Cellular internalization of doxorubicin loaded star-shaped micelles with hydrophilic zwitterionic sulfobetaine segments

Four arm star-shaped poly(ε-caprolactone)-b-poly((N,N-diethylaminoethyl methacrylate)-r-(N-(3-sulfopropyl)-N-methacryloxyethy-N,N-diethylammoniumbetaine)) (4sPCLDEAS) micelles were loaded with anticancer drug doxorubicin to track their endocytosis in Hela cancer cell line. The effects of mean diameters and surface charges of the drug loaded micelles on the cellular uptake were studied in details. The results demonstrated that the internalization of micelles was both time and energy dependent process. Endocytic pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis were all involved in the internalization; caveolae-mediated endocytosis was the main pathway for the internalization of 4sPCLDEAS micelles. The assays for cell apoptosis and growth inhibition of tumor spheroids identified that these doxorubicin loaded micelles could induce cell apoptosis and inhibit tumor spheroids growth efficiently, which was even equal to free DOX·HCl. This study provided a rational design strategy for fabricating diverse micellar drug delivery systems with high anticancer efficiency.     

Tartaric acid-based amphiphilic macromolecules with ether linkages exhibit enhanced repression of oxidized low density lipoprotein uptake

Cardiovascular disease initiates with the atherogenic cascade of scavenger receptor- (SR-) mediated oxidized low-density lipoprotein (oxLDL) uptake. Resulting foam cell formation leads to lipid-rich lesions within arteries. We designed amphiphilic macromolecules (AMs) to inhibit these processes by competitively blocking oxLDL uptake via SRs, potentially arresting atherosclerotic development. In this study, we investigated the impact of replacing ester linkages with ether linkages in the AM hydrophobic domain. We hypothesized that ether linkages would impart flexibility for orientation to improve binding to SR binding pockets, enhancing anti-atherogenic activity. A series of tartaric acid-based AMs with varying hydrophobic chain lengths and conjugation chemistries were synthesized, characterized, and evaluated for bioactivity. 3-D conformations of AMs in aqueous conditions may have significant effects on anti-atherogenic potency and were simulated by molecular modeling. Notably, ether-linked AMs exhibited significantly higher levels of inhibition of oxLDL uptake than their corresponding ester analogues, indicating a dominant effect of linkage flexibility on pharmacological activity. The degradation stability was also enhanced for ether-linked AMs. These studies further suggested that alkyl chain length (i.e., relative hydrophobicity), conformation (i.e., orientation), and chemical stability play a critical role in modulating oxLDL uptake, and guide the design of innovative cardiovascular therapies.     

Magnetic silica spheres with large nanopores for nucleic acid adsorption and cellular uptake

Template assisted fabrication of magnetic silica nanospheres with large nanopores (MSNLP) and their adsorption and delivery of nucleic acids are reported in this paper. Silica spheres with controlled particle diameter (∼400 nm) and large nanopore size (13-24 nm) are prepared by using Brij56 as a template of mesopore, enabling incorporation of magnetic nanocrystals into the particles under mild neutral synthesis conditions. High resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and field-dependent magnetisation measurements confirm that the magnetic nanocrystals have been encapsulated into the silica spheres. The saturation magnetisation values of the resulted magnetic-silica nanocomposites are tunable by adjusting the amount of Fe₃O₄ magnetic nanocrystals used in the synthesis process. The nitrogen sorption analysis reveals that mesopores with large pore size exist in the silica matrix. After functionalisation of the silica surface with poly-(l-lysine) (PLL), the nanoparticles show strong adsorption capacity (q_m ranging from 10 to 22.5 μg/mg) for CpG DNA. We have further demonstrated successful delivery of miRNA into rat proximal tubular epithelial cells, facilitated by efficient cellular uptake of the nanocomposites. This work provides a convenient strategy to prepare MSNLP which can offer a versatile platform for biological applications such as simultaneous drug delivery and magnetic resonance imagining under external magnetic field.     

Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin

Cellular uptake of sepiapterin resulted in an efficient accumulation of tetrahydrobiopterin. Tetrahydrobiopterin is much less permeable across the cell membrane than sepiapterin or dihydrobiopterin, the precursors of the tetrahydrobiopterin-salvage pathway. The uptake of sepiapterin by the cell was examined under metabolic arrest with N-acetylserotonin, an inhibitor of sepiapterin reductase. The release profile of previously accumulated sepiapterin was also analyzed. Two routes were clearly distinguishable, namely rapid and slow. Both were apparently bi-directional and equilibrating in type. Each route was connected to non-mixable pools somehow separated in the cell. The rapid process was too fast to analyze by the current methods of cell handling. The slower process was associated with conversion of sepiapterin to tetrahydrobiopterin in the absence of N-acetylserotonin, suggesting that this route opens into the cytosolic compartment where use of the salvage pathway was strongly driven by sepiapterin reductase and dihydrofolate reductase with a supply of NADPH which favors tetrahydrobiopterin accumulation. Consequently, sepiapterin was enforcedly taken up by the cell where it accumulated tetrahydrobiopterin in the cytosol in continuous manner.     

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer: Preparation,formation mechanism,improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-l-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of β-tricalcium phosphate (β-TCP) bioceramics by soaking β-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of β-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the β-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of β-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.     

Effects of mannose density on in vitro and in vivo cellular uptake and RNAi efficiency of polymeric nanoparticles

To evaluate the effects of mannose density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency of polymeric nanoparticles (NPs) in macrophages, mannose-modified trimethyl chitosan-cysteine (MTC) conjugates with mannose densities of 4%, 13%, and 21% (MTC-4, MTC-13, and MTC-21) were synthesized. Tumor necrosis factor-alpha (TNF-α) siRNA loaded MTC NPs with particle sizes of ∼150 nm exhibited desired structural stability and effectively protected siRNA from enzymatic degradation. Generally, cellular uptake and RNAi efficiency were affected by mannose density. As expected, MTC-21 NPs presented the maximum in vitro uptake and RNAi efficacy in Raw 264.7 cells among all NPs tested. However, MTC-4 NPs exhibited the optimal in vivo uptake by peritoneal exudate cell macrophages (PECs). In the inflammation model of acute hepatic injury, orally delivered MTC-4 and MTC-13 NPs worked better in silencing TNF-α expression and alleviating liver damage than MTC-21 NPs. As for the ulcerative colitis model, MTC-4 NPs outperformed MTC-13 and MTC-21 NPs with respect to TNF-α knockdown and therapeutic efficacy following oral administration. These results highlighted the importance of ligand density in cellular uptake and RNAi efficiency, which could serve as a guideline in the rational design of targeted nanocarriers for anti-inflammation therapy.     

The effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake

Nanoparticles engineered for biomedical applications are meant to be in contact with protein-rich physiological fluids. These proteins are usually adsorbed onto the nanoparticle's surface, forming a swaddling layer that has been described as a ‘protein corona’, the nature of which is expected to influence not only the physicochemical properties of the particles but also the internalization into a given cell type. We have investigated the process of protein adsorption onto different magnetic nanoparticles (MNPs) when immersed in cell culture medium, and how these changes affect the cellular uptake. The role of the MNPs surface charge has been assessed by synthesizing two colloids with the same hydrodynamic size and opposite surface charge: magnetite (Fe₃O₄) cores of 25–30 nm were in situ functionalized with (a) positive polyethyleneimine (PEI-MNPs) and (b) negative poly(acrylic acid) (PAA-MNPs). After few minutes of incubation in cell culture medium the wrapping of the MNPs by protein adsorption resulted in a 5-fold increase of the hydrodynamic size. After 24 h of incubation large MNP-protein aggregates with hydrodynamic sizes of ≈1500 nm (PAA-MNPs) and ≈3000 nm (PEI-MNPs) were observed, each one containing an estimated number of magnetic cores between 450 and 1000. These results are consistent with the formation of large protein-MNPs aggregate units having a ‘plum pudding’ structure of MNPs embedded into a protein network that results in a negative surface charge, irrespective of the MNP-core charge. In spite of the similar negative ζ-potential for both MNPs within cell culture, we demonstrated that PEI-MNPs are incorporated in much larger amounts than the PAA-MNPs units. Quantitative analysis showed that SH-SY5Y cells can incorporate 100% of the added PEI-MNPs up to ≈100 pg/cell, whereas for PAA-MNPs the uptake was less than 50%. The final cellular distribution showed also notable differences regarding partial attachment to the cell membrane. These results highlight the need to characterize the final properties of MNPs after protein adsorption in biological media, and demonstrate the impact of these properties on the internalization mechanisms in neural cells.     

