正常人精子和冷贮后精子的膜流动性及乙烯雌酚对其的影响

采用荧光探针DPH（1，6-diphenyl-1，3，5-hexatriene，1，6-二苯基-1，3，5-己三烯）标记人精子膜，测定正常人精子和冷贮与未冷贮精子膜流动性以及对乙烯雌酚的反应。结果表明乙烯雌酚可提高精子膜流动性，冷贮可降低精子膜流动性，但后者加入乙烯雌酚后膜流动性可恢复正常。提示采用冷贮精子人工授精时辅以雌激素有助于提高受精率。乙烯雌酚作用部位可能与精子雌激素受体有关。     

In Vitro Estimation of the Electrical Performance of Bipolar Pacing Electrode Systems

With the current interest in bipolar stimulation, there is a need for greater understanding of the electrical contributions of the two electrodes in this type of pacing. This has been investigated using a specially designed cell in which an electrode under test was immersed in a buffered saline solution (pH 7.42) at 37–38°C. Four electrode types were studied: a single stimulating electrode (6 mm² dish tip, Pt-black coated) and three indifferent electrodes. The indifferent electrodes were a 41 mm² polished Pt ring, a Pt-black coated version of the same electrode, and a titanium pacemaker enclosure which was included to allow comparison with the unipolar situation. These electrodes were tested individually against a chlorided silver reference electrode of negligible (1–2Ω) impedance, the results being processed in such a way as to allow estimation of the properties of the stimulating electrode taken in combination with each of the indifferents. Constant current pulses (1–10 mA amplitude, 0.5 and 1.0 ms duration) were applied and measurements made from the resulting voltage waveforms. These were V₁ and V₂, the potential at the leading and trailing edge of the pulse, and Va, the post-pulse potential at 20 ms following the trailing edge. The potential V₁ - V₂ is electrode dependent and allows the determination of the energy loss due to polarization to be calculated. Sinusoidal AC signals (0.1 Hz-10 kHz, 20 μA maximum amplitude) were also employed, allowing determination of sensing impedance. Under all conditions, the calculated performance of the stimulating electrode with the coated ring was nearly equivalent to that with the pacemaker enclosure. The performance with the uncoated ring was in all respects markedly inferior, displaying almost double the energy loss on pulsing, post-stimulus potentials an order of magnitude larger and sensing impedance increased by a factor of up to 8.     

Properties of a microcoaxial electrode designed for unit recording from human peripheral nerves

A new microcoaxial electrode is presented, which has been used successfully for recording single- and multiunit activity from myelinated and unmyelinated human nerve fibres. The main advantages (as compared with conventional needle electrodes) are electrical stability, mechanical durability, capability of being re-used and an insulation not easily scraped off, resulting in a low leakage capacitance. The AC polarization impedance and the potential transducing properties of the microelectrode have also been studied.     

1H-localized broadband 13C NMR spectroscopy of the rat brain in vivo at 9.4 T.

Localized (13)C NMR spectra were obtained from the rat brain in vivo over a broad spectral range (15-100 ppm) with minimal chemical-shift displacement error (<10%) using semi-adiabatic distortionless enhancement by polarization transfer (DEPT) combined with (1)H localization. A new gradient dephasing scheme was employed to eliminate unwanted coherences generated by DEPT when using surface coils with highly inhomogeneous B(1) fields. Excellent sensitivity was evident from the simultaneous detection of natural abundance signals for N-acetylaspartate, myo-inositol, and glutamate in the rat brain in vivo at 9.4 T. After infusion of (13)C-labeled glucose, up to 18 (13)C resonances were simultaneously measured in the rat brain, including glutamate C2, C3, C4, glutamine C2, C3, C4, aspartate C2, C3, glucose C1, C6, N-acetyl-aspartate C2, C3, C6, as well as GABA C2, lactate C3, and alanine C3. (13)C-(13)C multiplets corresponding to multiply labeled compounds were clearly observed, suggesting that extensive isotopomer analysis is possible in vivo. This unprecedented amount of information will be useful for metabolic modeling studies aimed at understanding brain energy metabolism and neurotransmission in the rodent brain.     

