Erythema multiforme associated with zonisamide in a dog.

This report describes a dog that developed erythema multiforme in temporal association with administration of the sulphonamide-based anticonvulsant drug zonisamide. Similar adverse drug reactions have been associated with sulphonamide antimicrobial drugs. Caution should be exercised when prescribing this medication for dogs with known hypersensitivity to sulphonamides.

Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I.

BACKGROUND: Treatment with intravenous enzyme replacement therapy and hematopoietic stem cell transplantation for mucopolysaccharidosis (MPS) type I does not address joint disease, resulting in persistent orthopedic complications and impaired quality of life. A proof-of-concept study was conducted to determine the safety, tolerability, and efficacy of intra-articular recombinant human iduronidase (IA-rhIDUA) enzyme replacement therapy in the canine MPS I model. METHODS: Four MPS I dogs underwent monthly rhIDUA injections (0.58 mg/joint) into the right elbow and knee for 6 months. Contralateral elbows and knees concurrently received normal saline. No intravenous rhIDUA therapy was administered. Monthly blood counts, chemistries, anti-rhIDUA antibody titers, and synovial fluid cell counts were measured. Lysosomal storage of synoviocytes and chondrocytes, synovial macrophages and plasma cells were scored at baseline and 1 month following the final injection. RESULTS: All injections were well-tolerated without adverse reactions. One animal required prednisone for spinal cord compression. There were no clinically significant abnormalities in blood counts or chemistries. Circulating anti-rhIDUA antibody titers gradually increased in all dogs except the prednisone-treated dog; plasma cells, which were absent in all baseline synovial specimens, were predominantly found in synovium of rhIDUA-treated joints at study-end. Lysosomal storage in synoviocytes and chondrocytes following 6 months of IA-rhIDUA demonstrated significant reduction compared to tissues at baseline, and saline-treated tissues at study-end. Mean joint synovial GAG levels in IA-rhIDUA joints were 8.62 ± 5.86 µg/mg dry weight and 21.6 ± 10.4 µg/mg dry weight in control joints (60% reduction). Cartilage heparan sulfate was also reduced in the IA-rhIDUA joints (113 ± 39.5 ng/g wet weight) compared to saline-treated joints (142 ± 56.4 ng/g wet weight). Synovial macrophage infiltration, which was present in all joints at baseline, was abolished in rhIDUA-treated joints only. CONCLUSIONS: Intra-articular rhIDUA is well-tolerated and safe in the canine MPS I animal model. Qualitative and quantitative assessments indicate that IA-rhIDUA successfully reduces tissue and cellular GAG storage in synovium and articular cartilage, including cartilage deep to the articular surface, and eliminates inflammatory macrophages from synovial tissue. CLINICAL RELEVANCE: The MPS I canine IA-rhIDUA results suggest that clinical studies should be performed to determine if IA-rhIDUA is a viable approach to ameliorating refractory orthopedic disease in human MPS I.

Zyflamend, a polyherbal mixture, down regulates class I and class II histone deacetylases and increases p21 levels in castrate-resistant prostate cancer cells.

BACKGROUND: Zyflamend, a mixture containing extracts of ten herbs, has shown promise in a variety of preclinical cancer models, including prostate cancer. The current experiments were designed to investigate the effects of Zyflamend on the expression of class I and II histone deacetylases, a family of enzymes known to be over expressed in a variety of cancers. METHODS: CWR22Rv1 cells, a castrate-resistant prostate cancer cell line, were treated with Zyflamend and the expression of class I and II histone deacetylases, along with their downstream target the tumor suppressor gene p21, was investigated. Involvement of p21 was confirmed with siRNA knockdown and over expression experiments. RESULTS: Zyflamend down-regulated the expression of all class I and II histone deacetylases where Chinese goldthread and baikal skullcap (two of its components) appear to be primarily responsible for these results. In addition, Zyflamend up regulated the histone acetyl transferase complex CBP/p300, potentially contributing to the increase in histone 3 acetylation. Expression of the tumor suppressor gene p21, a known downstream target of histone deacetylases and CBP/p300, was increased by Zyflamend treatment and the effect on p21 was, in part, mediated through Erk1/2. Knockdown of p21 with siRNA technology attenuated Zyflamend-induced growth inhibition. Over expression of p21 inhibited cell growth and concomitant treatment with Zyflamend enhanced this effect. CONCLUSIONS: Our results suggest that the extracts of this polyherbal combination increase histone 3 acetylation, inhibit the expression of class I and class II histone deacetylases, increase the activation of CBP/p300 and inhibit cell proliferation, in part, by up regulating p21 expression.

