Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.     

Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes

Background CD4⁺ and CD8⁺ T-lymphocytes are suggested to differentially affect airway inflammation in asthma. Agents which increase intracellular cAMP levels, such as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affect cytokine expression. Differences in the PDE isoenzyme profile between CD4⁺ and CD8⁺ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CD4⁺ and CD8⁺ T-lymphocytes. Methods CD4⁺ and CD8⁺ T-lymphocytes were purified (>98%) from peripheral blood mononuclear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4⁺ and CD8+ T-lymphocyte homogenates, PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0.5μM cyclic nucleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I, II and V activities were detected. About 20% of total eAMP hydrolysing capacity at 0.5 μM cAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE I-IV. The PDE isoenzyme pattern was not different between CD4⁺ and CDS⁺ T-lymphocytes. Moreover, representative inhibitors of PDE HI and IV activity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4^h and CD8⁺ T-lymphocytes. Conclusion Normal CD4⁺ and CD8⁺ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.     

ANP-mediated cGMP signaling and phosphodiesterase inhibition in the rat cervical spinal cord

Natriuretic peptides (NP) and the corresponding receptors are present in the rodent spinal cord. We have studied the structures which respond to atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide with an increased synthesis of cGMP. NP-responsive cGMP-producing structures were observed in laminae I–III, and X, and in addition in ependymal cells, astrocytes and a subpopulation of dorsal root ganglion cells. As the cGMP concentration is controlled by the rate of synthesis and the rate of breakdown by phosphodiesterases, we studied NP-responsive structures in spinal cord slices incubated in the presence of different phosphodiesterase inhibitors. We studied EHNA and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitors, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. Double immunostainings showed that cGMP-IR colocalized partial with the vesicular acetylcholine transporter molecule in lamina X, with Substance P in a subpopulation of neuronal fibers situated dorsolateral, and with a subpopulation of CGRP-IR dorsal root ganglion neurons. Colocalization of cGMP-IR was absent with parvalbumin, synaptophysin, and the vesicular transporter molecules for GABA and glutamate.

It is concluded that NPs in the spinal cord are probably involved in integrating intersegmental sensory processing in the spinal cord although the greater part of the NP-responsive cGMP-producing fibers could not be characterized. PDE2, 5, and 9 are involved in regulating NP-stimulated cGMP levels in the spinal cord. NPs may have a role in regulating cerebrospinal fluid homeostasis.     

Selective type IV phosphodiesterase inhibitors prevent IL-4-induced IgE production by human peripheral blood mononuclear cells

Background Selective type IV phosphodiesterase (PDE) inhibitors elicit anti-inflammatory and bronchodilatory activities in vitro and in vivo which suggest that these drugs could provide a new therapeutic approach for asthma treatment. Objective Regarding the role of IgE production in allergic and inflammatory reactions of the airways, we investigated the effect of selective PDE inhibitors on IL-4-driven IgE production by peripheral blood mononuclear cells (PBMC) or by purified B lymphocytes. Methods PBMC or purified B lymphocytes from non-allergic donors were stimulated for 13 days with IL-4 (100U/mL) in the presence or in the absence of selective PDE inhibitors. IgE production is evaluated by an ELISA technique. Results The selective PDE IV inhibitors, rolipram and Ro 20–1724 (10μM), inhibit lL-4-induced IgE production by PBMC. but not by purified B lymphocytes. No modification of the IgE production was noted with the selective PDE III inhibitors, milrinone and SK&F94-836, or the selective PDE V inhibitor, SK&F 96–231 (10 μM). Flow cytometry experiments showed that the effect of Rolipram could not be explained by the inhibition of the cell surface expression of the IL-4 receptor. Similarly, no significant effect of PDE IV inhibitors was observed on PHA-induced cell proliferation. The incubation of monocytes only with rolipram was sufficient to achieve a significant reduction of IgE production induced by IL-4. Conclusion Taken together, these results indicate that PDE IV inhibitors reduce lL-4-induced IgE production by PBMC and suggest that the inhibition of IgE production could be explained by a failure of monocytes to provide the necessary costimulatory signals.     

