Levels Of Lysosomal Enzymes Research Articles

Anti-inflammatory Effect of Total Saponin Fraction from Dioscorea nipponica Makino on Gouty Arthritis and Its Influence on NALP3 Inflammasome.

To investigate the mechanism of Chinese herbal medicine Dioscorea nipponica for the treatment of monosodium urate crystals-induced gouty arthritis (GA) in rats. Sixty male Wistar rats were divided into 6 groups: normal, model, indomethacin and three total saponin (900, 300 and 100 mg/kg) groups. The liver, kidney and serum levels of lysosomal enzymes, antioxidant capacities, and inflammatory factors were measured. In addition, the mRNA and protein levels of the NALP3 inflammasome components in the mononuclear cells of rats' peripheral blood were analyzed using real-time polymerase chain reaction and Western blotting methods, respectively. Total saponins groups could reduce the activities of β-galactosidase, β-N acetyl glucosamine enzyme, β-glucuronidase, acid phosphatase, and malonaldehyde as well as the contents of TNF-α, IL-1β and IL-8 (all P<0.05). They could also increase the activities of glutathione peroxidase and total superoxide dismutase (both P<0.05). Further studies showed that total saponins groups of high, middle and low doses could all increase the mRNA and protein levels of caspase-1, adapter apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and NALP3 in the mononuclear cells of peripheral blood (all P<0.05). Dioscorea nipponica may treat GA by regulating lysosomal enzymes, antioxidant capacities and the NALP3 inflammasome.

Clinical, biochemical and molecular characterization of Korean patients with mucolipidosis II/III and successful prenatal diagnosis

BackgroundMucolipidosis types II and III (ML II/III) are autosomal recessive disorders caused by a deficiency in the lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We investigated the molecular genetic characteristics of the GNPTAB gene, which codes for the alpha/beta subunits of a phosphotransferase, in Korean ML II/III patients. We included prenatal tests and evaluated the spectrum of mutations in East Asian populations with ML II/III through a literature review.MethodsSeven patients from six families were enrolled in the study including two prenatal tests using chorionic villi samples. A diagnosis of ML II/III was made based on clinical findings and increases in serum lysosomal enzyme levels. PCR and direct sequencing were performed to identify GNPTAB mutations.ResultsWe found 14 mutant alleles including seven known mutations of c.2189delT (p.Leu730fs*7), c.1090C > T (p.Arg364*), c.2681G > A (p.Trp894*), c.3565C > T (p.Arg1189*), c.310C > T (p.Gln104*), c.1071G > A (p.Trp357*) and c.2574_2575delGA (p.Asn859Glnfs*2). Four were novel variants of unknown significance: c.992A > G (p.Tyr331Cys), c.2666 T > A (p.Leu889*), c.637-6 T > G (p.Thr213Phefs*11), and c.471_472delTT (p.Tyr158Serfs*8). Family studies revealed the probands to be compound heterozygotes. The fetuses carried the same GNPTAB mutations as the mucolipidosis II/III probands in the prenatal diagnosis.ConclusionsWe identified GNPTAB mutations in all patients with ML II/III, but did not identify a hot spot in Korean patients. We successfully performed prenatal diagnosis using molecular investigation.

Efficacy of CoenzymeQ10 in inhibiting monosodium urate crystal-induced inflammation in rats

Gout is a type of arthritis, which could result from the deposition of monosodium urate crystals in joints. It can cause redness, burning pain, inflammation of joints especially in big toe. In this study, we have looked for anti-arthritic effect of coenzyme Q10 (CoQ10) on monosodium urate crystal-induced inflammation in rats and compared it with that of the non-steroidal anti-inflammatory drug, indomethacin. The evaluation was done by measuring the paw volume, antioxidant status, lipid peroxidation, lysosomal enzymes (β-glcuronidase, β-galactosidase, N-acetyl-β-d-glucosaminidase, acid phosphatase) activities and histopathological studies. Paw volume, the levels of lysosomal enzymes, lipid peroxidation were significantly (P<0.05) increased and the antioxidant activity status was in turn decreased in monosodium urate crystal-induced rats. CoQ10 (10mg/kg/b.w. orally) treated monosodium urate crystal-induced rats showed near normal activities of lysosomal enzymes, reduced levels of lipid peroxidation, near normal paw volume and antioxidant status. CoQ10 was also able to minimize mononuclear cell infiltration and damage to articular cartilage. Current study indicates that CoQ10 possesses anti-inflammatory effect against gouty arthritis and can be used to treat acute form of gouty arthritis.

Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation.

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG2), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.

Chitotriosidase and lysosomal enzymes as potential biomarkers of disease progression in amyotrophic lateral sclerosis: A survey clinic-based study

The aim of this study was to determine if blood chitotriosidase (Chit) activity and lysosomal enzyme levels might represent markers of disease activity and progression in amyotrophic lateral sclerosis (ALS).It is a survey clinic-based study performed in a tertiary ALS centre. Blood samples were obtained from 76 patients with ALS in different stages of the disease and from 106 healthy individuals serving as controls. Chit activity and the levels of acid alpha-glucosidase, acid alpha-galattosidase A, beta-glucocerebrosidase, and alpha-l-iduronidase were detected using the dried blood spots (DBS) technique. The CHIT1 genotype for exon 10 duplication and for the p.G102S variant was also determined. Chit activity was significantly higher in ALS patients than in healthy individuals. This difference was independent of the genotypes at CHIT1 functional variants. Chit were significantly higher in 34 rapidly progressing patients as compared to 42 with slowly progressive disease. Acid alpha-glucosidase was higher than normal and significantly correlated with the severity of the disease. Glucocerebrosidase and alpha-l-iduronidase activity were significantly lower in patients than in the controls. Alpha-galactosidase A was higher than normal only in rapidly progressing patients.We have employed a very simple and affordable laboratory test to measure blood Chit and lysosomal enzymes activity which could be easily included in the screening of ALS patients recruited in clinical trials. Remarkably, high levels of chitinase and alpha-galactosidase A could help to distinguish patients with fast progression from those with slow progression of the disease and possibly to follow the effects of treatments on neuroinflammation and autophagy.

Abstract 3185: Sub-lethal autolysis in livers of mice given phthalate esters precedes development of hepatic lesions

Abstract Primary prevention of human cancers through identification of potential environmental carcinogens is an important goal. Phthalates such as di(2-ethylhexyl)phthalate (DEHP) and di-n-octylphthalate (DOP), the most widely used industrial plasticizers, are low-level environmental contaminants. As plasticizers they impart flexibility to plastics and comprise a large portion of the final product. However, they are non-covalently bound and easily migrate out of the final products, leading to human exposure. Previous studies have shown that exposure to these compounds in the diet is associated with the development of hepatocellular carcinomas and adenomas in rodents. DEHP and DOP are most likely non-genotoxic, therefore the mechanism(s) by which these compounds induce changes in hepatocyte structure and function is unknown, although recently a peroxisome proliferator-activated receptor alpha mode of action has been suggested for DEHP. Bosmann (BBA, 264:339, 1972) proposed that sub-lethal autolysis at the cell surface resulting from secretion of lysosomal enzymes can alter cell-cell and cell-matrix interactions leading to loss of growth control and neoplastic transformation. Indeed, cultured 3T3 cells express increased levels of lysosomal enzymes following viral transformation and increased expression of lysosomal enzymes is found in many human malignancies including cancers of the breast, colon, larynx and prostate. To determine if there is an association between the expression of lysosomal enzymes and the carcinogenicity of phthalates, the effects of chronic exposure of male B6C3F1 mice to diets containing DEHP or DOP on liver lysosomal enzymes was evaluated. Mice were fed diets containing 0.12%, 0.60%, and 1.20% DEHP and 0.1%, 0.5% and 1.0% DOP. Groups of animals were sacrificed after 15, 30, 35, 52, 78 and 104 weeks of exposure. Expression of hepatic β-galactosidase, aryl sulfatase, n-acetyl-β-glucosaminidase, and cathepsin D was increased significantly following 15 weeks of treatment with either 0.6% or 1.2% DEHP. By 30 weeks of treatment animals in these groups had focal hepatic lesions and some liver tumors. Increased expression of β-glucuronidase was found only in animals receiving 0.6% and 1.2% DEHP for 52 weeks. Hepatomegaly was found in all the animals receiving the phthalates. The close association between early increases in expression of some lysosomal enzymes and later development of hepatic lesions following chronic exposure to non-genotoxic phthalate esters suggests that sub-lethal autolyis may be a contributing factor in carcinogenesis induced by these low-level environmental contaminants. Citation Format: Julia H. Carter, Steven Vogelpohl, Mary Kay Tucker, Robert Tagher. Sub-lethal autolysis in livers of mice given phthalate esters precedes development of hepatic lesions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2014-3185