Azoxymethane-induced colon carcinogenesis in mice occurs independently of de novo thymidylate synthesis capacity.

Folate metabolism affects DNA synthesis, methylation, mutation rates, genomic stability, and gene expression, which are altered in colon cancer. Serine hydroxymethyltransferase 1 (SHMT1) regulates thymidylate (dTMP) biosynthesis and uracil accumulation in DNA, and as such affects genome stability. Previously, we showed that decreased SHMT1 expression in Shmt1 knockout mice (Shmt1(-/+)) or its impaired nuclear localization, as occurs in mice over-expressing an Shmt1 transgene (Shmt1(tg+)), results in elevated uracil incorporation into DNA, which could affect colon cancer risk. We used these 2 models to determine the effect of altered SHMT1 expression and localization, and its interaction with folate insufficiency, on azoxymethane (AOM)-induced colon cancer in mice. Shmt1(-/+) and Shmt1(tg+) mice were weaned to a control or folate-and-choline-deficient (FCD) diet and fed the diet for 28 or 32 wk, respectively. At 6 wk of age, mice were injected weekly for 6 wk with 10 mg/kg AOM (w/v in saline). Colon uracil concentrations in nuclear DNA were elevated 2-7 fold in Shmt1(-/+) and Shmt1(tg+) mice. However, colon tumor incidence and numbers were not dependent on SHMT1 expression in Shmt1(-/+) or Shmt1(-/-) mice. The FCD diet reduced tumor load independent of Shmt1 genotype. In contrast, Shmt1(tg+) mice exhibited a 30% reduction in tumor incidence, a 50% reduction in tumor number, and a 60% reduction in tumor load compared with wild-type mice independent of dietary folate intake. Our data indicate that uracil accumulation in DNA does not predict tumor number in AOM-mediated carcinogenesis. Furthermore, enrichment of SHMT1 in the cytoplasm, as observed in Shmt1(tg+) mice, protects against AOM-mediated carcinogenesis independent of its role in nuclear de novo dTMP biosynthesis.

Islet Insulin Secretion, β-Cell Mass, and Energy Balance in a Polygenic Mouse Model of Type 2 Diabetes With Obesity

Abstract Type 2 diabetes (T2D) and obesity are polygenic metabolic diseases, highly prevalent in humans. The TALLYHO/Jng (TH) mouse is a polygenic model of T2D and obesity that encompasses many aspects of the human conditions. In this study, we investigated the key metabolic components including β-cell physiology and energy balance involved in the development of diabetes and obesity in TH mice. Glucose-stimulated insulin secretion from freshly isolated islets was significantly enhanced in TH mice compared with normal C57BL/6 (B6) mice, similar to the compensated stage in human T2D associated with obesity. This increased glucose responsiveness was accompanied by an increase in total β-cell mass in TH mice. Energy expenditure and locomotor activity were significantly reduced in TH mice compared with B6 mice. Food intake was comparable between the two strains but water intake was more in TH mice. Together, obesity in TH mice does not appear to be due to hyperphagia, and TH mice may be a genetic model for T2D with obesity, allowing study of the important signaling or metabolic pathways leading to compensatory increases in insulin secretion and β-cell mass in insulin resistance.

The involvement of endoplasmic reticulum stress in the suppression of colorectal tumorigenesis by tolfenamic acid.