Preferential differentiation of inflammatory cells by recombinant human interleukins

The effects of recombinant human interleukins (IL) on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical cord blood and bone marrow cells. The results showed that IL-5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with IL-5, over 90% of nonadherent cells in both bone marrow cell and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with IL-4 resulted in the selective growth of OKT-3+ lymphocytes. In suspension cultures of bone marrow cells and cord blood cells grown in the presence of IL-3, basophilic, eosinophilic, and neutrophilic myelocytes developed within 2 weeks. By 3 weeks, however, the majority of non-adherent cells became eosinophilic myelocytes. In contrast to mouse bone marrow cell cultures, neither IL-3 nor combination of IL-3 and IL-4 induced the differentiation of mast cells in human bone marrow or cord blood cell cultures.     

两步法分离人脐血单个核细胞的最佳条件

背景：如何获得较为纯化、高活性的干细胞,目前未见深入研究报告,也未见一个标准化操作流程方案。 目的：探讨两步法分离人脐血单个核细胞最佳分离条件。 方法：观察羟乙基淀粉在20,30,40,50,60,70 min不同时间沉淀脐血中红细胞的效果;使用人淋巴细胞分离液,分别在800,700,600,500,400 g/min,离心30,25,20 min的条件下分离人脐血单个核细胞。 结果与结论：6%羟乙基淀粉沉淀脐血60 min效果最好;使用密度为(1.077 0±0.000 1) g/mL人淋巴细胞分离液,在4 ℃条件下以700 g/min离心力,离心30 min,洗涤3次,这样获得的人脐血单个核细胞效果最好,所得细胞沉淀中混杂细胞如红细胞及其他细胞碎片较少,人脐血单个核细胞的细胞得率及活力比较高。提示应用羟乙基淀粉沉淀和人淋巴细胞分离液分离两步法,在最佳时间条件下可提高脐血干细胞的回收率。     

Cyclic nucleotide phosphodiesterase isoenzyme activities in human alveolar macrophages

Background Alveolar macrophages and their precursors, the monocytes are involved in airway inflammation in asthma. An increase in intraceliular cAMP by PDE inhibitors is known to suppress macrophage and monocyte functions. A comparison of the PDE-isoenzyine profiles of human alveolar macrophages from normal and atopic donors and of human peripheral blood monocytes might form a basis to differentially affect functions of these cells by PDE inhibitors. Objective The study compares the PDE isoenzyme activity profiles of human alveolar macrophages from normal and atopic asthmatic donors and human peripheral blood monocytes. In addition, the effect of in vitro maturation of monocytes on their PDE isoenzyme profile is studied. Methods Macrophages were purified (95-97%) by adherence to plastic, and blood monocytes were purified (88%) by counter-current elutriation. PDE isoenzyme activity profiles were investigated using isoenzyme selective inhibitors and activators. Results In macrophages substantial PDE I activity, which was significantly higher than PDE IIF-V activity was detected and PDE II was absent. PDE III was membrane-bound whereas PDE I, IV and V were soluble. No difference was found between alveolar macrophages of normal donors and atopic asthmatics. Monocytes exclusively contained PDE IV but their in vitro maturation led to a PDE isoenzyme profile similar to that of alveolar macrophages. Conclusion These results indicate that human monocytes and alveolar macrophages are distinct targets for the effects of selective PDE inhibitors while alveolar macrophages from normal and atopic individuals appear to be equally sensitive.     

