Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs

Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.     

Xenotransplantation literature update, May to June 2012

Schneider MKJ, Seebach JD. Xenotransplantation literature update, May to June 2012. Xenotransplantation 2012; 19: 265–268.. © 2012 John Wiley & Sons A/S.     

乳糖酶缺乏的间接诊断法（乙醇乳糖耐量试验血,尿半乳糖测定法）

乳糖酶缺乏(LD)是儿童和成人的常见疾病,确定诊断最可靠的方法是小肠粘膜活检直接测定酶活性,然而其侵入性难以作为常规手段应用。本文旨在通过血和尿半乳糖测定建立一LD的间接诊断法。在受试者口服含乳糖和乙醇150mg/kg溶液时,于空腹和乳糖负荷后40分钟分别采静脉血、毛细血管血和尿液各一次测定半乳糖浓度。血半乳糖<0.3mmol/L、尿半乳糖<2mmol/L诊断为LD。试验表明本法简便可信,可做为LD的间接诊断法供临床选用。     

Separation and characterization of two UTP-utilizing hexose phosphate uridylyltransferases from Entamoeba histolytica

Two UTP-utilizing uridylyltransferases which react with both glucose 1-phosphate and galactose 1-phosphate were isolated from cell-free extracts of Entamoeba histolytica. The more specific of these enzymes, glucose-1-phosphate uridylyltransferase, acts preferentially on glucose 1-phosphate, having a maximum velocity 20-fold greater with this substrate than with galactose 1-phosphate. It was purified 200 fold with a 25% yield and has a molecular weight of 45 000. This enzyme requires a reducing agent for stability. The less specific transferase reacts with both hexose phosphates, having a maximum velocity of 1.35 times greater with galactose 1-phosphate. It was purified 1000 fold with a 20% yield, and has a molecular weight of 40 000. The common Leloir enzyme, UDPglucose-hexose-1-phosphate uridylyltransferase (EC 2.7.7.12), was not found in this organism. To avoid confusion with the Leloir enzyme our experience suggests that the less specific enzyme, which is presently referred to in the literature as galactose-1-phosphate uridylyltransferase (EC 2.7.7.10), should be named UTP:hexose-1-phosphate uridylyltransferase (EC 2.7.7.?). The more specific enzyme (EC 2.7.7.9) should be more clearly named UTP:glucose-1-phosphate uridylyltransferase.     

Distribution of taurine in the crystalline lens of vertebrate species and in cataractogenesis

Marked heterogeneity was observed in the distribution of taurine in different regions of the lens in various species. In general low taurine pools were observed in the nucleus of all species except frog and human. The distribution of taurine in human senile cataractous lenses at different stages of maturation showed decreased contents in all the regions except capsule epithelium as compared to the normal human lenses. This decrease is progressive upto the 'mature' stage of cataract. In rat lenses with galactose cataracts taurine contents decreased by about 83-94% of the normal values in the equatorial, anterior, posterior cortical and nuclear regions.     

Aqueous flow in galactose-fed dogs

Dogs fed galactose develop diabetes-like ocular complications that include keratopathy, cataracts, and retinopathy. The purpose of this study was to investigate whether galactosemic dogs display reduced aqueous flow similar to that observed in patients with insulin-dependent diabetes mellitus. Twelve male beagles at 9 months of age were divided into three groups of four. The Galactose group was fed diet containing 30% galactose for 97 months and the Reversal group was fed the galactose diet for an initial 38 months then standard dog diet for the remaining period. The Control group was fed standard dog diet for 97 months. Aqueous flow was determined by fluorophotometry in one eye per dog at 96 and 97 months after the initiation of galactose feeding. Intraocular pressure (IOP) was measured once in the morning by pneumatonometry. Anterior chamber depth was measured by A-scan. At the end of the experiment, eyes were enucleated and processed for histological examination. Dogs fed galactose diet for 97 months had significantly (p<0.05) increased body weights but similar IOP and anterior chamber depth compared to the other groups, and significantly (p=0.05) reduced aqueous flow compared to the control group (4.4+/-2.2 vs. 6.8+/-2.4 microl/min, mean+/-standard deviation, respectively). Additionally, aqueous flow decreased in the Reversal group to 3.1+/-1.3 microl/min (p=0.002). This decrease correlated with morphological changes of the ciliary processes. Like patients with insulin-dependent diabetes mellitus, galactose-fed dogs demonstrate reduced aqueous flow. This reduction was irreversible and independent of the retinopathy present. This animal model may be useful for the study of aqueous humor dynamics in diabetes.     

A recombinant probiotic for treatment and prevention of cholera

BACKGROUND & AIMS: We have developed a therapeutic strategy based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. In the present study, this has been applied to the development of a recombinant probiotic for treatment and prevention of cholera, caused by Vibrio cholerae. METHODS: We expressed glycosyltransferase genes from Neisseria gonorrhoeae and Campylobacter jejuni in a harmless Escherichia coli strain, resulting in production of a chimeric lipopolysaccharide terminating in a mimic of the ganglioside GM(1). RESULTS: The recombinant bacterium was capable of binding cholera toxin, a sine qua non of virulence, with high avidity; when tested with purified cholera toxin, it was capable of adsorbing >5% of its own weight of toxin in vitro. Administration of the GM(1)-expressing probiotic also protected infant mice against challenge with virulent V cholerae, even when treatment was delayed until after establishment of infection. When treatment commenced 1 hour after challenge, 12 of 12 mice given the probiotic survived, compared with only 1 of 12 for control mice (P < .00001). CONCLUSIONS: Toxin-binding probiotics such as that described here have considerable potential for prophylaxis and treatment of cholera in humans.     

Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

Background Thyroid peroxidase (TPO) is an important autoantigen in Hashimoto’s thyroiditis (HT), and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, purified glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO.

Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays (ELISAs). The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs.

Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid.

Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.

     

Diagnosis & management of alpha-gal syndrome: lessons from 2,500 patients

ABSTRACT

Introduction

Alpha-gal Syndrome (AGS) is a unique allergy to non-primate mammalian meat (and derived-products) that is associated with tick bites and is due to a specific IgE antibody to the oligosaccharide galactose-α-1,3-galactose (alpha-gal). AGS has many novel features that broaden the paradigm of food allergy, including that reactions are delayed 3–6 hours after exposure and patients have frequently tolerated red meat for many years prior to the development of allergic reactions. Due to the ubiquitous inclusion of mammal-derived materials in foods, medications, personal products and stabilizing compounds, full avoidance is difficult to achieve.     

高剂量D- 半乳糖复制C57BL/6J 衰老小鼠模型的研究

目的 探讨高剂量D- 半乳糖复制C57BL/6J 衰老小鼠模型的效果。方法 选用30 只9 周龄雄 性C57BL/6J 小鼠,随机分为3 组：500 mg/（kg·d）D- 半乳糖组、1 000 mg/（kg·d）D- 半乳糖组及对照 组,持续皮下注射8 周后解剖小鼠,测定血液和组织中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSHPx） 及丙二醛（MDA）等,Western blotting 检测组织中血红素加氧酶-1（HO-1）的表达,并对部分组织行 苏木精- 伊红染色。结果 1 000 mg/（kg·d）D- 半乳糖组小鼠出现不同程度的脱毛现象。D- 半乳糖组小 鼠的SOD 和GSH-Px 低于对照组（P <0.05）,而MDA 和HO-1 高于对照组（P <0.05）。D- 半乳糖组小鼠肝、 脑组织有不同程度的衰老相关病理学改变。结论 D- 半乳糖可以成功诱导小鼠衰老,是复制小鼠衰老模型 的较好选择。