低分子质量壳聚糖修饰多壁碳纳米管的制备及细胞毒性研究

目的 利用低分子质量壳聚糖(CS)制备可在水溶液中稳定分散的壳聚糖/多壁碳纳米管(CS/MWCNTs)材料,并观察其与人脐静脉内皮细胞(HUVEC)的相互作用.方法 以物理吸附法对MWCNTs进行CS修饰,利用透射电镜、纳米粒度及Zeta电位分析仪对其进行表征.将CS/MWCNT进行荧光标记,以不同浓度与细胞作用24 h,通过激光共聚焦显微镜观察细胞摄入情况,并检测细胞毒性及细胞内活性氧自由基含量.结果 当低分子质量CS与MWCNTs的质量比大于10∶1时,可很好地将MWCNTs进行分散,CS/MWCNTs可在水相中稳定存在.细胞摄入实验显示,进入细胞内的碳纳米管主要位于胞浆内.毒性检测结果显示,在较高质量浓度(10、20 μg/ml)时,CS分散后的MWCNTs毒性较小.而与2种碳纳米管(MWCNTs与CS/MWCNTs)作用的细胞内的活性氧含量均随着浓度升高而显著提高,差别无统计学意义(P＞0.05).结论 水溶性的CS/MWCNTs材料拥有极好的分散性,性状稳定,细胞毒性低,这对后期将其应用于以MWCNTs为载体的治疗研究具有重要意义.     

Cellular uptake,elimination and toxicity of CdSe/ZnS quantum dots in HepG2 cells

In this work, the cellular uptake, elimination and toxicity of CdSe/ZnS QDs in HepG2 cells were comprehensively studied using inductively coupled plasma mass spectrometry (ICP-MS), MTT assay, AO/EB staining, and glutathione level and gene expression analysis. ICP-MS analytical results showed that the uptake efficiency of CdSe QDs by HepG2 cells was lower than that of Cd(II) and Se(IV), and the uptake was dose- and time-dependent. The uptake amount was related to the physicochemical properties of QDs, and NH₂-QDs with smaller size were more easily taken up by cells. In combination with various biochemical methodologies, a systematic and thorough quantitative analysis of the in vitro effects of CdSe/ZnS QDs with different coatings was conducted, along with that of Cd (II) and Se (IV). Although Cd(II) above 8.9 μM exhibited obvious toxicity to the cells, no obvious toxicity of four CdSe/ZnS QDs was observed within the tested concentration range (10–100 nM), most likely due to the protection of the ZnS shell and the PEG coating. From the molecular level's point of view, QDs at concentration of 100 nM exhibit obvious impact on the cells, such as increased gene expression (MT1A and CYP1A1), which was positively correlated with the intracellular concentration of QDs.     

叶酸靶向载紫杉醇磷脂-聚合物杂化纳米粒的制备及其体外细胞评价

目的 制备具有叶酸靶向性的载紫杉醇磷脂-聚合物杂化纳米粒(PTX-FLPNPs),并研究其对乳腺癌细胞EMT-6的细胞毒性及体外细胞吞噬.方法 以聚己内酯-聚乙二醇-聚己内酯(PCL-PEG-PCL)、二硬脂酰基磷脂酰乙醇胺-甲氧基聚乙二醇(DSPE-mPEG2000)和叶酸偶联的磷脂(Folate-PEG(2000)-DSPE)为药物载体,通过薄膜水化法自组装制备PTX-FLPNPs,并对其进行表征;使用激光扫描共聚焦显微镜观察比较叶酸受体高表达的乳腺癌细胞EMT-6对叶酸靶向及无靶向杂化纳米粒的吞噬作用;采用MTS法研究PTX-FLPNPs对EMT-6细胞的细胞毒性.结果 成功制备了PTX-FLPNPs,其呈球形,粒径均匀,具有明显的“核-壳”结构.投药量为30％的PTX-FLPNPs的平均粒径为(279.9±8.7)nm,多分散系数为0.173±0.021,Zeta电位为(-17.5±1.1)mV,载药量为(27.36±0.91)％,包封率为(91.16±1.12)％.细胞吞噬实验表明,叶酸受体高表达的EMT-6细胞对叶酸靶向的杂化纳米粒的吞噬作用明显强于无靶向的杂化纳米粒(P＜0.05).细胞毒性实验结果表明,PTX-FLPNPs的细胞毒性低于紫杉醇注射剂,且对肿瘤细胞的抑制效果优于无靶向的杂化纳米粒.结论 PTX-FLPNPs具有较高载药量及包封率,粒径均匀,可通过主动靶向作用介导肿瘤细胞内吞,并增加药物在肿瘤细胞内的浓度,是一种能有效抑制肿瘤的靶向载药纳米制剂.     