CM-Dil及DAPI联合标记示踪人脐血单核细胞细胞膜及细胞核的可行性

背景：氯甲基- 1,1 十八烷基-3,3,3’,3’-四甲基-吲哚-羧花青-高氯酸盐(chlormethylbenzamido-1, 1-dioctadecyl-3,3,3’,3’-tetramethylin-docarbocyamine,CM-DiI)和4’,6-联脒-2-苯基吲哚二盐酸盐 (4’,6-diamidino-2-phenylindole,DAPI)是常用的活细胞示踪剂,分别用来标记细胞膜和细胞核。 目的：观察应用细胞膜及细胞核标记物CM-DiI及DAPI联合示踪人脐血单核细胞的可行性及双标后人脐血单核细胞体外培养过程中细胞形态及活性的变化。 方法：将新鲜分离的人脐血单核细胞用示踪剂CM-DiI及DAPI进行双标记,体外培养双标后的人脐血单核细胞,倒置相差显微镜下观察细胞形态变化,锥虫蓝染色检测不同时间细胞活性,同时倒置荧光显微镜观察不同时间经CM-DiI和DAPI双标的人脐血单核细胞荧光染色阳性数。 结果与结论：人脐血单核细胞在CM-DiI和DAPI联合标记15 min后,荧光显微镜下可见标记细胞的细胞膜、细胞核分别在不同波长下分别呈现红色和蓝色的荧光。CM-DiI/DAPI双标后的人脐血单核细胞体外培养1,3,7,14,21 d,CM-DiI和DAPI双染的阳性细胞数各时间点比较差异无显著性意义。锥虫蓝染色观察人脐血单核细胞存活率为95.6%-98.8%。双标后的人脐血单核细胞体外培养过程中细胞形态变化与未经示踪标记的脐血单核细胞相比差异不明显,仍保持了良好的生长状态、贴壁能力和细胞增殖能力。由此证实,CM-DiI和DAPI可有效标记人脐血单核细胞,两种示踪剂对活细胞无毒不良反应,荧光衰减较慢,适用于干细胞标记及示踪。     

石菖蒲对脐血干细胞成骨分化的影响

背景:研究表明石菖蒲及其活性成分能促进成体神经的发生,对抗衰老和治疗相关神经退行性疾病有良好疗效。目的:探究中药石菖蒲浸提液对脐血干细胞增殖、成骨分化的影响,从中药学角度为促进干细胞的成骨分化提供新的思路。方法:采用溶剂提取法提取中药石菖蒲浸提液;流式细胞分选技术分离筛选脐血干细胞;电子显微镜观察脐血干细胞生长情况;CCK8法观察石菖蒲对脐血干细胞增殖的影响;ELISA方法检测石菖蒲对脐血干细胞培养上清液中骨钙素、骨形态发生蛋白2含量的影响;碱性磷酸酶染色试剂盒检测石菖蒲对脐血干细胞中碱性磷酸酶分布表达的影响。结果与结论:(1)分选出脐血单核细胞中的脐血干细胞纯度可达到(89.66±3.47)%;(2)加入石菖蒲浸提液低剂量、中剂量、高剂量培养脐血干细胞24,48,72 h后,干细胞的增殖率明显高于空白对照组(P<0.05),中剂量组的增殖率高于低剂量、高剂量组(P<0.05);(3)加入石菖蒲浸提液培养脐血干细胞5,10,15 d后,上清液中骨形态发生蛋白2、骨钙素的含量明显高于空白对照组(P<0.05),中剂量组的骨形态发生蛋白2、骨钙素含量高于低剂量、高剂量组(P<0.05);(4)碱性磷酸酶染色结果显示,加入石菖蒲浸提液培养干细胞10 d后碱性磷酸酶的分布表达明显高于空白对照(P<0.05),中剂量组和高剂量组的表达明显高于低剂量组(P<0.05)。(5)结果提示中药石菖蒲可以促进脐血干细胞的增殖以及其成骨分化。     

