Comparative study on mannose 6-phosphate residue contents of recombinant lysosomal enzymes.

As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT.

SARS-CoV-2 IgG seroprevalence in the Okinawa Main Island and remote islands in Okinawa, Japan, 2020-2021.

We estimated the seroprevalence of anti-SARS-COV-2 IgG in different island groups in Okinawa. A cross-sectional sero-survey was repeated in three periods between July 2020 and February 2021. A total of 2683 serum samples were collected from six referral medical centers, each covering a separate region in Okinawa. In the main island, the seroprevalence was 0.0% (0/392, 95% CI: 0.0-0.9), 0.6% (8/1448, 0.2-1.1), and 1.4% (8/582, 0.6-2.7) at the 1st, 2nd, and 3rd sero-survey, respectively. In the remote islands, the seroprevalence was 0.0% (0/144, 95% CI: 0.0-2.5) and 1.6% (2/123, 0.2-5.8) at the 2nd and 3rd survey, respectively. The case detection ratio was 2.7 (95% CI: 1.3-5.3) in the main island and 2.8 (0.7-11.1) in the remote islands during the 3rd survey. The case detection ratio was the highest in people aged 20-29 years (8.3, 95% CI: 3.3-21.4) in the main island and in those aged 50-59 years (14.1, 2.1-92.7) in the remote islands, suggesting under-reporting of clinical cases by the surveillance system in these subgroups. A sero-survey during an emerging infectious disease epidemic can be useful for validating the reliability of the surveillance system by providing the case detection ratio.

Hyperinsulinemia is an independent predictor for complex atherosclerotic lesion of thoracic aorta in non-diabetic patients.

OBJECTIVE: Hyperinsulinemia is a well known risk factor for cardiovascular event. However, it is not known whether hyperinsulinemia facilitates atherosclerotic complex lesions of aorta in non-diabetic patients. We investigated whether hyperinsulinemia is an independent marker of severity of atherosclerosis in thoracic aorta of non-diabetic patients using multiplane transesophageal echocardiography (TEE). RESEARCH DESIGN AND METHODS: Non-diabetic 90 patients with cardiovascular disease underwent TEE, and were analyzed for plasma insulin levels of oral glucose tolerance test, conventional atherosclerotic risk factors and coronary angiographic features. RESULTS: Thoracic aortic plaques were detected in 84 patients (93%). The complex atherosclerotic lesions were observed in 35 (39%) patients, most frequently at the part of aortic arch (p<0.005), showing the greatest atheroma score in thoracic aorta (p<0.05). Univariate analysis showed age, male gender, smoking, coronary artery disease, HDL-cholesterol, insulin levels in glucose tolerance test and homeostasis model assessment insulin resistance index (HOMA index) were found to be significant predictors of complex atherosclerotic lesions. Multivariate regression analysis revealed that HOMA index was an independent predictor of complex atherosclerotic lesions (odds ratio 1.93, p=0.006). There was a significant positive correlation between HOMA index and the atheroma score of thoracic aorta (p<0.001). CONCLUSIONS: Hyperinsulinemia is an independent predictor of complex atherosclerotic lesions detected by TEE in the thoracic aorta of non-diabetic patients.

Thermal treatment attenuates neointimal thickening with enhanced expression of heat-shock protein 72 and suppression of oxidative stress.

BACKGROUND: The beneficial effects of thermal therapy have been reported in several cardiovascular diseases. However, it is unknown whether the thermal treatment has some beneficial roles against the development of atherosclerosis. METHODS AND RESULTS: The inflammatory arterial lesion was introduced by placement of a polyethylene cuff on femoral arteries of male Sprague-Dawley rats for 4 weeks. Thermal-treated group underwent daily bathing in 41 degrees C hot water for 15 minutes. Neointimal thickening along with immunohistochemical expression of heat-shock proteins (HSPs), monocyte chemoattractant protein-1 (MCP-1), and NADPH oxidase were compared with those of a thermally untreated (Control) group. Morphometric analysis demonstrated a significant suppression of neointimal thickening in thermal-treated group compared with the Control group (intimal/medial area ratios, 0.01+/-0.01 versus 0.31+/-0.04, P<0.01). Expression of MCP-1 and infiltration of ED-positive cells were enhanced in the adventitial layer of Control. More importantly, expression of HSP72 in media was enhanced by thermal treatment. Expression of p22-phox, the major membrane subunit of NADPH oxidase, and MCP-1 was augmented in cuff-injured adventitia of the Control but not the thermal-treated groups. CONCLUSIONS: Thermal treatment significantly attenuated infiltration of inflammatory cells in adventitia and suppressed neointimal thickening in cuff-injured arteries with the enhancement of HSP72 expression and suppression of oxidative stress.

