Effective modulation of CD4+CD25+high regulatory T and NK cells in malignant patients by combination of interferon-α and interleukin-2

Overinduced CD4⁺CD25^+high regulatory T cells (Treg) and downregulated NK cells contribute to tumor-relevant immune tolerance and interfere with tumor immunity. In this study, we aimed to design a novel strategy with cytokine combination to correct the dysregulated Treg and NK cells in malignant patients. Initially, a total of 58 healthy individuals and 561 malignant patients were analyzed for their cellular immunity by flow cytometry. The average percentages of CD4⁺CD25^+high/lymphocyte were 1.30?±?1.19?% ( $ \bar{x} $ ?±?SD) in normal adults and 3.274?±?4.835?% in malignant patients (p?<?0.001). The ratio of CD4⁺CD25^+high to CD4⁺ was 3.58?±?3.19?% in normal adults and 6.01?±?5.89?% to 13.50?±?23.60?% in different kinds of malignancies (p?<?0.001). Of normal adults, 15.52?% had >3?% Treg and 12.07?% had <10?% NK cells. In contrast, the Treg (>3?%) and NK (<10?%) percentages were 40.82 and 34.94?% in malignant patients, respectively. One hundred and ten patients received the immunomodulation therapy with IFN-?? and/or IL-2. The overinduced Treg in 86.3?% and the reduced NK cells in 71.17?% of the patients were successfully modulated. In comparison, other lymphocyte subpopulations in most patients were much less affected by this treatment. No other treatment-relevant complications except slight pyrexia, fatigue, headache, and myalgia were observed. In conclusion, dysregulated Treg and/or NK cells were common in malignant patients. Different from any regimens ever reported, this strategy was simple and effective without severe complications and will become a basic regimen for other cancer therapies.     

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.     

上海及邻近地区果蝇（Drosophila albomicans）的迁入及其线粒体DNA多态性研究

本文报道１９９１年秋果蝇（Ｄ．ａｌｂｏｍｉｃａｎｓ）突然迁入上海的事实。我们用９种限制性核酸内切酶对上海、嘉定、青浦和南汇４个群体的２９个单雌系的线粒体ＤＮＡ进行了酶切片段长度多态性研究,发现每个单雌系各有其特征的酶切图谱。在与广东、昆明、台湾珊瑚潭、日本洲本及马来西亚吉隆坡群体比较后,我们认为上海及邻近地区突然发现的Ｄ．ａｌｂｏｍｉｃａｎｓ来自中国大陆南部的多个群体。这一结论支持该种目前正处于向北迁移的分化阶段的观点。我们还对线粒体ＤＮＡ的种内多态性进行了讨论     

CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1-infected individuals

The basis for the extensive variability seen in the reconstitution of CD4(+) T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4(+) T cells/mm(3). The CCL3L1-CCR5 genotypes favoring CD4(+) T cell recovery are similar to those that blunted CD4(+) T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4(+) cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution.     

Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli

Atrial natriuretic peptide (ANP), a small peptide consisting of 28 amino acids, has been applied in clinical treatment for heart failure, but it can encounter proteolytic degradation during its expression in host cells. Therefore, it is usually reported that ANP was expressed as a part of fusion protein. The aim of our study was to use an overexpression system to express the fusion protein REF-ANP and to optimize a purification method. First, Escherichia coli DH5alpha was transformed with constructed expression vector containing two tandem copies of ref-anp gene and the fusion protein REF-ANP was overexpressed in shaking flask culture. Subsequently, the inclusion bodies were purified with reverse phase chromatography and pooled fractions were lyophilized. After this step, REF-ANP can be solubilized under native conditions without urea. After cleavage reaction, the sample was subjected to size exclusion chromatography and then rANP was polished with reverse phase chromatography. The final purity of rANP was more than 98% and the recovery of rANP per liter of shaking flask culture was more than 3mg. Such methods as mass spectrometry, capillary isoelectrofocusing analysis, and N-terminal amino acid sequence were used to identify rANP. The capillary isoelectrofocusing analysis showed that the pI of ANP was about pH 9.7. In this study, an efficient refolding and purification process should make scaling-up procedures easier and more successful than earlier reports. Moreover, it is possible that the refolding and purification method along with the overexpression system described in this article may offer new ideas on optimizing expression and purification of other kinds of short peptides.     

