Organogenesis involves a series of dynamic morphogenesis and remodeling processes. Since feathers exhibit complex forms, we have been using the feather as a model to analyze how molecular pathways and cellular events are used. While several major molecular pathways have been studied, the roles of matrix degrading proteases and inhibitors in feather morphogenesis are unknown. Here we addressed this knowledge gap by studying the temporal and spatial expression of proteases and inhibitors in developing feathers using mammalian antibodies that cross react with chicken proteins. We also investigated the effect of protease inhibitors on feather development employing an in vitro feather bud culture system. The results show that antibodies specific for mammalian MMP2 and TIMP2 stained positive in both feather epithelium and mesenchyme. The staining co-localized in structures of E10-E13 developing feathers. Interestingly, MMP2 and TIMP2 exhibited a complementary staining pattern in developing E15 and E20 feathers and in maturing feather filaments. Although they exhibited a slight delay in feather bud development, similar patterns of MMP2 and TIMP2 staining were observed in in vitro culture explants. The broad spectrum pharmacological inhibitors AG3340 and BB103 (MMP inhibitors) but not Aprotinin (a plasmin inhibitor) showed a reversible effect on epithelium invagination and feather bud elongation. TIMP2, a physiological inhibitor to MMPs, exhibited a similar effect. Markers of feather morphogenesis showed that MMP activity was required for both epithelium invagination and mesenchymal cell proliferation. Inhibition of MMP activity led to an overall delay in the expression of molecules that regulate either early feather bud growth and/or differentiation and thereby produced abnormal buds with incomplete follicle formation. This work demonstrates that MMPs and their inhibitors are not only important in injury repair, but also in development tissue remodeling as demonstrated here for the formation of feather follicles.

2010-06-01·Neurological Research4区 · 医学

Adenovirus mediated transfer of p53, GM-CSF and B7-1 suppresses growth and enhances immunogenicity of glioma cells

4区 · 医学

Article

作者: Minpei Liu ; Xinli Feng ; Sizhe Feng ; Xiao Tian ; Dongsheng Pan ; Xiang Zhang ; Xuezhong Wei

OBJECTIVESMalignant gliomas are good targets for gene therapy because they have been proven incurable with conventional treatments. However, malignant gliomas are genetically and physiologically highly heterogeneous, and current gene therapy interventions have been designed to target only a few variations of this kind of disease. Hence, we developed a combined gene therapy approach using a recombinant adenovirus carrying human wild-type p53 (WT-p53), granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes (designated BB-102) to combat the disease.METHODSHuman malignant glioma cells U251 and U87 were transduced with BB-102. Expression of WT-p53, GM-CSF and B7-1 genes were determined by Western blot, enzyme linked immunosorbent assay and flow cytometric analysis, respectively. Growth rates were determined by serial cell counts. Apoptosis was detected by flow cytometric analysis. Proliferation of autologous peripheral blood lymphocytes (PBLs) and cytotoxicity against primary glioma cells were assessed by cell proliferation and cytotoxicity assay kits, respectively.RESULTSBy the transduction of BB-102, high expression levels of the three exogenesis genes were detected in glioma cells. Cell growth was inhibited and apoptosis was induced. Significant proliferation of autologous PBLs and specific cytotoxicity against primary glioma cells were also induced by the infection of BB-102 in vitro, with the effect being more evident than that of Ad-p53.CONCLUSIONThese results suggest that glioma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a feasible and effective immunotherapeutic approach in glioma treatments.

2003-03-01·Marine Environmental Research2区 · 环境科学与生态学

Serum antibody levels against select bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from Beaufort NC USA and Charleston Harbor, Charleston, SC, USA

2区 · 环境科学与生态学

Article

作者: C.D Rice ; B.M Beck

Concern over the emergence of zoonotic diseases in marine organisms is growing. In response to this concern, this study set out to measure antibody activities against bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from the coastal estuaries of NC and SC, USA. Individuals from Charleston SC harbor, a heavily industrialized shipping harbor estuary, and from Beaufort NC, a non-shipping estuary, were examined. Purified IgG was obtained from pooled sera using ammonium sulfate precipitation steps and protein-G procedures, which was then used to generate a panel of IgG-specific monoclonal antibodies. Two of these antibodies, mAbs BB-10-2 (IgG1) and BB-32-2 (IgG2b), were then used to determine total serum IgG concentrations using a sandwich capture ELISA. Circulating IgG levels were variable between individuals and between the two pods. MAb BB-10-2 was then used in an indirect ELISA to determine serum antibody activities against several common marine bacteria as well as the human pathogens E. coli and E. coli strain 0157:H7, Vibrio parahemolyticus, V. vulnificus, V. cholerae, Mycobacteria marinum, M. fortuitum, and M. chelonae. The highest antibody activities were against mycobacteria, two of which are zoonotic pathogens. Males had the highest antibody activities, thus suggesting low cell-mediated immunity against intracellular pathogens in these individuals. T-cell proliferation in response to Con-A, an indicator of cell-mediated immune function, was then measured in the Beaufort population. Males had the lowest proliferation responses, however a negative correlation between antibody activities and T-cell proliferation in individuals could not be established for either of the Mycobacteria species. Overall, antibody activities against all bacteria, including innocuous species such as V. anguillarum, V. natrigens, and M. xenopi were highly variable between individual dolphins and the two pods, with some animals exhibiting very high activities. These studies suggests that dolphin populations should be monitored by following the health and seroprevalence of pathogens of interest in select individual animals over time.