Impaired glutathione redox system paradoxically suppresses angiotensin II-induced vascular remodeling.

BACKGROUND: Angiotensin II (AII) plays a central role in vascular remodeling via oxidative stress. However, the interaction between AII and reduced glutathione (GSH) redox status in cardiovascular remodeling remains unknown. METHODS: In vivo: The cuff-induced vascular injury model was applied to Sprague Dawley rats. Then we administered saline or a GSH inhibitor, buthionine sulfoximine (BSO, 30 mmol/L in drinking water) for a week, subsequently administered 4 more weeks by osmotic pump with saline or AII (200 ng/kg/minute) to the rats. In vitro: Incorporation of bromodeoxyuridine (BrdU) was measured to determine DNA synthesis in cultured rat vascular smooth muscle cells (VSMCs). RESULTS: BSO reduced whole blood GSH levels. Systolic blood pressure was increased up to 215 ± 4 mmHg by AII at 4 weeks (p<0.01), which was not affected by BSO. Superoxide production in vascular wall was increased by AII and BSO alone, and was markedly enhanced by AII+BSO. The left ventricular weight to body weight ratio was significantly increased in AII and AII+BSO as compared to controls (2.52 ± 0.08, 2.50 ± 0.09 and 2.10 ± 0.07 mg/g respectively, p<0.05). Surprisingly, the co-treatment of BSO totally abolished these morphological changes. Although the vascular circumferential wall stress was well compensated in AII, significantly increased in AII+BSO. The anti-single-stranded DNA staining revealed increasing apoptotic cells in the neointima of injured arteries in BSO groups. BrdU incorporation in cultured VSMCs with AII was increased dose-dependently. Furthermore it was totally abolished by BSO and was reversed by GSH monoethyl ester. CONCLUSIONS: We demonstrated that a vast oxidative stress in impaired GSH redox system totally abolished AII-induced vascular, not cardiac remodeling via enhancement of apoptosis in the neointima and suppression of cell growth in the media. The drastic suppression of remodeling may result in fragile vasculature intolerable to mechanical stress by AII.

Analysis of genetic and predisposing factors in Japanese patients with atypical hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Approximately 10% of cases are classified as atypical due to the absence of Shiga toxin-producing bacteria as a trigger. Uncontrolled activation of the complement system plays a role in the pathogenesis of atypical HUS (aHUS). Although many genetic studies on aHUS have been published in recent years, only limited data has been gathered in Asian countries. We analyzed the genetic variants of 6 candidate genes and the gene deletion in complement factor H (CFH) and CFH-related genes, examined the prevalence of CFH autoantibodies and evaluated the genotype-phenotype relationship in 10 Japanese patients with aHUS. We identified 7 causative or potentially causative mutations in CFH (p.R1215Q), C3 (p.R425C, p.S562L, and p.I1157T), membrane cofactor protein (p.Y189D and p.A359V) and thrombomodulin (p.T500M) in 8 out of 10 patients. All 7 of the mutations were heterozygous and four of them were novel. Two patients carried CFH p.R1215Q and 3 other patients carried C3 p.I1157T. One patient had 2 causative mutations in different genes. One patient was a compound heterozygote of the 2 MCP mutations. The patients carrying mutations in CFH or C3 had a high frequency of relapse and a worse prognosis. One patient had CFH autoantibodies. The present study identified the cause of aHUS in 9 out of 10 Japanese patients. Since the phenotype-genotype correlation of aHUS has clinical significance in predicting renal recovery and transplant outcome, a comprehensively accurate assessment of molecular variation would be necessary for the proper management of aHUS patients in Japan.