The nonsteroidal anti-inflammatory drug tolfenamic acid has been shown to suppress cancer cell growth and tumorigenesis in different cancer models. However, the underlying mechanism by which tolfenamic acid exerts its antitumorigenic effect remains unclear. Previous data from our group and others indicate that tolfenamic acid alters expression of apoptosis- and cell-cycle arrest-related genes in colorectal cancer cells. Here, we show that tolfenamic acid markedly reduced the number of polyps and tumor load in APC(min)(/+) mice, accompanied with cyclin D1 downregulation in vitro and in vivo. Mechanistically, tolfenamic acid promotes endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) signaling pathway, of which PERK-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) induces the repression of cyclin D1 translation. Moreover, the PERK-eIF2α-ATF4 branch of the UPR pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells, as silencing ATF4 attenuates tolfenamic acid-induced apoptosis. Taken together, these results suggest ER stress is involved in tolfenamic acid-induced inhibition of colorectal cancer cell growth, which could contribute to antitumorigenesis in a mouse model.

Zyflamend, a combination of herbal extracts, attenuates tumor growth in murine xenograft models of prostate cancer.

Prostate cancer (PrC) is the second deadliest cancer of males in the United States Hormone deprivation therapy (HDT), a common therapy for advanced forms of the disease, results in tumor regression; unfortunately, tumors inevitably become castrate-resistant. Diet is not an appropriate primary therapy for refractory forms of the disease; however, diet may be effective as an adjuvant to HDT, potentially extending the latency period and delaying relapse and/or inhibiting refractory growth. Zyflamend® is a combination of extracts from multiple herbs, each with reported anticancer properties. Zyflamend can inhibit growth of various PrC cell lines, but no studies have investigated its potential use in vivo using a model of castrate-resistant PrC. In this study, oral doses of Zyflamend at human equivalent doses inhibited androgen-dependent and castrate-resistant tumor growth in a mouse model that mimics advanced stages of the disease, and reduced the expression of a number of biomarkers linked to PrC progression including pAKT, prostate specific antigen, histone deacetylases, and androgen receptor. In summary, this is the first article to report that Zyflamend, when provided at human equivalent doses, can potentiate the effects of hormone deprivation on tumor regression and growth inhibition of androgen-dependent and castrate-resistant PrC tumors in vivo.

Zyflamend reduces the expression of androgen receptor in a model of castrate-resistant prostate cancer.

Prostate cancer is the most commonly diagnosed solid malignancy, and tumor cells eventually transform to castrate resistance through multiple pathways including activation of the androgen receptor via insulin-like growth factor receptor (IGF-1R) signaling involving phospho-AKT (pAKT). In this study, a mixture of herbal extracts, Zyflamend®, was used as a treatment in a model of castrate-resistant prostate cancer using CWR22Rv1 cells. Zyflamend reduced androgen receptor and IGF-1R expression along with a reduction of IGF-1-mediated proliferation of CWR22Rv1 cells. IGF-1 induced downstream AKT phosphorylation; however, the induction of pAKT was not associated with androgen receptor expression. Further, constitutively active form of AKT had no effect on nuclear expression of androgen receptor, indicating that upregulation of pAKT did not promote androgen receptor expression or nuclear translocation in castrate-resistant CWR22Rv1 cells. Conversely, Zyflamend reduced androgen receptor expression following IGF-1 stimulation and in cells overexpressing pAKT. These results demonstrated that Zyflamend inhibited IGF-1-stimulated cell growth, IGF-1R expression, and androgen receptor expression and its nuclear localization, but these effects were not dependent upon phosphatidylinositol 3-kinase/pAKT signaling. In conclusion, Zyflamend decreased cell proliferation and inhibited IGF-1R and androgen receptor expression in a phosphatidylinositol 3-kinase/pAKT independent manner.

Anthropometric variables accurately predict dual energy x-ray absorptiometric-derived body composition and can be used to screen for diabetes.