Pitfalls in the prenatal diagnosis of mucolipidosis II alpha/beta: A case report

Mucolipidosis II alpha/beta is an autosomal recessive disorder caused by deficient activity of GlcNAc-1-phosphotransferase. We report the prenatal diagnosis of a fetus who was found to exhibit normal levels of lysosomal enzymes in the amniotic fluid but low levels in amniocytes, and who was found to be heterozygous for the most common GNPTAB mutation. As in some carriers of Mucolipidosis II biochemical abnormalities may hinder prenatal diagnosis, we suggest DNA analysis should be performed whenever possible.

Protective effect of administration of Withania somifera against bromobenzene induced nephrotoxicity and mitochondrial oxidative stress in rats

Background: The present study was conducted to elucidate the protective role of Withania somnifera against bromobenzene induced nephrotoxicity and mitochondrial dysfunction in rats. Methods: In this study, Wistar albino rats of either sex were divided into six groups consisting of six animals each. The first one was control, those in group II received bromobenzene (10 mmol/kg, intragastric intubation) once, but group III and IV animals received W. somnifera (250 and 500 mg/kg, orally), respectively for 8 days followed by bromobenzene once on the 8th day and silymarin (100 mg/kg, orally) was given for 8 days to group V animals and then bromobenzene on the last day. Group VI animals received only W. somnifera (500 mg/kg) for 8 days. Results: The levels of renal lipid peroxidation, lysosomal enzymes and glycoprotein were increased significantly (p < 0.05) in the bromobenzene alone treated rats and antioxidant status and mitochondrial enzymes were found to be decreased, when compared to the control group. The levels of kidney functional markers (urea, uric acid and creatinine) were also found to be abnormal in serum of bromobenzene alone treated rats. On the other hand, administration of W. somnifera (250 and 500 mg/kg) along with bromobenzene offered a significant dose-dependent protection to the biochemical alterations as observed in the bromobenzene alone treated rats, which was also evidenced by histopathology. Conclusion: Thus, the oral administration of W. somnifera significantly protected against the bromobenzene induced nephrotoxicity and renal mitochondrial dysfunction in rats.

Urinary lysosomal enzyme excretion in pregnant women with hypertensive disorders

Background: The authors assessed proximal renal tubular dysfunction and/or damage in pregnant women with various types of hypertension by measuring the three urinary lysosomal enzyme levels: N-acetyl-β-d-glucosaminidase (NAG), arylsulfatase A and β-glucuronidase. Methods: The study consisted of 120 pregnant women divided into four groups: 41 women in 20th week of gestation or more, with pregnancy-induced hypertension (PIH group), 28 pregnant women after 20 weeks of pregnancy with pre-eclampsia (PE group), 21 pregnant women with chronic hypertension, identified before 20th week of pregnancy (CH group) and 30 healthy, pregnant women (healthy controls (HC) group). Results: Statistical analysis showed significantly higher levels of all the three of lysosomal enzymes in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and the ones with chronic hypertension. Additionally, significantly higher values of NAG were found in the group of pregnant women with PIH compared with healthy pregnancies. No correlation was found between the concentration of enzymes in urine and values of blood pressure in any of the analyzed groups of pregnant women. Conclusions: The authors conclude that higher values of all the studied enzymes in PE group, in the comparison with the other groups, indicate proximal tubular damage at the cellular level. The lack of correlation between the concentration of lysosomal enzymes and blood pressure suggests that the damage to these parts of kidney is complex. In addition, mechanisms other than hypertension realizing intracellular enzymes may be involved in this process.