儿童急性淋巴细胞白血病化疗前后细胞免疫的变化及与人巨细胞病毒感染的关系

目的 探讨儿童急性淋巴细胞白血病(ALL)化疗前后细胞免疫的变化及与人巨细胞病毒(HCMV)感染的关系.方法 103例接受化疗的ALL患儿设为ALL组,34例健康儿童设为对照组,流式细胞术检测对照组、ALL组初治时、化疗结束时、化疗后3个月T细胞亚群.化疗结束时采用PCR检测HCMV病毒,并根据检测结果分为感染组和未感染组,对比分析两组T细胞亚群差异,多因素Logistic回归分析HCMV感染的影响因素.结果 ALL组(初治时)T细胞亚群CD3+、CD4+ 、CD8+、CD4 +/CD8+均低于对照组,差异有统计学意义(P ＜0.05);ALL组化疗前后CD3+、CD4+、CD4+/CD8+表现出先降低后升高的趋势,差异具有统计学意义(P＜0.05);化疗结束时共18例患者HCMV病毒检测为阳性,感染组化疗结束时CD3+、CD4+、CD4+/CD8+低于未感染组,差异有统计学意义(P＜0.05);多因素Logistic回归分析显示,疾病危险度是ALL患儿HCMV感染的危险因素(AOR=1.543,95％ CI:1.213～1.809,P=0.028),而CD3+、CD4+、CD4+/CD8+、最低PLT是HCMV感染的保护因素.结论 ALL自身以及化疗后细胞免疫功能降低,增加了HCMV感染风险,细胞免疫功能下降与HCMV感染可能互为因果.     

Tryptophan-induced lowering of blood pressure and changes of serotonin uptake by platelets in patients with essential hypertension

Summary Oral tryptophan loading and serotonin (5-HT) uptake by platelets were investigated as an approach to the study of central serotonergic mechanisms in patients with essential hypertension. Single oral doses of L-tryptophan (50 mg/kg body weight) lowered blood pressure significantly 90–120 min after administration in 14 patients with essential hypertension, but not in normotensive controls. Baseline measurements (without tryptophan loading) of 5-HT uptake by platelets did not differ between hypertensive and normotensive persons. Whereas L-tryptophan changed the uptake kinetics and increased 5-HT uptake in normal controls, these effects were not observed — or occurred to a much lesser degree — in hypertensive patients. It is suggested that in human essential hypertension central serotonergic mechanisms are involved in pathogenetic mechanisms. The tryptophan-in-duced lowering of blood pressure could be attributable to the enhancement of central 5-HT synthesis.Abbreviations 5-HT 5-hydroxytryptamine, i.e. serotonin - PRP platelet rich plasma Dedicated to Professor Dr. W. Kaufmann on the occasion of his 60th birthday     

硬膜外阻滞联合全身麻醉对肺癌根治术患者细胞免疫功能的影响

目的研究硬膜外阻滞联合全身麻醉对肺癌根治术患者细胞免疫功能的影响,为手术方案提供参考。方法选取2014年3月至2016年4月在我院接受肺癌根治手术的患者116例为观察对象。根据麻醉方案分为观察组与对照组各58例,两组术前均进行静脉全身麻醉,观察组在此基础上联合硬膜外阻滞,对比两组效果。结果观察组手术时间、苏醒时间、拔管时间、恢复自主呼吸时间均显著优于对照组;术前两组各项免疫指标无明显差异,术后2d观察组CD4+、NK水平均显著高于对照组。结论硬膜外阻滞联合全身麻醉对肺癌根治术患者细胞免疫功能影响较小,可缩短患者手术时间以及拔管时间,促进其早日康复。     

Sociodemographic,psychosocial and clinical factors associated with uptake of genetic counselling for hereditary cancer: a systematic review

