Damage-associated molecular pattern (DAMP) activation in melanoma: investigation of the immunogenic activity of 15-deoxy, Δ12,14 prostamide J2

Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, Δ^12,14 prostamide J₂ (15dPMJ₂). As such, the current study investigates whether 15dPMJ₂ induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ₂ caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ₂ also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ₂ to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ₂-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ₂-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ₂ and the ER stress pathway. These results demonstrate that 15dPMJ₂ is a tumor-selective inducer of DAMP signaling in melanoma.     

Host MyD88 signaling protects against acute graft-versus-host disease after allogeneic bone marrow transplantation

Recent experimental strategies to reduce graft-versus-host disease (GVHD) have focused largely on modifying innate immunity. Toll-like receptor (TLR)-driven myeloid differentiation primary response 88 (MyD88)-dependent signalling pathways that initiate adaptive immune function are also critical for the pathogenesis of GVHD. This study aimed to delineate the role of host MyD88 in the development of acute GVHD following fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT). When myeloablated BALB/c MyD88 knock-out recipients were transplanted with C57BL/6 (B6) donor cells, they developed significantly more severe GVHD than wild-type (WT) BALB/c hosts. The increased morbidity and mortality in MyD88^–/– mice correlated with increased serum levels of lipopolysaccharide and elevated inflammatory cytokines in GVHD target organs. Additionally, MyD88 deficiency in BMT recipients led to increased donor T cell expansion and more donor CD11c⁺ cell intestinal infiltration with apoptotic cells but reduced proliferation of intestinal epithelial cells compared with that in WT BMT recipients. Decreased expression of tight junction mRNA in epithelial cells of MyD88^–/– mice suggested that MyD88 contributes to intestinal integrity. Cox-2 expression in the GVHD-targeted organs of WT mice is increased upon GVHD induction, but this enhanced expression was obviously inhibited by MyD88 deficiency. The present findings demonstrate an unexpected role for host MyD88 in preventing GVHD after allogeneic BMT.     

负载膀胱癌冻融抗原对树突状细胞分泌IL-12的影响

目的 探讨负载膀胱癌冻融抗原对树突状细胞（DCs）分泌IL-12的影响。方法 反复冻融法获得膀胱癌细胞株BIU-87细胞抗原；联合应用重组人的GM-CSF、IL-4和TNF-α体外诱导健康志愿者DCs并负载膀胱癌肿瘤抗原；用ELISA法检测第3、6和9天培养细胞的上清液，评价DCs分泌IL-12 p70的能力。结果 联合应用重组人的GM-CSF、IL-4和TNF-α可在体外诱导出DCs；经ELISA方法检测，不同时期的DCs分泌IL-12的量不同，而且负载抗原DCs较未负载抗原DCs有更强的分泌能力（P〈0．05）。结论 IL-12 p70的分泌量受DCs的成熟状态及某些刺激信号的影响，膀胱癌冻融抗原是其刺激信号之一。     

树突状细胞激活的肿瘤浸润性淋巴细胞体外对自体肝癌细胞杀伤活性的研究

目的探讨树突状细胞(DC)激活的肿瘤浸润性淋巴细胞(TIL)体外对自体肝癌细胞杀伤活性.方法从肝癌患者外周血获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)和肿瘤抗原激活DC,然后用DC来激活TIL,观察TIL在体外对自体肝癌细胞的杀伤活性.结果DC激活的TIL具有很高的对自体肝癌细胞杀伤活性,杀伤率为(89.39±3.05)%,明显高于未经DC激活的TIL、CD激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体肝癌细胞的杀伤率.结论肝癌患者外周血DC能诱导TIL产生高效而特异的抗肝癌免疫.     

Bacterial ghosts (BGs)—Advanced antigen and drug delivery system

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2–3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.     

Primary Alloproliferative TH1 Response Induced by Immature Plasmacytoid Dendritic Cells in Collaboration with Myeloid DCs

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.     

Synaptic organization of cortico-cortical connections from the primary visual cortex to the posteromedial lateral suprasylvian visual area in the cat

The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.     

脂多糖刺激树突状细胞前后TLR2、3、4基因表达的检测

目的 检测脂多糖作用于树突状细胞前后TLR2、3、4基因的表达情况。方法 从外周血分离单核细胞，加入rhGM-CSF及rhIL-4，培养的第6天，实验组加入脂多糖刺激24h，对照组不加。分别提取细胞总RNA，行半定量RT-PCR，产物进行琼脂糖凝胶电泳分析。结果 单核细胞经GM-CSF及IL-4诱导7d后的DC表达较高水平TLR2、3、4。脂多糖刺激24h后，TLR2、3、4表达下降，TLR4几乎测不到。结论 树突状细胞在受脂多糖刺激前后TLRs的表达有显著变化，推测和它的功能成熟有关。     

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory‐tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis. Objective To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. Methods Sputum cells were induced from steroid‐naïve, allergen‐challenged and allergen‐naïve subjects (atopic asthmatics, atopic non‐asthmatics and non‐atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. Results hRTDC stained HLA‐DR⁺ (negative for markers of other cell lineages) were predominantly myeloid and comprised ∼0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4⁺ naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC‐dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05–P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c⁻CD123^high hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. Conclusion Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.     

人端粒酶逆转录酶在肿瘤免疫治疗中的应用

寻找有效的抗原表位是近期肿瘤免疫治疗的热点．人端粒酶逆转录酶(hTERT)的免疫学特性使其成为肿瘤免疫治疗中一个吸引人的目标。从人和鼠系统获得的数据证实，细胞毒淋巴细胞(CTL)能识别hTERT特异的肽并杀死多种组织类型hTERT表达的肿瘤细胞。由于hTERT在人肿瘤组织中的广泛表达，在极少正常组织的低水平表达，临床试验已开始检验将hTERT作为肿瘤免疫治疗目标的可行程度。近期树突状细胞(DC)转运系统及其相关技术的发展和成熟提供了一个快速有效筛选抗原肽的方法。