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2021-03-13

·CPhI制药在线

一文盘点慢性乙型肝炎抗病毒新药研究进展

慢性乙型肝炎（chronic hepatitis B，CHB）是由乙型肝炎病毒（hepatitis B virus，HBV）引起的慢性进展性疾病，如果没有得到及时有效的抗病毒治疗，疾病最终将进展为肝硬化、肝衰竭或肝细胞癌（HCC）而导致死亡。抗病毒治疗是治疗CHB重要和有效的方法。大量临床研究和临床实践证明，无论采用核苷（酸）类似物（NAs），还是聚乙二醇干扰素-α（Peg-IFN-α），通过长期有效的抗病毒治疗，清除或抑制HBV后，可以明显改善患者的肝脏生物化学指标，并使肝组织炎症和纤维化得到缓解或逆转，阻止和延缓疾病进展为肝硬化、肝衰竭或肝细胞癌，从而改善患者的长期预后，降低患者的病死率。自从1999年第一个NAs抗病毒药物问世以来，乙肝抗病毒治疗的脚步就没有停止过。下面我们一起看下目前全球慢性乙型肝炎抗病毒新药研究进展。 1病毒进入抑制剂 Myrcludex B 它是MYR Pharma 公司自主研发的乙肝病毒进入抑制剂，在对照干扰素治疗期间，观察到Myrcludex B联合干扰素（IFN）在治疗慢性乙肝和慢性丁肝合并感染的HDV-RNA具有高效抑制作用。基于这项多中心、放开标签的二期临床试验，有少部分慢性乙肝患者实现了表面抗原消失。目前Myrcludex B正处于临床Ⅱ期试验中，具体上市时间仍未知。同时Blank等证实Myrcludex B对当前一线的NAs药物替诺福韦(TDF)的代谢无明显影响，且在临床I期试验中，无不耐受或严重并发症报道。 2衣壳抑制剂莫非赛定（GLS4） GLS4是我国自主研制的核心蛋白变构调节剂类乙肝新药（class I），由广东东阳光药业有限公司研发，可干扰HBV核衣壳的组装。在EASL2020年会上，报告了一项关于GLS4的Ⅱb期、多中心临床研究，在HBeAg阳性的慢性HBV感染者中对GLS4/利托那韦（RTV）联合恩替卡韦治疗及恩替卡韦单药治疗的有效性和安全性进行了评估。研究纳入250例慢性HBV感染者，分为初治组125人（未经治疗或最近6个月内未接触过抗HBV药物的患者）和经治组125人（服用恩替卡韦1年以上，HBV DNA＜50 IU/mL）。每组患者按4:1随机分配，接受120 mg GLS4/100 mg RTV（3次/日）联合0.5 mg 恩替卡韦（1次/日）联合治疗96周或0.5 mg恩替卡韦（1次/日）单药治疗96周。截止至报道时，研究纳入229名患者，有110名治疗长达24周以上。与基线时相比，在初治队列中，联合治疗组HBV DNA下降均值为6.04 log10 IU/mL，恩替卡韦单药组为4.92 log10 IU/mL；就HBV pgRNA而言，联合治疗组和恩替卡韦单药组下降的均值分别为3.53和0.73 log10 IU/mL；联合治疗组和恩替卡韦单药组，HBsAg下降的均值分别为0.69 log10 IU/mL和0.40 log10 IU/mL；HBeAg下降均值分别为0.89和0.58 log10 IU/mL。HBsAg下降≧0.5 log IU/mL的患者比例在联合治疗组和恩替卡韦单药组分别为46.9%（15/32）和28.6%（6/21），并且联合用药组中有3名患者（9.4%）HBsAg下降≧1.5 log IU/mL（分别为：从75110 IU/mL降至1089 IU/mL; 从＞128000 IU/mL降至2559 IU/mL; 从73300 IU/mL降至242.9 IU/mL）。在经治队列中，与基线时相比，联合治疗组和恩替卡韦单药组HBV pgRNA的下降均值分别为1.55 log10 IU/mLl和0.16 log10 IU/mL，HBsAg的下降均值分别为0.10 log10 IU/mL和0.05 log10 IU/mL；HBeAg的下降均值分别为0.23 log10 IU/mL和0.14 log10 IU/ml。联合治疗组和恩替卡韦单药治疗组HBsAg下降≧0.1log IU/mL的患者比例分别为46.9% （15/32）和8.0%（2/25）。 GLS4/RTV联合恩替卡韦具有良好的安全性和耐受性，最常见的不良反应是丙氨酸氨基转移酶（ALT）升高和高甘油三脂血症，这种不良反应通常可自行缓解，或在短暂停药后获得缓解。联合用药组的抗病毒治疗效果明显优于恩替卡韦单药组。 3核酸干扰药物或其他核酸药物 ARO-HBV（JNJ3989） ARO-HBV（JNJ3989）是由Arrowhead公司研发的核酸干扰药物。在临床研究中，在HBeAg阳性或阴性，以及未经治或用核苷（酸）类似物治疗患者中，每月1次皮下给药，共给药3次，剂量分别为25、50、100、200、300、400 mg。