防水型氢扩散电极的制备和性质

用蓝色氧化钨和仲钨酸铵分别制取碳化钨。把碳化钨和聚四氟乙烯乳液、碳黑相混和,加工成防水型氢扩散多孔电极。测定了电极在4mol HCl/(kgH_2O)溶浓中的稳态极化曲线。发现超电位在8mV之间的弱极化区,超电位与总表观电流密度呈线性关系;在阳极极化中等程度的区域,有两条塔费尔直线。电位较高区塔费尔直线的斜率为电位较低区直线斜率的一倍。这结果与气体扩散电极的薄层平板模型理论推论相符合,从而阐明了电极在各电位区内极化的性质。X射线衍射照相证明,制得的碳化钨为α-WC,属六方晶系。     

Secretion of adipocytokines by perivascular adipose tissue near stenotic and non-stenotic coronary artery segments in patients undergoing CABG

Objective

Perivascular adipose tissue (pvAT) may induce a local pro-inflammatory environment, possibly contributing to coronary atherosclerosis. We investigated whether there is a difference in adipocytokine production by pvAT near stenotic and non-stenotic coronary artery segments in patients with coronary artery disease (CAD).

Methods

In patients undergoing CABG with or without valve replacement (n = 38) pvAT near stenotic and near non-stenotic coronary segments was harvested. PvAT was incubated ex vivo for 24 h. Concentrations of 23 adipocytokines were measured in the supernatants with a Multiplex assay. The number of macrophages (CD68, CD11c, CD206) and lymphocytes (CD45) in pvAT was determined. Differences between stenosis and control pvAT were tested with Wilcoxon signed rank test corrected for multiple comparisons.

Results

Production of IL-5, IL-1α, IL-17, IL-18 and IL-23 was higher in control than stenosis pvAT samples (p < 0.0021). Macrophages were more abundant in stenosis than in control pvAT (median n/400× field: 2.3 IQR: 0.3–4.5 versus 1.2 IQR: 0.1–2.5). There was a predominance of M2 macrophages in both stenosis and control pvAT (median n/400× field: macrophages stenosis: M1: 0.0; M2: 1.0 p = 0.004; control: M1: 0.0; M2: 0.6 p = 0.013). The relation between adipocytokine production and macrophage infiltration was not different in stenosis and control pvAT.

Conclusion

In patients with CAD, multiple adipocytokines were secreted at higher levels by pvAT near non-stenotic than near stenotic coronary artery segments. Furthermore, pvAT macrophages are associated with stenosis of the adjacent vessel. M2 macrophages were more abundant than M1 macrophages in pvAT.     

Autocapture compatibility in patients with the MembraneEX lead and Affinity pulse generators.

The first Autocapture generation worked well with all recommended leads. The newer Autocapture generation provides a more sensitive resolution for evoked response testing and its implementation in a dual-chamber device. The purpose of the study was to evaluate the performance of the Affinity SR/DR pacemaker with the new Autocapture algorithm in combination with the small surface area pacing lead MembraneEX in 129 patients. Autocapture ventricular threshold, sensing threshold, lead impedance, evoked response (ER) and polarization signals were determined at implantation and discharge, as well as after 1 and 3 months. Autocapture recommendation rate was based on the ER sensitivity test. The median pacing threshold was 0.38, 0.50, 0.75, 0.75 V at implant, discharge, 1 and 3 months post-implant, respectively. The respective data for median lead impedance were 744, 605, 649 and 691 ohms; median sensing threshold was 12.5 mV at all visits. The median ER amplitude was 9.0, 10.1, 9.9 and 10.1 mV and the median polarization signal 0.39 mV at all visits. The frequency of recommended Autocapture activation was 98.3%, 99.2%, 98.3% and 96.2% of all patients at implant, at discharge, 1 and 3 months post-implant respectively. In conclusion, the studied pulse generator enabled, in combination with this pacing lead, in >95% of all patients activation of Autocapture.     

Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival

Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.Macrophages are phagocytic cells of our immune system that are found in almost all tissues. Originating from progenitor cells in bone marrow, circulating blood monocytes extravasate into tissues, where they differentiate into macrophages and can subsequently be shifted to diverse functional phenotypes by local environmental cues (1). These polarization states are broadly categorized as classically activated M1 macrophages or alternatively activated M2 macrophages. The M1 macrophage phenotype is induced by mediators, such as IFN-γ or LPS, resulting in a proinflammatory and microbicidal functional phenotype (2). In contrast, M2 macrophages are activated by IL-4 and IL-13, and they possess functional roles in resolution of inflammation and tissue remodeling (3).There are several chronic pathological findings associated with increased tissue levels of M2 macrophages, including cancer, late-stage atherosclerosis, fibrosis, and asthma (4). In cancer, populations of tumor-associated macrophages (TAMs) mediate immunosuppression and possess M2-like qualities, such as poor antigen presentation, promotion of angiogenesis, and tissue remodeling and repair, although there is heterogeneity in pathways and phenotypes in different tumors (5). TAMs also secrete the factors milk fat globule-EGF factor 8 and IL-6 that lead to tumorigenicity and drug resistance of cancer stem/initiating cells (6). The extent of TAM accumulation within tumors generally correlates with poor disease prognosis (7, 8). Indeed, the role of M2 macrophages in chronic inflammation and disease is increasingly appreciated, and the ability to modulate specific subsets of macrophage populations is a major focus of macrophage-targeted therapy (4). However, there is a dearth of available targeting entities that are specific for M2 macrophages.Several ligands have been used to target macrophage populations. The small molecules mannose and folate, which are ligands for the mannose receptor and folate receptor β, respectively, have been conjugated to drugs or drug carriers for macrophage delivery (9–11). However, receptors for both molecules are highly expressed in other cell types. Mannose receptor is a pathogen recognition receptor that is also used by dendritic cells (12). In addition to activated macrophages, folate binds to receptors on normal epithelial cells and tumor cells (13). Segers et al. (14) reported a peptide that binds the scavenger receptor-A on macrophages found within atherosclerotic plaques, but scavenger receptor-A is also expressed on all dendritic cells.Our goal is to develop an M2-targeted construct that can be used to ablate TAMs selectively but not other leukocytes. To achieve this goal, an M2-selective ligand was first identified by a library selection approach. Peptide phage library screening is a common method of identifying novel targeting ligands, and it has been used to select peptides for targeting tumor vasculature (15) and colon cancer (16) among several others (17). We therefore designed a subtractive phage panning strategy that uses bone marrow-derived, in vitro-activated macrophage subpopulations to increase binding specificity of a selected phage to the M2 macrophage subpopulation. We report here the discovery and evaluation of a peptide, called M2pep, which shows selective binding and internalization in M2 macrophages but with minimal M1 macrophage interaction. We demonstrate this peptide’s ability to identify murine M2-like TAMs within mixed cell populations isolated from mouse colon carcinoma tumors and to accumulate in TAMs after i.v. injection into tumor-bearing mice. We show that i.v. administration of M2pep fusion peptides with KLAKLAKKLAKLAK (KLA), a proapoptotic peptide, to tumor-bearing mice selectively reduces TAM populations and prolongs survival.     

