealing, our results seem to indicate that this approach is only slightly successful for improving neuroinflammat ion. In contrast to the attenuating effects on neuroinflammation described in a mouse model of traumatic brain injury and electroconvulsive induced seizures (Ch rzaszcz, 2010) this study found Minozac to be wholly ineffective in mitigating levels of neural proinflammatory cytokines. The aforementioned Minozac trial administered the drug intraperitoneally and acutely. Our trial administered the drug subcutaneously and chronically. Our failure to observe an decrease in levels of proinflammatory cytokines may be caused by the mode of administration of the drug or, perhaps, by the development of tolerance to the drug. Fosteum, a form of Genistein, additionally fortified with zinc bisglycinate and cholecalc iferol (vitamin D3), increased concentrations of neural proinflammatory cytokines across all cytokines measured. It is speculated that the cholecalciferol infused in the Fosteum may have had an adverse effect on the murine species. Increased levels of chol ecalciferol aid in calcium absorption, which can cause hypercalcemia and, consequently, calcification throughout their organ systems (Marshall, 1984). It is possible that sub toxic levels of cholecalciferol may have exacerbated stress and inflammation in t he brains of MPS IIIB mice. Sanfilippo s yndrome is a disease with a multifaceted and unclear pathology. T reatment must not only target the underlying problem (i.e. NAGLU deficiency) but also the secondary effects of neuroinflam mation. This research aimed to assess the ability of Genistein, Minozac, Fosteum and a combination of Genistein and Minozac on the levels of proinflammatory cytokines in the brains of MPS IIIB mice. Our study has shown that Genistein has a small effect in decreasing proinflammatory cytokines, but has failed to identify Minozac or Fosteum as viable treatment options.

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