人外周血与脐血内皮祖细胞生物学特性的比较

背景：内皮祖细胞可从外周血与脐血中获取,是修复各种疾病所致损伤的血管内皮细胞不可或缺的细胞来源。 目的：比较人外周血与脐血来源的内皮祖细胞经体外培养后生物学特性的差异。 方法：通过密度梯度离心法和6%羟乙基淀粉结合密度梯度离心法分别分离人外周血与脐血中的单个核细胞,分别设为脐血源组和外周血源组,计数各组单个核细胞数量,按1.0×106/cm2接种于大鼠尾胶包被的培养皿中,用内皮细胞培养基进行诱导,共培养7 d。 结果与结论：外周血与脐血体外培养分离出内皮祖细胞具有类似的形态学特征。光学显微镜下观察,随着培养天数的增加,大多数细胞由早期的贴壁圆形转变为梭形。外周血源组内皮祖细胞有细胞集落形成,脐血源组可见梭形细胞自行排列生长为典型的线样结构。锥虫蓝染色及绘制细胞生长曲线后发现,外周血源组单个核细胞及内皮祖细胞数量、内皮祖细胞活率及增殖能力均低于脐血源组(P < 0.05)。外周血源组和脐血源组内皮祖细胞在接种后第3天增殖速度达到峰值,在随后的培养中细胞增殖呈衰减态。流式细胞仪及免疫荧光染色检测结果显示,外周血源组和脐血源组内皮祖细胞均可表达具有内皮祖细胞表型的CD133、CD34和血管内皮细胞因子受体2表面标志物,两组既摄取Dil标记乙酰化低密度脂蛋白,也能标记体外内皮祖细胞的标志物荆豆凝集素Ⅰ。结果证实,脐血来源的内皮祖细胞与外周血来源的内皮祖细胞生物学特性相近,脐血来源的内皮祖细胞增殖能力更强。     

Selective phosphodiesterase inhibitors for the treatment of bronchial asthma and chronic obstructive pulmonary disease

Theophylline is commonly used in the treatment of obstructive airway diseases. The identification and functional characterization of different phosphodiesterase (PDE) isoenzymes has led to the development of various isoenzyme-selective inhibitors as potential anti-asthma drugs. Considering the distribution of isoenzymes in target tissues, with high activity of PDE3 and PDE4 in airway smooth muscle and inflammatory cells, selective inhibitors of these isoenzymes may add to the therapy of chronic airflow obstruction. However, initial data from clinical trials with selective PDE3 and PDE4 inhibitors have been somewhat disappointing and have tempered the expectations considerably since these drugs had limited efficacy and their use was clinically limited through side effects. The improved understanding of the molecular biology of PDEs enabled the synthesis of novel drugs with an improved risk/benefit ratio. These 'second generation' selective drugs have produced more promising clinical results not only for the treatment of bronchial asthma but also for the treatment of chronic obstructive pulmonary disease.     

The role of protein kinase C isoenzymes in the regulation of calcineurin activity in human peripheral blood mononuclear cells

It is known that PMA (phorbol-12-myristate-13-acetate) can activate the classical and novel protein kinase C isoenzymes (cPKC alpha, beta, gamma and nPKC delta, epsilon, eta, theta), while the calcium ion can induce only the activity of cPKC. Calcineurin binding protein (Cabin 1) belongs to the group of endogenous inhibitors of calcineurin. Cabin 1 becomes hyperphosphorylated in response to PKC activation and may play a negative role in calcineurin signalling. It was observed that both PMA treatment and the increase in intracellular Ca2+ contributed to the reduction of calcineurin activity in human peripheral blood mononuclear cells without modulating the mRNA and the protein levels of calcineurin. PMA and Ca-ionophore (A23187), the activating agents of PKC, applied alone or in combination, significantly increased the phosphorylation state of Cabin 1 as revealed by immunoprecipitation of Cabin 1 detecting its phospho-Ser content by specific antibodies. GF109203X, an inhibitor of the classic and the novel protein kinase C isoenzymes, and G?6976, the selective inhibitor of the classical cPKC isoenzymes were able to abolish the effect of PMA or/and Ca-ionophore on the calcineurin activity with concomitant reversal of the hyperphosphorylation of Cabin 1. The calcineurin/Cabin 1 system was not influenced by Rottlerin, an inhibitor of PKC delta isoenzyme either in the absence or in the presence of Ca-ionophore and PMA. We presented evidence for the prominent role of cPKC alpha, beta, gamma isoenzymes in the inhibition of calcineurin as induced by PMA and Ca-ionophore. We demonstrated also that hyperphosphorylation of Cabin 1 by PMA/Ca2+-activated cPKC isoenzymes resulted in a simultaneous inhibition of calcineurin in peripheral blood mononuclear cells. These results suggest a negative regulatory role for Cabin 1 in calcineurin signalling and provide a possible mechanism of feedback inhibition through cross-talk between PKC and calcineurin.     