在给药后的113 d，HBsAg平均降低值（log IU/ml）分别为1.00 (25 mg组, n=8)、1.18 (50 mg组, n=8)、1.54 (100 mg组, n=8)、1.71 (200 mg, n=7)、1.48 (300 mg, n=8)、1.75 (400 mg, n=8)。所有患者都达到了HBsAg降低1个log的目标，并且该效果能在最后一次给药后持续4个月以上。治疗后80%的患者HBsAg最低值≤100 IU/ml，35%的患者HBsAg最低值≤10 IU/ml，12.5%的患者HBsAg最低值≤1 IU/ml。后续增加给药频次，即每两周或每周100 mg剂量，或者每周剂量200 mg或300 mg, 研究结果都证实更频繁的用药并没有增加HBsAg降低的速度和程度。在该研究中，HBV DNA、HBV RNA、HBeAg和HBcrAg水平也有一定降低。Arrowhead和Janssen公司也探索了JNJ3989联合用药的潜力，在最新公布的JNJ3989+JNJ6379联合核苷（酸）类似物药物的队列研究结果中，所有12例患者都达到了HBsAg降低1.0 log IU/ml的目标。并且在HBeAg阳性（n=4, 平均最低值为-1.52 log IU/ml）或阴性患者（n=8, 平均最低值为-1.76 log IU/ml）中这一效果无差异。所有患者的基线HBsAg＞100 IU/ml，但在治疗12周后，所有患者的HBsAg＜100 IU/ml。目前该品种可能会扩大研究病例数，继续探索在CHB患者中的有效性和安全性。此外，目前在研的核酸干扰品种还有AB729、VIR-2218、RG6346、SR016、STSG-002、BB-103等。 4免疫调节剂 Vesatolimod（GS-9620）免疫调节剂目前在研项目（除疫苗外）主要集中的靶点为TLR7、TLR8、PD-L1以及PD-1等。针对TLR7靶点，Gilead公司曾开发Vesatolimod（GS-9620）。在一项临床2期多中心、随机双盲、安慰剂对照研究中评估了Vesatolimod在初治CHB患者中的有效性和安全性。研究纳入192例患者，根据HBeAg状态和转氨酶水平按照2∶2∶2∶1的比例随机分为4组，分别接受Vesatolimod 1 mg、2 mg、4 mg或安慰剂治疗，每周1次给药，共12周。同时所有受试者使用每日1次的替诺福韦300 mg，共48周。第24周时，给药组中只有10例患者（6%）HBsAg经历了0.5 log的降低，而安慰剂组降低的比例为11%，没有患者HBsAg消失。3例患者在第48周时发生了HBeAg消失和血清学转换，其中2例发生在安慰剂组。由于Vesatolimod对于HBV病毒学指标的改变并不明显，治疗HBV感染相关研究已终止。总之，尽管抗病毒治疗已经走过了近20个年头，临床研究的证据在不断增加，药物的疗效强且不良反应小，但仍有一些尚待解决和需要进一步研究的问题。如不能简单的从单一指标的改变来评判孰优孰劣等，虽然CHB的治愈之路依然充满挑战，但曙光就在前方。最后，让我们共同期待未来有更多的抗病毒药物研发上市，造福病患。参考来源： 1. Mouzannar K, Liang TJ. Hepatitis B virus - recent therapeutic advances and challenges to cure. J Hepatol. 2020;S0168-8278(20)30227-0. doi:10.1016/j.jhep.2020.04.015. 2. Seto, Wai-Kay, Ying-Ru Lo, Jean-Michel Pawlotsky, and Man-Fung Yuen. Chronic hepatitis B virus infection [J]. The Lancet.2018, 392 (10161): 2313-24. 3. EASL2020：SAT452. 4. Kitamura K, Que L, Shimadu M, et al. Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus[J]. PLoS Pathog, 2018, 14(6): e1007124. DOI: 10.1371/journal.ppat.1007124. 5.Fanning et al., (2019). Therapeutic strategies for hepatitis B virus infection: towards a cure. Nature Reviews Drug Discovery.

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