Chromatographic finger print analysis and lysosomal membrane stabilisation activity of active fraction of Alstonia scholaris leaf extract in arthritic rats

Object: The present study was aimed to assess the anti‑arthritic activity of chloroform fraction of Alstonia scholaris leaf extract against Freund’s complete adjuvant (FCA)‑induced arthritis in rats. Materials and Methods: The anti‑inflammatory activity of various fractions of ethanolic extract of Alstonia scholaris at concentration of 100 mg/kg was studied using the carrageenan‑induced inflammatory models. The chloroform fraction shows significant anti‑inflammatory activity. The chloroform fraction was further studied for anti‑arthritic activity and HPTLC fingerprint analysis. For anti‑arthritic activity, the active chloroform fraction was administered at the concentrations of 50 and 100 mg/kg body weight. The effect of chloroform fraction on liver ALP, ACP and LDH levels of lysosomal enzymes of FCA arthritic animals were studied. Indomethacin and prednisolone (10 mg/kg) was used as standard. HPTLC studies were carried out using CAMAG HPTLC system equipped with linomat IV applicator, TLC scanner; Reprostar 3 and WIN CATS‑4 software were used. Results: The chloroform fraction at 100 mg/kg, showed maximum inhibition (34.16%) of inflammation induced by carrageenan. In FCA‑induced arthritis, the chloroform fraction showed a highly significant reduction in paw volume (50 mg/kg-72.71%; 100 mg/kg-74.35%). The levels of lysosomal enzymes were significantly decreased in the chloroform fraction‑treated groups. Conclusion: The possible mechanism of action of the chloroform fraction of Alstonia scholaris leaf extract may be through its stabilising action on lysosomal membranes. Future studies will provide new insights into the anti‑arthritic activity of Alstonia scholaris and isolation of compound from it may eventually lead to development of a new class of anti‑arthritic agent. Key words: Alstonia scholaris, athritis, chloroform fraction, lysosomal membrane stabilisation

Therapeutic effect of Ananus comosus peel on breast cancer induced by 7, 12- dimethylbenz(α)anthracene

Ananus comosus , has demonstrated a wide variety of biological activities which make it a good plant source for the treatment of many oxidative stress mediated diseases. The present study was aimed to evaluate therapeutic potential by assaying the activities of liver marker and lysosomal enzymes in 7, 12 dimethylbenz(α)anthracene induced mammary cancer bearing rats. Animals were divided into five groups of six numbers each. Group 1 served as control, group 2 induced mammary carcinogenesis, group 3 and 4 were treated with plant extract and tamoxifen and group 5 served as animals treated with plant extract alone. All the animals were sacrificed after 30 days treatment, serum and mammary tissue are used for the analysis of liver marker and lysosomal marker enzymes using standard protocols. The rats were induced with mammary cancer by DMBA showed altered the levels of liver markers and lysosomal marker enzymes in serum and mammary tissue. On the other hand, oral administrations of ethanolic extract of Ananus comosus peel and standard drug tamoxifen to mammary cancer bearing rats for 30 days, those levels were brought back to near normal. The histology of mammary tissues inevitably supports the biochemical alterations and this was attributed due to the interaction of Ananus comosus peel through the induction or inhibition of metabolism and also the modulating property in the marker and lysosomal enzymes.

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.

Anti-inflammatory and anti-arthritic effects of 3-hydroxy, 2-methoxy sodium butanoate from the leaves of Clerodendrum phlomidis L.f.