The role of phosphodiesterase isoforms 2, 5, and 9 in the regulation of NO-dependent and NO-independent cGMP production in the rat cervical spinal cord

NO-responsive, cGMP-producing structures are abundantly present in the cervical spinal cord. NO-mediated cGMP synthesis has been implicated in nociceptive signaling and it has been demonstrated that cGMP has a role establishing synaptic connections in the spinal cord during development. As cGMP levels are controlled by the activity of soluble guanylyl cyclase (synthesis) and the phosphodiesterase (PDE) activity (breakdown), we studied the influence of PDE activity on NO-stimulated cGMP levels in the rat cervical spinal cord.

cGMP-immunoreactivity (cGMP-IR) was localized in sections prepared from slices incubated in vitro. A number of reported PDE isoform-selective PDE inhibitors was studied in combination with diethylamineNONOate (DEANO) as a NO-donor including isobutyl-methylxanthine (IBMX) as a non-selective PDE inhibitor. We studied 8-methoxy-IBMX as a selective PDE1 inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitor, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. cGMP-IR structures (nerve fibers, axons, and terminals) were characterized using the following neurochemical markers: vesicular transporter molecules for acetylcholine, GABA, and glutamate (type 1 and type 2), parvalbumin, glutamate transporter molecule EAAT3, synaptophysin, substance P, calcitonin gene-related peptide, and isolectin B4. Most intense cGMP-IR was observed in the dorsal lamina. Ventral motor neurons were devoid of cGMP-IR. cGMP-IR was observed in GABAergic, and glutamatergic terminals in all gray matter laminae. cGMP-IR was abundantly colocalized with anti-vesicular glutamate transporter 2 (vGLUT2), however not with the anti-vesicular glutamate transporter 1 (vGLUT1), suggesting a functional difference between structures expressing vGLUT1 or vGLUT2. cGMP-IR did not colocalize with substance P- or calcitonin-gene related peptide-IR structures, however did partially colocalize with isolectin B4 in the dorsal horn. cGMP-IR in cholinergic structures was observed in dorsal root fibers entering the spinal cord, occasionally in laminae 1–3, in laminae 8 and 9 in isolated boutons and in the C-type terminals, and in small cells and varicosities in lamina 10. This latter observation suggests that the proprioceptive interneurons arising in lamina 10 are also NO-responsive.

No region-specific nor a constant co-expression of cGMP-IR with various neuronal markers was observed after incubation of the slices with one of the selected PDE inhibitors. Expression of the mRNA of PDE2, 5, and 9 was observed in all lamina. The ventral motor neurons and the ependymal cells lining the central canal expressed all three PDE isoforms.

Incubation of the slices in the presence of IBMX, DEANO in combination with BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase, provided evidence for endogenous NO synthesis in the slice preparations and enhanced cGMP-IR in all lamina. Under these conditions cGMP-IR colocalized with substance P in a subpopulation of substance P-IR fibers.

It is concluded that NO functions as a retrograde neurotransmitter in the spinal cord but that also postsynaptic structures are NO-responsive by producing cGMP. cGMP-IR in a subpopulation of isolectin B4 positive fibers and boutons is indicative for a role of NO-cGMP signaling in nociceptive processing. cGMP levels in the spinal cord are controlled by the concerted action of a number of PDE isoforms, which can be present in the same cell.     