The current world-wide epidemic of obesity has stimulated interest in developing simple screening methods to identify individuals with undiagnosed diabetes mellitus type 2 (DM2) or metabolic syndrome (MS). Prior work utilizing body composition obtained by sophisticated technology has shown that the ratio of abdominal fat to total fat is a good predictor for DM2 or MS. The goals of this study were to determine how well simple anthropometric variables predict the fat mass distribution as determined by dual energy x-ray absorptometry (DXA), and whether these are useful to screen for DM2 or MS within a population. To accomplish this, the body composition of 341 females spanning a wide range of body mass indices and with a 23% prevalence of DM2 and MS was determined using DXA. Stepwise linear regression models incorporating age, weight, height, waistline, and hipline predicted DXA body composition (i.e., fat mass, trunk fat, fat free mass, and total mass) with good accuracy. Using body composition as independent variables, nominal logistic regression was then performed to estimate the probability of DM2. The results show good discrimination with the receiver operating characteristic (ROC) having an area under the curve (AUC) of 0.78. The anthropometrically-derived body composition equations derived from the full DXA study group were then applied to a group of 1153 female patients selected from a general endocrinology practice. Similar to the smaller study group, the ROC from logistical regression using body composition had an AUC of 0.81 for the detection of DM2. These results are superior to screening based on questionnaires and compare favorably with published data derived from invasive testing, e.g., hemoglobin A1c. This anthropometric approach offers promise for the development of simple, inexpensive, non-invasive screening to identify individuals with metabolic dysfunction within large populations.

Glycosaminoglycan storage in neuroanatomical regions of mucopolysaccharidosis I dogs following intrathecal recombinant human iduronidase.

Intrathecal (IT) recombinant human α-l-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in mucopolysaccharidosis I (MPS I) dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean: 2.36 ± 0.54 µg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following IT treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared with untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.

Arterial pathology in canine mucopolysaccharidosis-I and response to therapy.

Mucopolysaccharidosis-I (MPS-I) is an inherited deficiency of α-L-iduronidase (IdU) that causes lysosomal accumulation of glycosaminoglycans (GAG) in a variety of parenchymal cell types and connective tissues. The fundamental link between genetic mutation and tissue GAG accumulation is clear, but relatively little attention has been given to the morphology or pathogenesis of associated lesions, particularly those affecting the vascular system. The terminal parietal branches of the abdominal aorta were examined from a colony of dogs homozygous (MPS-I affected) or heterozygous (unaffected carrier) for an IdU mutation that eliminated all enzyme activity, and in affected animals treated with human recombinant IdU. High-resolution computed tomography showed that vascular wall thickenings occurred in affected animals near branch points, and associated with low endothelial shear stress. Histologically these asymmetric 'plaques' entailed extensive intimal thickening with disruption of the internal elastic lamina, occluding more than 50% of the vascular lumen in some cases. Immunohistochemistry was used to show that areas of sclerosis contained foamy (GAG laden) macrophages, fibroblasts and smooth muscle cells, with loss of overlying endothelial basement membrane and claudin-5 expression. Lesions contained scattered cells expressing nuclear factor-κß (p65), increased fibronectin and transforming growth factor ß-1 signaling (with nuclear Smad3 accumulation) in comparison to unaffected vessels. Intimal lesion development and morphology was improved by intravenous recombinant enzyme treatment, particularly with immune tolerance to this exogenous protein. The progressive sclerotic vasculopathy of MPS-I shares some morphological and molecular similarities to atherosclerosis, including formation in areas of low shear stress near branch points, and can be reduced or inhibited by intravenous administration of recombinant IdU.

Shmt1 heterozygosity impairs folate-dependent thymidylate synthesis capacity and modifies risk of Apc(min)-mediated intestinal cancer risk.

Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate, and S-adenosylmethionine, the primary cellular methyl donor. Impairments in folate metabolism diminish cellular methylation potential and genome stability, which are risk factors for colorectal cancer (CRC). Cytoplasmic serine hydroxymethyltransferase (SHMT1) regulates the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis. Therefore, changes in SHMT1 expression enable the determination of the specific contributions made by thymidylate and S-adenosylmethionine biosynthesis to CRC risk. Shmt1 hemizygosity was associated with a decreased capacity for thymidylate synthesis due to downregulation of enzymes in its biosynthetic pathway, namely thymidylate synthase and cytoplasmic thymidine kinase. Significant Shmt1-dependent changes to methylation capacity, gene expression, and purine synthesis were not observed. Shmt1 hemizygosity was associated with increased risk for intestinal cancer in Apc(min)(/+) mice through a gene-by-diet interaction, indicating that the capacity for thymidylate synthesis modifies susceptibility to intestinal cancer in Apc(min)(/+) mice.