The leaves of Clerodendrum phlomidis L.f. have been used in the Indian traditional system of medicine to treat several inflammatory diseases and arthritis. The aim of the present study was to assess the anti-inflammatory and anti-arthritic activities of the leaves of C. phlomidis and to isolate the active principle by bioactivity guided fractionation. To find the anti-inflammatory constituents from this plant, fractionations were performed with concurrent bioassays. Carrageenan-induced inflammation and Freund complete adjuvant (FCA)-induced arthritic rat models were used. The anti-inflammatory and anti-arthritic activities of the isolated compound were studied by assessing the histology of the joints, levels of lysosomal enzymes, protein-bound carbohydrates, acute phase protein, etc., in plasma, as well as by estimating the levels and expression of pro-inflammatory cytokines in the joints. Repeated fractionations and bioassays yielded a novel bioactive compound: 3-hydroxy, 2-methoxy-sodium butanoate. Treatment with this compound reduced the paw edema induced by carrageenan and FCA dose dependently. The levels of lysosomal enzymes and protein-bound carbohydrates decreased significantly upon treatment with the compound. The level of plasma acute phase protein was also decreased compared with control animals. Protein levels and mRNA expression of pro-inflammatory cytokines TNF, IL-1 and IL-6 in the joints were decreased significantly in a dose-dependent manner and the histopathological data also added evidence of the anti-arthritic property of the compound. The 3-hydroxy,2-methoxy sodium butanoate isolated from plant leaves displays considerable potency in anti-inflammatory action and has a prominent anti-arthritic effect. This is the first report of this natural compound with bioactivity.

Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a prevalent cause of end-stage renal disease, but the mechanisms underlying progression are unresolved. Lysosomal protein accumulation in the proximal tubule, mediated by megalin and cubilin endocytosis of increased amounts of filtered protein, is thought to result in inflammation and fibrosis. Here we determine whether release of inflammatory and fibrotic mediators in response to protein overload in the proximal tubule is caused by lysosomal enzyme deficits and insufficient proteolysis. As a model of FSGS, we used inducible podocyte-specific podocin-knockout mice analyzed at different time points. The content of megalin and cubilin ligands increased in the lysosomes after onset of proteinuria; however, protein and mRNA levels of megalin and cubilin showed only minor changes. To determine if the elevated lysosomal ligand content was caused by deficiency of enzymes, we analyzed protein and mRNA levels of lysosomal enzymes and found increased endogenous synthesis. Injection of dye-quenched fluorescent and iodinated albumin showed that proteolytic turnover in lysosomes of knockout mice adapted to the increased protein load. Inflammatory and fibrotic signals were increased early in disease, although the majority of lysosomes degraded endocytosed proteins effectively. Thus, insufficient lysosomal degradation in FSGS is not the cause of the inflammation and fibrosis during kidney disease.

P-Coumaric acid, a common dietary polyphenol, protects cadmium chloride-induced nephrotoxicity in rats

The present study was conducted to elucidate the protective role of p-coumaric acid, a common dietary polyphenol against cadmium induced nephrotoxicity in rats. For the purpose of comparison, a standard reference drug silymarin (50 mg/kg b. wt) was used. In this experiment, the animals were divided into four groups, with each consisting of six animals. The animals in Group I animals received saline and served as a control group and those in Group II received cadmium chloride (3 mg/kg b. wt) subcutaneously once daily for 3 weeks, but Group III and IV animals received cadmium chloride followed by p-coumaric acid (100 mg/kg b. wt, oral) and silymarin (50 mg/kg b. wt, oral), respectively, daily for 3 weeks. At the end of the treatment, the animals were sacrificed, and the blood and kidney samples were collected. The results obtained in this study revealed the fact that the levels of lipid peroxidation, lysosomal enzymes, glycoprotein, cadmium and metallothionein were increased in the cadmium chloride alone treated rats and antioxidant status was found to be decreased, when compared to the control group. The levels of kidney functional markers (urea, uric acid and creatinine) were also found to be abnormal in serum and urine of cadmium chloride alone treated rats. On the other hand, the administration of p-coumaric acid along with cadmium chloride significantly protected the biochemical alterations as observed in the cadmium chloride alone treated rats as evidenced by histopathology. Thus, the oral administration of p-coumaric acid significantly protected the cadmium-induced nephrotoxicity in rats.

Trikatu, a herbal compound that suppresses monosodium urate crystal-induced inflammation in rats, an experimental model for acute gouty arthritis.