Elements in erythrocytes of population with different thyroid hormone status

The contents of elements K, Ca, Fe, Cu, Zn, Se, and Rb in erythrocytes of 78 cases with different thyroid hormone status have been measured by proton-induced X-ray emission and neutron activation analysis. According to the status of thyroid hormones T₃, T₄, TSH, FT₃, and FT₄ detected by radioimmunoassay, the experiment subjects were divided into four groups (i.e., hyperthyroid, hypothyroid, critical [one of thyroid hormones was abnormal], and normal). Elements contents and hormones levels of four groups were analyzed by one-way analysis of variance and correlation using an SPSS/PC statistical package. The results showed that the Se contents of four groups were not significantly different (p<0.05). Zn content of hypothyroid group was significantly higher than those of hyperthyroid and critical groups. The Zn content of the normal group was higher than that of the hypothyroid group and lower than that of the hyperthyroid and critical groups. In the hyperthyroid group, there were significant correlations between elements contents and thyroid hormones levels (except TSH), but not between elements contents and levels of thyroid hormones. However, in the hypothyroid group, relatively strong correlations have been found between elements contents and thyroid hormones levels, especially between Zn and the T₃/T₄ ratio, and between Zn and TSH.     

A freshwater ecoregion delineation approach based on freshwater macroinvertebrate community features and spatial environmental data in Taizi River Basin, northeastern China

The ecoregion is currently widely used as the basic geospatial unit in freshwater biodiversity conservation. The popularly used delineation is usually based on the assumption that attributes of aquatic ecosystems are influenced by landscape-scale environmental variables. However, few ecoregion delineations attempt to establish the local validity of this assumption prior to delineation, and few studies check the correspondence of the derived ecoregion boundaries with the distributions of attributes of aquatic biota. In this study, we established an approach to overcome these shortcomings. The notable features of the approach are: (1) the delineation variables were filtered through a series of analytical steps to select those that best represented the aquatic community traits, and which avoided redundancy in the data; (2) the method was quantitative and repeatable; and (3) the derived ecoregion boundaries were checked for consistency with the spatial attributes of aquatic biota. The approach was applied in the Taizi River Basin, northeast China. The procedure proposed here filtered out altitude and annual precipitation as the best variables to include in the freshwater ecoregion delineation. Then, using the quantitative ISODATA classification method, the basin was classified into three ecoregions. A test of accuracy indicated that freshwater ecoregions matched well with the spatial distribution pattern of macroinvertebrate community attributes. Statistical analysis showed that natural geographical attributes and river attributes were different in the three ecoregions, and indices representing macroinvertebrate community attributes are significantly different as a whole among the three ecoregions. The case study proved this approach effective on ecoregion delineation.     

The Leu72Met polymorphism of the GHRL gene prevents the development of diabetic nephropathy in Chinese patients with type 2 diabetes mellitus

The preproghrelin (GHRL) Leu72Met polymorphism (rs 696217) is associated with obesity, reduced glucose-induced insulin secretion in healthy or diabetic subjects, and reduced serum creatinine (Scr) levels in type 2 diabetes. We evaluated the association of the Leu72Met polymorphism with measures of insulin sensitivity in non-diabetic control individuals and type 2 diabetics, and whether this variation contributes to the development of diabetic nephropathy (DN) in type 2 diabetes. A case–control study was performed of 291 non-diabetic control subjects and 466 patients with type 2 diabetes, of whom 238 had DN with overt albuminuria (DN group; albuminuric excretion rate [AER] ≥ 300 mg/24 h) and 228 did not have DN, but had diabetes for more than 10 years (non-DN group). Genotyping was performed using a TaqMan PCR assay. The Leu/Leu, Leu/Met, and Met/Met genotype frequencies were significantly different between the non-DN and DN groups (p = 0.011). The frequency of the variant genotypes (Leu/Met, Met/Met) was significantly lower in the DN group than the non-DN group (23.5 vs. 36.0 %, p = 0.003). Met/Met non-diabetic control subjects had lower BMI and Scr levels and higher eGFR level than Leu/Leu or Leu/Met individuals (p < 0.05). Leu/Met and Met/Met type 2 diabetics had significantly lower AER and Scr levels and higher eGFR level than Leu/Leu type 2 diabetics (all p < 0.001). The GHRL Leu72Met polymorphism may help to maintain normal renal function and may protect against the development of DN by reducing albuminuria and improving renal function in Chinese patients with type 2 diabetes.     

Resonance light scattering study on the interaction between quinidine sulfate and congo red and its analytical application

The interaction between quinidine sulfate (QDS) and congo red (CR) was studied using resonance light scattering (RLS) technique, ultraviolet–visual spectrophotometry and fluorimetry. In weak acidic medium, QDS reacts with CR to form a supermolecular complex which results in the enhanced RLS intensity. Some important interacting parameters, such as the solution acidity and CR concentration, salt effect and addition order of the reagents, were investigated and optimized. Under the optimum conditions, it was found that the enhanced RLS intensity was in proportion to the concentration of QDS in the range 0.2–8.4 µg mL^?1. The corresponding detection limit was 12.0 ng mL^?1. The results showed that this new method enabled simple, sensitive and rapid determination of QDS and was used for the determination of QDS in urine and simulated huamn serum samples. Copyright © 2009 John Wiley & Sons, Ltd.