Evidence suggests that a significant proportion of individuals referred to cancer genetic counselling (GC) do not attend, and thus may not be engaged in adequate cancer risk management. We aimed to review the literature to better understand barriers to accessing GC and how they may be overcome. We conducted a systematic literature search for articles examining factors influencing cancer GC uptake as well as motivators and barriers to GC attendance. Factors were categorised as sociodemographic, psychosocial or clinical. The literature search identified 1413 citations, 35 of which met the inclusion criteria. GC uptake ranged from 19% to 88%. With the exceptions of education level, socioeconomic status, cancer‐specific distress, personal cancer diagnosis and actual and perceived risk of cancer, support was lacking for most sociodemographic, clinical and psychosocial factors as predictors of GC uptake. Cost and logistical barriers, emotional concerns, family concerns and low perceived personal relevance were reported as important considerations for those declining GC. We conclude that there is poor understanding of GC and a lack of decision support among those referred to GC. Research into ways of providing education and support to referred individuals will be important as the scope and availability of GC and genetic testing broaden.     

Calcium ion uptake by cultures of skin fibroblasts from normal subjects and from patients with systemic connective tissue disorders

The uptake of radioactive calcium (⁴⁵Ca) and the effect of adrenalin on this process were studied in suspensions of fibroblasts from monolayer cultures of skin fibroblasts from healthy persons and from patients with systemic scleroderma and rheumatic fever. A considerable increase in calcium uptake was found in systemic scleroderma compared with normal and with rheumatic fever. Adrenalin, in concentrations of 2·10^–7–2·10^–5 M, increased the rate of calcium accumulation by normal fibroblasts but had the opposite action in scleroderma. The results point to a disturbance of the function of the fibroblast membrane in systemic scleroderma, and this may lie at the basis of changes in the metabolic activity of these cells.Laboratory of Functional Diagnosis and Connective Tissue Laboratory, Institute of Rheumatism, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 3, pp. 287–289, March, 1978.     

Reproducibility of onset and recovery oxygen uptake kinetics in moderately impaired patients with chronic heart failure

Oxygen (O2) kinetics reflect the ability to adapt to or recover from exercise that is indicative of daily life. In patients with chronic heart failure (CHF), parameters of O2 kinetics have shown to be useful for clinical purposes like grading of functional impairment and assessment of prognosis. This study compared the goodness of fit and reproducibility of previously described methods to assess O2 kinetics in these patients. Nineteen CHF patients, New York Heart Association class II-III, performed two constant-load tests on a cycle ergometer at 50% of the maximum workload. Time constants of O2 onset- and recovery kinetics (tau) were calculated by mono-exponential modeling with four different sampling intervals (5 and 10 s, 5 and 8 breaths). The goodness of fit was expressed as the coefficient of determination (R2). Onset kinetics were also evaluated by the mean response time (MRT). Considering O2 onset kinetics, tau showed a significant inverse correlation with peak- VO2 (R = -0.88, using 10 s sampling intervals). The limits of agreement of both tau and MRT, however, were not clinically acceptable. O2 recovery kinetics yielded better reproducibility and goodness of fit. Using the most optimal sampling interval (5 breaths), a change of at least 13 s in tau is needed to exceed normal test-to-test variations. In conclusion, O2 recovery kinetics are more reproducible for clinical purposes than O2 onset kinetics in moderately impaired patients with CHF. It should be recognized that this observation cannot be assumed to be generalizable to more severely impaired CHF patients.     

Cellular senescence of human mammary epithelial cells (HMEC) is associated with an altered MMP-7/HB-EGF signaling and increased formation of elastin-like structures

The extracellular matrix (ECM) and a complex interplay of cell-to-cell and cell-to-matrix (ECM) interactions provide important platforms to determine cellular senescence and a potentially tumorigenic transformation of normal human mammary epithelial cells (HMEC). An enhanced formation of extracellular filaments, consisting of elastin-like structures, in senescent post-selection HMEC populations was paralleled by a significantly increased expression of its precursor protein tropoelastin and matched with a markedly elevated activity of the cross-linking enzyme family of lysyl oxidases (LOX). RNAi experiments revealed both the ECM metalloproteinase MMP-7 and the growth factor HB-EGF as potential effectors of an increased tropoelastin expression. Moreover, co-localization of MMP-7 and HB-EGF as well as a concomittant downstream signaling via Fra-1 indicated a possible association between the reduced MMP-7 enzyme activity and an impaired HB-EGF processing, resulting in an enhanced tropoelastin synthesis during senescence of HMEC. In agreement with previous work, these findings suggested an important influence of the extracellular proteinase MMP-7 on the aging process of HMEC, affecting both extracellular remodeling as well as intracellular signaling pathways.     