钠-葡萄糖共转运体２抑制剂通过调节巨噬细胞极化和旁分泌改善心肌纤维化

目的 验证钠-葡萄糖共转运体2抑制剂(SGLT2i)能否在缺氧/复氧(H/R)条件下通过改变巨噬细胞旁分泌来调节心脏成纤维细胞(CFs)的激活,从而改善心肌纤维化。方法 将27只C57BL/6雄性小鼠按随机数表法随机分为3组,每组9只:(1)假手术组(sham组);(2)缺血再灌注损伤(IRI)组;(3)缺血再灌注损伤+SGLT2i组(IRI+SGLT2i组)。28d后采用超声心动图和免疫荧光染色检测心肌纤维化和不良重构。培养RAW264.7细胞,分为以下3组:(1)阴性对照(NC组);(2)缺氧/复氧组(H/R组);(3)缺氧/复氧+SGLT2i组(H/R+SGLT2i组)。采用免疫荧光染色、Western blotting(WB)检测M2极化,使用酶联免疫吸附试验(ELISA)检测转化生长因子β(TGF-β)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-ɑ)、白细胞介素6(IL-6)、血小板衍生生长因子a(PDGF-a)的分泌情况。提取小鼠CFs分为以下3组:(1)阴性对照(NC组);(2)缺氧/复氧组(H/R组);(3)缺氧/复氧+SGLT2i组(H/R+SGLT2i组)。在各组中加入相应的巨噬细胞上清液,检测CFs的激活情况。采用GraphPad Prism 8软件进行数据分析。多组间比较采用单因素方差分析,2组间比较采用Tukey′s事后检验。结果 动物模型术后28d超声心动图结果显示:与IRI组相比,IRI+SGLT2i组表现出较高的左室射血分数和左室缩短分数,而左室收缩末期容积、左室舒张末期容积、左室收缩期内径和左室舒张期内径都较小(均 P<0.05)。免疫荧光染色结果显示:与IRI组相比,IRI+SGLT2i组的Ⅰ型和Ⅲ型胶原蛋白含量降低( P<0.05),且天狼星红染色结果显示Ⅰ型和Ⅲ型胶原蛋白比例明显下降( P<0.05)。细胞模型WB结果显示:与H/R组相比,H/R+SGLT2i组的M2标志物精氨酸酶1(Arg1)含量较多,M1标志物诱导型一氧化氮合酶(iNOS)含量较少( P<0.05)。ELISA实验证实:TGF-β、IL-1β、TNF-ɑ、IL-6及PDGF-a在H/R+SGLT2i组的含量相对H/R组更少( P<0.05)。WB结果证实:H/R+SGLT2i组CFs表达的ɑ-平滑肌肌动蛋白和胶原蛋白的表达量低于H/R组( P<0.05)。结论 SGLT2i可改善IRI诱导的心肌纤维化。SGLT2i抑制M1极化,促进M2极化,抑制炎症和纤维化相关细胞因子的分泌。SGLT2i处理RAW264.7细胞上清液可抑制CFs激活,证实SGLT2i通过调节巨噬细胞的旁分泌来抑制CFs的激活,改善心室不良重构。     

LXRα is expressed at higher levels in healthy people compared to atherosclerosis patients and its over-expression polarizes macrophages towards an anti-inflammatory MΦ2 phenotype

Objectives: (1) To investigate the expression patterns of MΦ1 and MΦ2 phenotype markers of peripheral blood monocyte (PBMC)-derived macrophages in atherosclerosis patients and healthy controls, as well as the expression correlation among these genes. (2) To elucidate whether a high level of liver X receptor α (LXRα) expression is associated with anti-inflammatory MΦ2-type polarization.

Design: Peripheral blood monocytes (PBMCs) were obtained from 28 patients with carotid artery plaques and 10 normal persons, who did not have carotid artery plaques. M1 and M2 phenotype markers were analyzed after cellular differentiation into macrophages. Human macrophages derived from healthy donors were transfected with plasmid DNA encoding LXRα and control null-plasmids. Gene expression levels were quantified after further differentiation.

Results: Three genes (LXRα, CD68, and CD36) were expressed at a significantly lower rate in the atherosclerotic group than normal patients. There were correlations between the expression of LXRα, CD68, and peroxisome proliferator-activated receptor (PPARγ), and between CD163, CD36 and scavenger receptor class A (SRA1). Macrophages over-expressing LXRα exhibited enhanced expression level of MΦ2-type genes and decreased expression level of MΦ1-type genes.

Conclusions: PBMCs from healthy persons were predisposed to the MΦ2 differentiation phenotype, which exhibits elevated cholesterol uptake and anti-inflammatory properties. LXRα over-expression polarizes macrophages towards the anti-inflammatory MΦ2 phenotype.