Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

Background

While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.

Methods

We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.

Findings

Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.

Conclusions and Hypothesis

Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.     

The B Cell Adaptor Molecule Bam32 Is Critically Important for Optimal Antibody Response and Resistance to Trypanosoma congolense Infection in Mice

Background

Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection.

Methodology/Principal Findings

We found that T. congolense-infected Bam32^-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32^-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32^-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32^-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32^-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32^-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32^-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32^-/- mice to T. congolense infection.

Conclusions/Significance

Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.     

Retracted: Screening of candidate genes and fine mapping of drought tolerance quantitative trait loci on chromosome 4 in rice (Oryza sativa L.) under drought stress

Due to severe water resource shortage, genetics of and breeding for DT (drought tolerance) in rice (Oryza sativa L.) have become one of the hot research topics. Identification of grain yield QTLs (quantitative trait loci) directly related to the DT trait of rice can provide useful information for breeding new drought‐resistant and water‐saving rice varieties via marker‐assisted selection. A population of 105 advanced BILs (backcross introgression lines) derived from a cross between Zhenshan97B and IRAT109 in Zhenshan97B background were grown under drought stress in a field experiment and phenotypic traits were investigated. The results showed that in the target interval of RM273‐RM255 on chromosome 4, three main‐effect QTLs related to panicle length, panicle number, and spikelet number per panicle were identified (LOD [logarithm of the odds] > 2.0). The panicle length‐related QTL had two loci located in the neighboring intervals of RM17308‐RM17305 and RM17349‐RM17190, which explained 18.80% and 20.42%, respectively, of the phenotypic variation, while the panicle number‐related QTL was identified in the interval of RM1354‐RM17308, explaining 11.47% of the phenotypic variation. As far as the spikelet number per panicle‐related QTL was concerned, it was found to be located in the interval of RM17308‐RM17305, which explained 28.08% of the phenotypic variation. Using the online Plant‐GE query system, a total of 13 matched ESTs (expressed sequence tags) were found in the target region, and of the 13 ESTs, 12 had corresponding predicted genes. For instance, the two ESTs CB096766 and CA765747 were corresponded to the same predicted gene LOC_Os04g46370, while the other four ESTs, CA754286, CB000011, CX056247, and CX056240, were corresponded to the same predicted gene LOC_Os04g46390.     

Expression of TLR2/4 in the sperm‐storing oviduct of the Chinese soft‐shelled turtle Pelodiscus sinensis during hibernation season

The initiation of innate immunology system could play an important role in the aspect of protection for sperms long‐term storage when the sperms got into oviduct of turtles and come into contact with epithelium. The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable. The objective of this study was to examine the gene and protein expression profiles in Chinese soft‐shelled turtle during hibernation from November to April in the next year. The protein distribution of TLR2/4 was investigated in the magnum, isthmus, uterus, and vagina of the turtle oviduct using immunohistochemistry, and the gene expression of TLR2/4 was analyzed using quantitative real‐time PCR (qRT‐PCR). The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct. TLR4 immunoreactivity was widely observed in almost every part of the oviduct, particularly in the epithelium and secretory gland membrane. Analysis of protein, mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus, uterus, and vagina during hibernation. The protein and mRNA expression of TLR2 in the magnum, isthmus, uterus, and vagina was decreased in April compared with that in November. TLR4 protein and mRNA expression in the magnum, isthmus, uterus and vagina was decreased in November compared with that in April. These results indicated that TLR2/4 expression might protect the sperm from microbial infections. In contrast to the function of TLR2, which protects sperm during the early stages of hibernation, TLR4 might play a role in later stages of storage. The present study is the first to report the functions of TLR2/4 in reptiles.     

苜蓿夜蛾保幼激素酯酶cDNA的克隆、序列分析及表达研究

【目的】研究苜蓿夜蛾Heliothis viriplaca保幼激素酯酶(Juvenile hormone esterase,JHE)的功能和作用机理。【方法】本试验提取苜蓿夜蛾的总RNA,并利用RT-PCR和RACE技术,扩增得到苜蓿夜蛾保幼激素酯酶基因的全长c DNA序列,命名为Hvjhe(Gen Bank登录号:JQ901384)。【结果】该基因含有3 106 bp,包括一个1 746 bp的开放阅读框,编码一个含581个氨基酸的多肽,分子量约为63.9 ku,多肽的等电点为5.11,且氨基酸序列具含有5个保幼激素酯酶特有的氨基酸保守模块。推导的氨基酸序列与烟芽夜蛾Heliothis virescens和棉铃虫Helicoverpa armigera中获得的保幼激素酯酶相似性较高,分别达到83%和82%。荧光定量PCR结果表明,该基因在苜蓿夜蛾不同发育时期和不同组织中都有m RNA水平的特异性表达,且在预蛹期表达量相对较高,取食期、蛹期期和成虫期表达量相对较低;在中肠和脂肪体内的表达量相对于其它组织较高。该基因在大肠杆菌Escherich coli表达系统中进行了诱导表达,经SDS-PAGE和Western blot检测结果表明,表达出与预测的蛋白分子量相符的融合蛋白。【结论】研究结果表明本试验获得了苜蓿夜蛾中一个新的保幼激素酯酶基因的c DNA序列。     