Gout is an inflammatory joint disorder characterized by hyperuricaemia and precipitation of monosodium urate crystals in the joints. In the present study, we aimed to investigate the anti-inflammatory effect of trikatu, a herbal compound in monosodium urate crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Paw volume and levels/activities of lysosomal enzymes, lipid peroxidation, anti-oxidant status and histopathological examination of ankle joints were determined in control and monosodium urate crystal-induced rats. In addition, analgesic (acetic acid-induced writhing response), anti-pyretic (yeast-induced pyrexia) and gastric ulceration effects were tested. The levels of lysosomal enzymes, lipid peroxidation and paw volume were significantly increased, and anti-oxidant status was found to be reduced in monosodium urate crystal-induced rats, whereas the biochemical changes were reverted to near normal levels upon trikatu (1000 mg/kg b.wt) administration. The trikatu has also been found to exhibit significant analgesic and anti-pyretic effects with the absence of gastric damage. In conclusion, the present results clearly indicated that trikatu exert a potent anti-inflammatory effect against monosodium urate crystal-induced inflammation in rats in association with analgesic and anti-pyretic effects in the absence of gastrointestinal damage.

Gene Therapy for Mucopolysaccharidosis Type VI Is Effective in Cats Without Pre-Existing Immunity to AAV8

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

Pikfyve-Deficient Platelets Mediate Inflammation and Thrombosis by Releasing Aberrant Granules

Abstract 262Platelets play key roles in hemostasis and thrombosis, as well as in non-hemostatic processes, including atherosclerosis and inflammation. Although platelet secretory products are thought to be essential mediators in these processes, the mechanism of cross-talk between platelets and cells of both the vascular and immune systems is poorly understood. Here, we report that the inactivation of a phosphoinositide kinase, PIKFyve, specifically in platelets, leads to the secretion of aberrant platelet granules and tissue infiltration of vacuolated macrophages, thus promoting both inflammation and thrombosis in mice.We recently generated a mouse model with the intent to analyze the role of phosphoinositide synthesis in platelet granule biogenesis. These mice were genetically engineered to ablate platelet-specific PIKFyve kinase activity (PF4Cre+ PIKFyvefl/fl) necessary to synthesize phosphatidylinositol(3,5)P2. The PF4Cre+ PIKFyvefl/fl mice formed aberrant platelet granules, but unexpectedly developed hair loss, body-swelling, osteopenia, and multiorgan failure, leading to death by 5–6 months of age. Necropsy revealed extensive tissue infiltration of large vacuolated macrophages. These findings were associated with accelerated arterial thrombosis in vivo. Because the targeting strategy was not anticipated to directly affect macrophages, we analyzed tissue expression of the PF4-Cre transgene in the PF4Cre+ PIKFyvefl/fl mouse by crossing it with a Cre-dependent LacZ reporter mouse. The PF4-Cre expression was confirmed to be limited to platelets and megakaryocytes, and not in any other cells, including the vacuolated macrophages. This suggests that the macrophage-driven phenotype is mediated by macrophage-extrinsic factors, likely by PIKFyve-null platelets.To examine whether the macrophage phenotype is mediated by hematopoietic cells, PF4 Cre+ PIKFyvefl/fl bone marrow cells were transplanted into lethally irradiated WT mice. The recipient mice engrafted with PF4 Cre+ PIKFyvefl/fl donor cells developed tissue infiltration of vacuolated macrophages in multiple organs, recapitulating the phenotype of PF4 Cre+ PIKFyvefl/fl mice. Conversely, the lethally irradiated PF4 Cre+ PIKFyvefl/fl recipient mice repopulated with WT donor cells completely reverted their hair loss and body swelling, and resolved their tissue accumulation of vacuolated macrophages. Moreover, while PF4 Cre+ PIKFyvefl/fl mice survive for only 5–6 months, the PF4 Cre+ PIKFyvefl/fl mice that were recipients of normal hematopoietic progenitor cells appear to have a normal lifespan. Together, our data demonstrate that the macrophage-driven phenotype is exclusively mediated by hematopoietic cells.To determine whether the released granules from PIKFyve-null platelets mediate the macrophage phenotype, PF4 Cre+ PIKFyvefl/fl mice were crossed with Bloc1−/− mice. The Bloc1 complex is required for vesicle docking and fusion during the biogenesis and secretion of platelet granules. The PF4 Cre+ PIKFyvefl/fl mice that were also homozygous for the Bloc1 mutation did not develop the macrophage-driven pleomorphic phenotype typically seen in PF4 Cre+ PIKFyvefl/fl mice. This suggests that the secretion of granules from PIKFyve-null platelets is required to drive the macrophage response in PF4 Cre+ PIKFyvefl/fl mice. Notably, PIKFyve-null megakaryocytes displayed numerous enlarged vacuoles in the cytoplasm by electron microscopy. In addition, despite normal secretion of the alpha and dense granule cargos, PIKFyve-null platelets contained and released elevated levels of lysosomal enzymes. Although ex vivo study of PIKFyve-null platelet aggregation was normal, in vivo analysis of platelet thrombus formation with a FeCl3-induced carotid artery injury model showed an accelerated formation of arterial thrombus indicating that PF4 Cre+ PIKFyvefl/fl mice were prothrombotic.Collectively, our data show that the loss of PIKFyve kinase activity in platelets results in the formation of aberrant platelet granules with increased release of lysosomal enzymes, and promotes the development of macrophage-driven inflammation and thrombosis. This study provides further evidence that secretion of platelet granules modulates primary immunity, and contributes to a wide range of biological processes that extend beyond hemostasis. Disclosures:No relevant conflicts of interest to declare.

