ARMD与转铁蛋白受体基因单核苷酸多态性相关性研究

目的：探讨中国东北地区汉族人群中年龄相关性黄斑变性(ARMD)与转铁蛋白受体2(TFR2)基因单核苷酸多态性的相关性。

方法：选取200例ARMD患者(干性100例,湿性100例)和100名健康志愿者(对照组),采集所有研究对象的外周静脉血并以EDTA抗凝处理,提取基因组DNA,根据相关文献提供的引物序列,扩增TFR2基因多态性位点rs2075674,进行聚合酶链式反应(PCR)。比较各组间的基因型频率,并按照Hardy-Weinberg平衡原理确定样本的群体代表性。

结果：TFR2基因的多态性位点rs2075674 ARMD组与对照组比较有差异(χ²=6.494,P=0.011); 湿性ARMD组与对照组比较有差异(χ²=11.054,P=0.001),而干性ARMD组与对照组无差异(χ²=1.418,P=0.234)。

结论：中国东北地区汉族人群中TFR2基因多态性rs2075674与ARMD具有相关性,且与湿性ARMD相关性较大。     

Solubilization of flurbiprofen into aptamer-modified PEG–PLA micelles for targeted delivery to brain-derived endothelial cells in vitro

Abstract

Novel aptamer-functionalized polyethylene glycol–polylactic acid (PEG–PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer’s disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

The Burden of Iron Deficiency in Heart Failure: Therapeutic Approach

Heart failure (HF) is highlighted by its burdening symptom-limited exercise capacity and recurrent hospitalizations. Despite substantial advances regarding disease-modifying drugs in HF with reduced ejection fraction, additional therapeutic strategies to improve quality of life are invaluable. Currently, iron deficiency (ID) is overwhelmingly recognized in over 30% to 50% of patients with stable chronic HF, which worsens prognosis. The established pathophysiological mechanisms of progressive HF may be intertwined with increasing myocardial iron scarcity, wherein one begets the other. Most importantly, ID constitutes a novel target for symptom relief in carefully selected patients. In this regard, intravenous iron may be a safe and efficacious intervention, potentially reducing HF hospitalizations. We discuss the evidence and gaps in knowledge concerning iron therapy in HF and propose a practical, comprehensive, clinically oriented algorithm for timely adequate iron replenishment in different clinical scenarios. Finally, we further debate imperative decision-making before intervention and the drawbacks of such a strategy.     

Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich''s ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased “free iron” for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich''s ataxia.     

Erythropoietin production and erythropoiesis in compensated and anaemic states of hereditary spherocytosis

A compensated haemolytic state is defined by decreased red cell life-span without anaemia, i.e. by increased erythropoiesis in the absence of the physiological stimulus for erythropoietin (Epo) production. We evaluated s-Epo levels and the expansion of erythropoiesis (as measured by circulating transferrin receptor, s-TfR) in 32 patients with hereditary spherocytosis (HS) with the aim of verifying whether the enhanced erythropoiesis of compensated haemolysis was Epo-dependent. 20 of the patients (62.5%) had normal Hb values (> 12 g/dl in females and > 13 g/dl in males). Their compensated haemolytic state was the result of up to 8.2 times normal s-Epo and up to 3.9 times normal s-TfR levels, which were maintained by physiological regulation of erythropoiesis, as documented by the inverse dependence of Hb on s-Epo levels. Considering that patients with iron-deficiency anaemia represented the predicted physiological Epo response to anaemia, the observed/predicted ln s-Epo ratio (O/P ratio) was calculated in HS patients with anaemia and was used as an index of the adequateness of Epo production. All the anaemic HS patients had an O/P ratio > 1, documenting inappropriately high s-Epo levels. This work demonstrates that the compensated haemolytic state of HS patients is produced by an inappropriately high s-Epo level, and that the pattern of Epo overproduction is a biological characteristic of the disease.     

Beta‐2 transferrin is detectable for 14 days whether refrigerated or stored at room temperature

Background

The effect of time and temperature on beta‐2 transferrin stability in cerebrospinal fluid (CSF) is not well established. After collecting nasal CSF for testing, beta‐2 transferrin has been found to be stable and detectable for 1 week, whether being refrigerated or stored at room temperature. The purpose of this study was to determine if beta‐2 transferrin remained detectable longer than 1 week and whether refrigeration improved its detectability.

Methods

In patients undergoing therapeutic CSF diversion, 2‐mL CSF samples were collected from 18 patients. The samples were divided and stored either at room temperature, or at 4°C, and tested for beta‐2 transferrin at 7 and 14 days. CSF was collected from external ventricular drains (EVDs) (n = 15), lumbar drains (n = 2), and subdural drains (n = 1).

Results

Of the 18 CSF samples originally testing positive for beta‐2 transferrin, none turned negative at 7 or 14 days, in both the refrigerated and room temperature groups (95% confidence interval [CI], 0% to 18.5%).

Conclusion

Beta‐2 transferrin remained detectable for 14 days in all CSF samples, regardless of being stored at 4°C or room temperature.

     

The alternate iron transport pathway: mobilferrin and integrin in reticulocytes

Iron transport in reticulocytes is known to occur via the well-described transferrin-receptor–endosome pathway. An alternative pathway for iron transport independent of transferrin has been postulated in reticulocytes and other cells. Transport of iron into reticulocytes from ferric citrate solutions was shown to be saturable and independent of transferrin. During transport of iron from ferric citrate, both cell surface integrins, and a soluble protein, mobilferrin, were labelled. This demonstrated that the reticulocyte transferrin independent pathway for iron transport involved integrins and mobilferrin similar to intestinal absorptive cells. This pathway would be expected to transport iron into cells under conditions of iron overload and was capable of providing iron for haemoglobin synthesis. Mobilferrin was also radiolabelled when radioiron labelled transferrin was incubated with reticulocytes and this occurred with a different time course than was observed following reticulocyte exposure to radiolabelled ferric citrate. This suggested that mobilferrin may serve as an intermediary in both pathways.