柑橘大实蝇生物、生态学及辐照不育技术最新研究进展

柑橘大实蝇是柑橘类果树的一种重要害虫,近几年在我国柑橘种植区的危害呈上升趋势,给柑橘产业造成了巨大损失。本文概述了环境因素包括温度、光周期、营养状况(饥渴)以及水淹对柑橘大实蝇生物学特性和成虫行为的影响,柑橘大实蝇成虫和幼虫人工饲养技术参数,柑橘大实蝇蛹滞育机制,辐照不育关键技术包括最佳辐照剂量和辐照时期,及其在湖北柑橘园柑橘大实蝇防控中的应用与示范情况等,并指出了尚需进一步开展的研究内容。     

不同产地丁香挥发性成分分析

采用顶空固相微萃取-气相色谱-质谱联用技术对产自国内外4个地区的丁香(Eugenia caryophyllata Thunb.)挥发性成分进行了检测,并以峰面积归一化法计算了各组分的相对含量。结果显示,从4个产地13批次丁香中共鉴定出挥发性成分72种(匹配度均高于75),其中,主要成分含量排名前两位的均为丁香酚、(-)-α-芹子烯,排名第三位的挥发性成分分别为:顺式-α-没药烯(马达加斯加塔马塔夫,平均为3.75%)、罗勒烯(中国广东,平均为4.21%)、亚麻三烯(中国广西,平均为3.74%)、丁香烯(印度尼西亚爪哇岛,平均为3.60%)。表明同一产地的丁香挥发性成分具有一定的相似性,不同产地丁香挥发性成分也有一定的差异,这对丁香产地的鉴别具有重要意义。主成分分析和聚类分析的结果既可以将4个产地的丁香很好地区分开来,又能反映出它们之间的亲缘关系。本研究采用HS-SPME-GC-MS方法检测不同产地丁香挥发性成分并结合主成分分析(PCA)及聚类分析法,能有效区分4个产地的丁香,该方法可作为丁香产地的鉴别方法,也为进一步比较不同产地丁香挥发性成分的差异及质量控制提供了新思路。     

黑曲霉柠檬酸工业菌株原生质体制备与转化

黑曲霉是柠檬酸工业化生产的主要发酵菌株。尽管现代遗传操作技术在黑曲霉的实验室菌株、蛋白质生产菌株等的应用中获得了成功,但是柠檬酸工业菌株遗传转化异常困难,成为柠檬酸工业持续升级的重要限制因素。以柠檬酸工业生产实际应用的黑曲霉菌株为研究对象,对其原生质体的制备与再生以及PEG介导的转化等条件进行了细致优化。结果表明,柠檬酸高产工业菌株原生质体的制备需选取丰富培养基中培养48 h的年轻菌丝体,在1.5%裂解酶-0.5%蜗牛酶-0.2%溶菌酶的复合酶解体系下裂解2.5 h,原生质体的制备浓度可达106个/m L以上,原生质体再生效率可达90%以上。原生质体的浓度是原生质体-PEG介导转化方法的关键,当原生质体浓度达到106个/m L以上时,高产柠檬酸菌株的转化效率大幅提高。成功建立了由原生质体-PEG所介导的高产柠檬酸黑曲霉菌株的遗传转化体系。     

A Network Meta-Analysis of Efficacy and Evaluation of Safety of Subcutaneous Pegylated Interferon Beta-1a versus Other Injectable Therapies for the Treatment of Relapsing-Remitting Multiple Sclerosis

Subcutaneous pegylated interferon beta-1a (peginterferon beta-1a [PEG-IFN]) 125 μg every two or four weeks has been studied in relapsing-remitting multiple sclerosis (RRMS) patients in the pivotal Phase 3 ADVANCE trial. In the absence of direct comparative evidence, a network meta-analysis (NMA) was conducted to provide an indirect assessment of the relative efficacy, safety, and tolerability of PEG-IFN versus other injectable RRMS therapies. Systematic searches were conducted in MEDLINE, Embase, and the Cochrane Library, and conference proceedings from relevant annual symposia were hand-searched. Included studies were randomized controlled trials evaluating ≥1 first-line treatments including interferon beta-1a 30, 44, and 22 μg, interferon beta-1b, and glatiramer acetate in patients with RRMS. Studies were included based on a pre-specified protocol and extracted by a team of independent reviewers and information scientists, utilizing criteria from NICE and IQWiG. In line with ADVANCE findings, NMA results support that PEG-IFN every 2 weeks significantly reduced annualized relapse rate, and 3- and 6-month confirmed disability progression (CDP) versus placebo. There was numerical trend favoring PEG-IFN every 2 weeks versus other IFNs assessed for annualized relapse rate, and versus all other injectables for 3- and 6-month CDP (6-month CDP was significantly reduced versus IFN beta-1a 30 μg). The safety and tolerability profile of PEG-IFN beta-1a 125 μg every 2 weeks was consistent with that of other evaluated treatments. Study limitations for the NMA include variant definitions of relapse and other systematic differences across trials, assumptions that populations were sufficiently similar, and inability to perform NMA of adverse events. With similar efficacy compared to other RRMS treatments in terms of annualized relapse rate and 3- and 6-month CDP, a promising safety profile, and up to 93% reduction in number of injections (which may improve adherence), PEG-IFN every 2 weeks offers a valuable alternative treatment option for patients with RRMS.