Analogous effects of serum lipids from patients with nonthyroidal illness and normal subjects on the uptake of thyroxine and its conversion to triiodothyronine by rat hepatocytes in culture

Summary The low level of triiodothyronine (T3) in nonthyroidal illnesses (NTI) has been attributed to the decreased peripheral conversion of thyroxine (T₄) to T₃; patient's serum lipids decreased the conversion in a cell-free system. The objective of our study was to determine whether patients' serum lipids, whose content was elevated 2.5-fold above the reference serum value, and oleic acid affected the uptake of T₄ and its conversion to T₃ by rat hepatocytes in culture, thereby providing information on the cell's response to these processes. Serum ether extracts and oleic acid (0.1 mol/l) were incubated with cells followed by assessment of T₄ uptake and conversion of T₄ to T₃. The mean T4 uptake in the presence of ether extracts of NTI patients' or normals' sera were similar (112±15% and 110±24%, respectively). There was no difference in the T₄ to T₃ conversion between the patient and normal groups (90 ±14%); oleic acid also did not influence the conversion (96.7 ± 1.6%). Uptake and conversion in the absence of either extracts and oleic acid were controls. These results suggest that serum lipids from NTI patients and normal subjects exercise qualitatively and quantitatively almost similar influences on T₄ uptake and its conversion to T₃; oleic acid is not an inhibitor of T₄ uptake and T₄ to T₃ conversion in the rat hepatocyte. Since hepatocytes actively process fatty acids, their influence on intracellular conversion of T₄ is not equitable with T₄ conversion using the cell-free system. Our results do not support the hypothesis that abnormal lipid metabolism in NTI impairs hepatic T₄ to T₃ conversion.Abbreviations NTI nonthyroidal illness - T₃ triiodothyro-nine - TT₃ total T₃ - rT₃ reverse T₃ - T₄ thyroxine - TT₄ total T₄ - FT₄ free T4     

Comparison of genetically engineered hypoallergenic rBet v 1 derivatives with rBet v 1 wild-type by skin prick and intradermal testing: results obtained in a French population

BACKGROUND: Bet v 1, the major allergen in birch pollen, is recognized by more than 90% of patients allergic to birch in northern and central Europe. Immunotherapy is commonly performed with birch pollen extracts. Recently, hypoallergenic derivatives of Bet v 1 (rBet v 1 fragments, rBet v 1 dimer and trimer) were constructed and purified. OBJECTIVE: Our aim was to compare the allergenic activity of wild-type rBet v 1 with recombinant Bet v 1 derivatives (rBet v 1 fragments, dimer and trimer) with potentially reduced anaphylactic activity by skin testing in a French population. METHODS: Among the 36 birch pollen allergic patients included in the study, 29 were tested by skin prick testing and 30 by intradermal injections with purified monosubstances: rBet v 1 fragments (F1: aa1-74 and F2: aa75-160), Bet v 1 dimer and trimer. Intradermal tests were performed by the end-point intradermal titration method. Eight of the intradermally-tested patients were previously hyposensitized. Tests were performed over a period of 6 months (before, during and after birch pollen season); Bet v 1-specific IgE and IgG4 subclass responses were measured by immunoblotting and ELISA. RESULTS: All patients showed lower reactivity with the modified rBet v 1 allergens, both in skin prick and intradermal tests. In 25 and 23 out of 29 patients the lowest concentration of fragment 1 and 2, respectively, resulting in a positive prick test was 100-fold higher than the lowest concentration of monomer resulting in a positive prick test. For dimer it was 100-fold or more in 25 out of 29 patients, and for trimer it was 100-fold or superior in 26 out of 29 patients. By intradermal testing, the end-point concentration was 160-fold higher for trimer than for monomer in 24 patients and 40-fold higher in five patients. For the two fragments the end-point concentration was 160-fold higher in 20 out of 22 patients. CONCLUSION: Genetically modified hypoallergenic derivatives of the major birch pollen allergen, Bet v 1 showed reduced capacity to induce immediate type skin reactions. They may represent candidate molecules for immunotherapy of birch pollen allergy with reduced risk of anaphylactic side-effects.