脐带血CD4~+和CD8~+ T细胞中T细胞特异转录因子Elf-1表达特点

目的:了解脐带血单个核细胞(CBMCs)及其CD4+和CD8+ T细胞亚群中转录因子Elf-1的表达特点。方法:采用SYBR GreenⅠ荧光定量PCR和相对定量分析法检测12例脐带血CBMCs、CD4+和CD8+ T细胞亚群中Elf-1基因的表达情况,以β2微球蛋白基因(β2M)作为内参照,10例健康成人作为对照,采用相对定量公式:2-△Ct×100%,计算Elf-1mRNA相对表达量。结果:脐带血和健康成人外周血单个核细胞(PBMC)、CD4+和CD8+ T细胞均表达Elf-1,具体表达量呈现明显的个体差异性。Elf-1 mRNA相对表达量在CBMCs为18.55%±2.48%(其中最高为36.22%,最低为6.45%),在脐带血CD8+ T细胞为3.52%±0.45%(其中最高为6.75%,最低为1.71%),均明显高于健康成人PBMC(9.16%±1.92%)及其CD8+ T细胞(2.02%±0.27%)(P0.01,P0.05),而脐带血CD4+T细胞Elf-1 mRNA相对表达量为3.83%±0.61%,与健康成人外周血CD4+ T细胞(2.73±0.52%)比较,无显著差异。结论:本研究率先报道脐带血T细胞亚群中Elf-1 mRNA表达特点,结果提示Elf-1基因在CBMCs及其CD8+ T细胞亚群中的高表达可能是T细胞的免疫学特点之一。     

混合脐血血浆对植物血凝集素诱导的脐血源性T淋巴细胞增殖、分化的影响

目的:探讨混合脐血血浆(CBP)在植物血凝集素(PHA)诱导脐血源性T淋巴细胞巾的作用.方法:收集脐血血浆和脐血单个核细胞;将脐血单个核细胞分为CBP PHA和PHA两组.检测两组细胞增殖率及免疫表型.结果:CBP PHA组细胞增殖指数低于PHA组.但无统计学意义;两组细胞表型都以CD3 、CD4 为主,两组差异无统计学意义.结论:混合脐血血浆对脐血源性T淋巴细胞增殖、分化影响不明显,可能与RPMI1640培养体系具有相似的效果.     

脐血单个核细胞移植治疗失代偿期肝硬化

背景：原位肝移植是目前治疗终末期肝病的最有效手段,但是供肝来源匮乏、免疫排斥、无法有效控制反复感染等问题限制了其应用。干细胞移植技术的应用为该病种的治疗提供了新的思路和研究方法,许多研究证实可以通过各种不同的方法在体外成功诱导脐血来源的间充质干细胞向肝细胞转化。 目的：探讨人脐血单个核细胞移植治疗失代偿期肝硬化的临床疗效及可行性。 方法：异体人脐血单个核细胞移植治疗23例肝硬化失代偿期患者,检测移植后2,4,8,24周的血清丙氨酸氨基转移酶、白蛋白、胆碱酯酶、总胆红素和凝血酶原时间,并观察患者临床症状体征改善情况以及不良反应。 结果与结论：人脐血单个核细胞移植后2周,肝功能各项指标较治疗前无明显改善(P > 0.05);移植后4周,谷丙转氨酶有显著改善(P < 0.05)、其余指标无明显改善;移植后12周肝功能各项指标均有改善(P < 0.05),且肝脏硬度有所改善(P < 0.05);移植后24周各项指标有显著性改善(P < 0.01)。移植后4周时患者临床症状有明显改善,20例乏力好转(87%)、21例食欲改善(91%)、19例腹水减轻(83%);所有患者移植期间及治疗后随访24周无严重不良反应。结果显示异体人脐血单个核细胞移植治疗肝硬化失代偿期患者临床疗效肯定,安全性好,可作为中晚期肝硬化患者的临床治疗手段。     

Phosphodiesterase Type 4 Inhibitors

Phosphodiesterases (PDEs) are involved in the regulation of intracellular levels of the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). These enzymes hydrolyse the cyclic nucleotides to the corresponding nucleoside 5'-monophosphates. Nine PDE subtypes have been identified; these differ in their substrate specificity and mode of activation. The type 4 PDE (PDE(4)) hydrolyses cAMP, is activated by elevated levels of cAMP, and is inhibited by rolipram. Inhibition of enzyme activity has been shown to modulate the activity of cells of the immune system. The production of tumour necrosis factor (TNF)(alpha) by activated monocytes and macrophages is inhibited, and cytokine secretion and proliferation of type 1 T helper cells are suppressed. Both immune cell activation and their concomitant induction of cytokine secretion are implicated in multiple sclerosis (MS), which is the major demyelinating disease of the central nervous system. Studies with the selective PDE(4) inhibitor rolipram in experimental autoimmune encephalomyelitis (an animal model of MS) in mice, rats and nonhuman primates have demonstrated the efficacy of the compound in this disease model, suggesting that PDE(4) inhibitors could ameliorate the clinical course of MS. Unfortunately, clinical trials with PDE(4) inhibitors revealed the major adverse effects of these drugs, namely nausea and vomiting. However, novel PDE(4) inhibitors, which target only a subpopulation of PDE(4) enzymes, may provoke fewer adverse effects. The efficacy of a PDE(4) inhibitor in MS still needs to be demonstrated in a well designed clinical trial.     