Ameliorative effect of Drynaria quercifolia (L.) J. Sm., an ethnomedicinal plant, in arthritic animals

Drynaria quercifolia (L.) J. Sm., is an ethnomedicinal plant used widely in Tamil Nadu to treat arthritis. The present study was aimed to evaluate the traditional claim of D. quercifolia rhizome water extract in adjuvant induced arthritic animals. Anti-arthritic effect was studied by assessing the levels of lysosomal enzymes, protein bound carbohydrates, urinary degradative collagen and serum cytokines on control and adjuvant induced arthritis. The paw swelling and body weight were also analyzed. The levels of ROS and lysosomal enzymes in neutrophils of control and adjuvant induced animals were also estimated. D. quercifolia rhizome water extract at doses of 100 and 200mg/kg reduced the paw thickness and elevated the mean body weight of arthritic rats. The treatment with extract showed a significant reduction in the levels of plasma and liver lysosomal enzymes as well as protein bound carbohydrates and urinary degradative collagen levels. The treatment reduced the levels of ROS and lysosomal enzymes in neutrophils significantly. The significant reduction in the levels of serum pro-inflammatory cytokines (TNF-α and IL-1β) and the increment in the levels of anti-inflammatory cytokine (IL-10) were also observed by the treatment. The present study supports the traditional claim of using D. quercifolia to treat rheumatism.

Protective Effect of Hesperidin, a Citrus Flavanoglycone, Against γ-Radiation-Induced Tissue Damage in Sprague–Dawley Rats

The present study was designed to evaluate the radioprotective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced cellular damage in the liver, heart, and kidney of rats. Whole-body γ-radiation exposure (5 Gy) of healthy adult rats resulted in cellular damage and oxidative stress manifested as increased levels of serum marker enzymes, lipid peroxidation, and fibrosis in the tissues, accompanied by depletion of cellular glutathione and abnormal alteration in the levels of lysosomal enzymes. Treatment with hesperidin (50 and 100 mg/kg, p.o.) for 7 days was found to offer significant protection against γ-radiation-induced toxicity in the tissues, which was evident by the improved status of most of the parameters investigated. Further, the histological examination of periodic acid-Schiff-stained tissue sections of animals treated with hesperidin following radiation exposure showed minimal necrotic damage with a recovery pattern in a dose-dependent manner compared with radiation-exposed animals. The results of our study show that administration of hesperidin offers effective protection against γ-radiation-induced cellular damage and oxidative stress in rats.