Ocular lesions in canine mucopolysaccharidosis I and response to enzyme replacement therapy.

PURPOSE: Mucopolysaccharidosis I (MPS I) is an inherited metabolic disorder resulting from deficiency of α-L-iduronidase and lysosomal accumulation of glycosaminoglycans (GAG) in multiple tissues. Accumulation of GAG in corneal stromal cells causes corneal opacity and reduced vision. The purpose of this study was to determine the extent of ocular GAG accumulation and investigate the effectiveness of intravenous enzyme replacement therapy (ERT) on corneal GAG accumulation in dogs. METHODS: Ocular tissues were obtained from 58 dogs with mucopolysaccharidosis I and four unaffected controls. Affected dogs received either low-dose ERT, high-dose ERT, or no treatment; some low-dose dogs also received intrathecal treatments. Histologic severity of corneal stromal GAG accumulation was scored. RESULTS: Accumulation of GAG was found in corneal stromal cells and scleral fibroblasts but not in corneal epithelium, endothelium, ciliary epithelium, choroid, retina, retinal pigment epithelium, or optic nerve. Corneal GAG accumulation increased in severity with increasing age. Although low-dose ERT did not significantly reduce corneal stromal GAG accumulation in comparison with untreated animals, high-dose ERT did result in significantly less GAG accumulation compared with the untreated dogs (adjusted P = 0.0143) or the low-dose ERT group (adjusted P = 0.0031). Intrathecal treatments did not significantly affect GAG accumulation. Dogs that began ERT shortly after birth also had significantly less (P < 0.0001) GAG accumulation in the corneal stroma than dogs with a later onset of treatment. CONCLUSIONS: These data suggest that high-dose, intravenous ERT is effective at preventing and/or clearing corneal stromal GAG accumulation, particularly if initiated early after birth.

Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.

The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.

Replacing the enzyme alpha-L-iduronidase at birth ameliorates symptoms in the brain and periphery of dogs with mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by loss of activity of α-l-iduronidase and attendant accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current treatments are suboptimal and do not address residual disease including corneal clouding, skeletal deformities, valvular heart disease, and cognitive impairment. We treated neonatal dogs with MPS I with intravenous recombinant α-l-iduronidase replacement therapy at the conventional 0.58 mg/kg or a higher 1.57 mg/kg weekly dose for 56 to 81 weeks. In contrast to previous results in animals and patients treated at a later age, the dogs failed to mount an antibody response to enzyme therapy, consistent with the induction of immune tolerance in neonates. The higher dose of enzyme led to complete normalization of lysosomal storage in the liver, spleen, lung, kidney, synovium, and myocardium, as well as in the hard-to-treat mitral valve. Cardiac biochemistry and function were restored, and there were improvements in skeletal disease as shown by clinical and radiographic assessments. Glycosaminoglycan levels in the brain were normalized after intravenous enzyme therapy, in the presence or absence of intrathecal administration of recombinant α-l-iduronidase. Histopathological evidence of glycosaminoglycan storage in the brain was ameliorated with the higher-dose intravenous therapy and was further improved by combining intravenous and intrathecal therapy. These findings argue that neonatal testing and early treatment of patients with MPS I may more effectively treat this disease.

Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I.

Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.

MCC-555-induced NAG-1 expression is mediated in part by KLF4.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in cell differentiation, metabolism and tumorigenesis. We have investigated the therapeutic properties of 5-[[6-[(2-fluorophenyl)-methoxy]-2-napthalenyl]-methyl]-2,4-thiazolidinedione (MCC-555) a PPARgamma agonist in human colorectal cancer cells. To elucidate the molecular mechanism(s), by which MCC-555 exerts its effects on the human colorectal cancer cells, we have analyzed the expression of two pro-apoptotic proteins, Krüppel-like factor 4 (KLF4) and nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1). MCC-555-induced expression of the transcription factor, KLF-4 was blocked by a PPARgamma specific antagonist GW9662 in PPARgamma-dependent manner in HCT-116 cells. We further identified a new KLF4 target gene NAG-1, which shows a pro-apoptotic activity. We confirmed that PPARgamma agonists-induced NAG-1 expression was abolished using KLF4 siRNA in HCT-116 cells. Subsequently, KLF4 expression enhances the NAG-1 promoter activity in HCT-116 cells, and functional KLF4 binding sites in the NAG-1 promoter were also identified. MCC-555, a PPARgamma agonist induced the expression of Klf4 mRNA and protein in murine intestinal tumors from MCC-555-treated mice, as assessed by RT-PCR and immunohistochemistry. This study shows that PPARgamma agonists up-regulate KLF4 expression in receptor-dependent manner, and KLF4 was identified as a novel transcription factor that controls NAG-1 promoter activity in human and mouse colorectal cancers.