人脐血单个核细胞移植治疗急性一氧化碳中毒后迟发脑病1年随访

背景：研究报道在特定的体内外环境下,脐血间充质干细胞能够诱导分化成为包括神经干细胞在内的多种组织细胞。 目的：评价人脐血单个核细胞经腰穿途径移植后治疗急性一氧化碳中毒后迟发性脑病的疗效及安全性。 方法：一氧化碳中毒后迟发性脑病患者60例随机分为2组。对照组给予高压氧及药物治疗;治疗组采用鞘内注射法将经密度梯度离心法分离出的人脐血单个核细胞移植到一氧化碳中毒性脑病患者的蛛网膜下腔,余治疗方法同对照组。分别于人脐血单个核细胞移植前、移植后3,9,12个月对患者进行简易精神状态检查法、改良Asworth肌肉痉挛程度分级及日常生活量表评分检查;比较两组患者MRI变化;同时对随诊患者行胸片、心电图及血生化检查,客观评价人脐血单个核细胞移植的安全性。 结果与结论：人脐血单个核细胞移植3,9,12个月,治疗组Asworth肌肉痉挛程度分级评分均显著低于对照组(P=0.032);移植后9,12个月简易智力状况检查法及日常生活量表评分均显著高于对照组(P < 0.05);两组患者神经功能在各时间点的变化趋势相似。人脐血单个核细胞移植后12个月MRI检查结果显示,治疗组患者MRI改善程度较对照组明显。提示鞘内注射移植人脐血单个核细胞治疗一氧化碳中毒后迟发脑病疗效优于高压氧治疗。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

盐霉素联合脐血间充质干细胞干预对乳腺癌MCF-7细胞的影响及作用机制

BACKGROUND:Mesenchymal stem cells-released factors can form a microenvironment inhibiting malignant tumor cell proliferation, and moreover, salinomycin also inhibits the growth of tumor cells. OBJECTIVE:To explore the effect of salinomycin with umbilical cord blood mesenchymal stem cells in breast cancer MCF-7 cells and its action mechanism, in order to seek a new target and therapeutic strategy to treat breast cancer. METHODS:Logarithmically growing MCF-7 cells were randomly divided into control, umbilical cord blood mesenchymal stem cell group and combination group. Cells in the control group were given no treatment, while those in the other two groups were given umbilical cord blood mesenchymal stem cell suspension or umbilical cord blood mesenchymal stem cell suspension combined with salinomycin, respectively. RESULTS AND CONCLUSION:Forty-eight hours after the intervention, the proliferation and invasion of MCF-7 cells and expression of POSTN protein in cells were significantly reduced in the combination group compared with the other two groups. These findings indicate that the combination of umbilical cord blood mesenchymal stem cell suspension and salinomycin effectively reduces the proliferation and migration of MCF-7 cells, and this combined use provides a new insight into the treatment of breast cancer.     

Immunological aspects of phosphodiesterase inhibition in the respiratory system

Phosphodiesterases (PDEs) are known as a super-family of 11 isoenzymes, which can exert various functions based on their organ distribution. Aside from non-selective PDE inhibitors (methylxanthines, e.g. theophylline) used many years in clinical settings, increasing attention is focused on the involvement of selective PDE inhibitors in therapy of obstructive airway diseases associated with chronic inflammation. There are mostly PDE3, PDE4, and PDE7 isoforms present in the respiratory system. This paper describes the mechanisms of action, adverse effects, and potential clinical use of both non-selective and selective PDE inhibitors. The focus of the review is on the influence of PDE inhibitors on the immune system.