Peroxisome proliferator-activated receptor ligand MCC-555 suppresses intestinal polyps in ApcMin/+ mice via extracellular signal-regulated kinase and peroxisome proliferator-activated receptor-dependent pathways.

A large body of studies has suggested that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, such as thiazolidinedione, are potent candidates for chemopreventive agents. MCC-555 is a PPARgamma/alpha dual agonist and has been shown previously to induce apoptosis in vitro; however, the molecular mechanisms by which MCC-555 affects antitumorigenesis in vivo are poorly understood. In this study, we explored the antitumorigenic effects of MCC-555 both in cell culture and in Apc-deficient mice, an animal model for human familial adenomatous polyposis. MCC-555 increased MUC2 expression in colorectal and lung cancer cells, and treatment with the PPARgamma antagonist GW9662 revealed that MUC2 induction by MCC-555 was mediated in a PPARgamma-dependent manner. Moreover, MCC-555 increased transcriptional activity of human and mouse MUC2 promoters. Subsequently, treatment with MCC-555 (30 mg/kg/d) for 4 weeks reduced the number of small intestinal polyps to 54.8% of that in control mice. In agreement with in vitro studies, enhanced Muc2 expression was observed in the small intestinal tumors of Min mice treated with MCC-555, suggesting that MUC2 expression may be associated at least in part with the antitumorigenic action of MCC-555. In addition, highly phosphorylated extracellular signal-regulated kinase (ERK) was found in the intestinal tumors of MCC-555-treated Min mice, and inhibition of the ERK pathway by a specific inhibitor markedly suppressed MCC-555-induced Muc2 expression in vitro. Overall, these results indicate that MCC-555 has a potent tumor suppressor activity in intestinal tumorigenesis, likely involving MUC2 up-regulation by ERK and PPARgamma pathways.

Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I.

Mucopolysaccharidoses (MPSs) are lysosomal storage diseases caused by a deficit in the enzymes needed for glycosaminoglycan (GAG) degradation. Enzyme replacement therapy with recombinant human alpha-L-iduronidase successfully reduces lysosomal storage in canines and humans with iduronidase-deficient MPS I, but therapy usually also induces antibodies specific for the recombinant enzyme that could reduce its efficacy. To understand the potential impact of alpha-L-iduronidase-specific antibodies, we studied whether inducing antigen-specific immune tolerance to iduronidase could improve the effectiveness of recombinant iduronidase treatment in canines. A total of 24 canines with MPS I were either tolerized to iduronidase or left nontolerant. All canines received i.v. recombinant iduronidase at the FDA-approved human dose or a higher dose for 9-44 weeks. Nontolerized canines developed iduronidase-specific antibodies that proportionally reduced in vitro iduronidase uptake. Immune-tolerized canines achieved increased tissue enzyme levels at either dose in most nonreticular tissues and a greater reduction in tissue GAG levels, lysosomal pathology, and urinary GAG excretion. Tolerized MPS I dogs treated with the higher dose received some further benefit in the reduction of GAGs in tissues, urine, and the heart valve. Therefore, immune tolerance to iduronidase improved the efficacy of enzyme replacement therapy with recombinant iduronidase in canine MPS I and could potentially improve outcomes in patients with MPS I and other lysosomal storage diseases.

A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer.

BACKGROUND & AIMS: Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer. METHODS: We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment. RESULTS: The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression. CONCLUSIONS: The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.