黑素瘤B16-F1体外诱导淋巴管增生的研究

目的诱导建立小鼠淋巴管内皮细胞构成的良性肿瘤模型,观察小鼠黑素瘤细胞系B16F1体外对淋巴管增生的影响。方法8周龄C57BL/6小鼠腹腔注射弗氏不完全佐剂,对诱导出的小鼠腹腔淋巴管瘤进行病理组织学观察,用免疫组化方法检测淋巴管内皮细胞标志性抗原VEGFC和Flt4。分离、分切肿物后,于纤维蛋白凝胶内用B16F1细胞条件培养液培养,倒置显微镜下观察。结果实验小鼠中膈肌腹腔面、肝脏表面及侧腹壁腹腔面可见边界清楚的散在分布白色肿瘤样组织,常规和超微病理发现为由内皮细胞构成的多管腔囊性结构,并表达淋巴管内皮细胞标志性抗原VEGFC和Flt4。倒置显微镜下可观察到从淋巴管瘤块长入纤维蛋白凝胶内的微淋巴管,B16F1细胞条件培养液可促进淋巴管的生成。结论小鼠腹腔注射弗氏不完全佐剂可稳定诱导小鼠腹腔淋巴管瘤,B16F1细胞对淋巴管的生成有促进作用,黑素瘤的转移与淋巴管生成的关系有必要进一步研究。     

Tumor regression of human and murine melanoma after intratumoral injection of IL-12-encoding plasmid DNA in mice

DNA coding for murine interleukin 12 (IL-12) prevents the formation of B16-melanoma metastasis when administered intramuscularly. Here, the antitumor effect of IL-12-encoding DNA on established mouse B16 melanoma and human melanoma tumors was investigated in vivo using two animal models: B16 melanoma in C57B/6 mice and human melanoma in nude mice. In B16 melanoma, intratumoral injections of IL-12-encoding DNA resulted in highly significant growth retardation when compared with mice injected with control vector. In the case of the human melanoma model, treatment with DNA coding for IL-12 induced regression of tumors in all cases, with complete disappearance of the tumor in two out of five animals. DNA treatment did not induce systemic side-effects. In the animals injected with control vector the human melanoma tumors grew expansively. The therapeutic effect of the DNA injection was mediated in part by natural killer (NK) cells as shown by NK-depletion experiments. An antivascular effect of IL-12 treatment was evident in histological examination with endothelial thickening and abrupt changes in vessel diameters. These results suggest that intratumoral plasmid DNA coding for IL-12 holds some promise as a new therapeutic tool for accessible melanoma lesions and should be tested in clinical trial.     

B16F10黑素瘤细胞培养上清对相同基因小鼠腹腔巨噬细胞的抑制作用

目的探讨B16F10黑素瘤细胞培养上清对相同基因小鼠腹腔巨噬细胞的抑制作用。方法制备B16F10黑素瘤细胞培养上清,并作用于相同基因小鼠腹腔巨噬细胞,经细菌脂多糖(LPS)刺激,通过中性红吞噬试验检测巨噬细胞吞噬功能和MTT法检测巨噬细胞活性。结果在B16F10黑素瘤细胞培养上清的作用下,LPS诱导的相同基因小鼠腹腔巨噬细胞吞噬中性红的能力为0.552±0.060,巨噬细胞活性为0.411±0.035,与对照组(1.247±0.164和0.861±0.208)相比,差异均有统计学意义(P均<0.005)。结论 B16F10黑素瘤细胞培养上清对相同基因小鼠腹腔巨噬细胞具有抑制作用。     

光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤MIF表达的影响

目的:探讨光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤巨噬细胞移动抑制因子(MIF)表达的影响。方法:选取BALB/c裸鼠,皮下注射人宫颈癌Caski细胞,10d后将达到荷瘤标准的40只裸鼠采用随机数字表法分为四组,即A组、B组、C组和D组,各10只。A组作为阴性对照、B组给予单次光动力疗法、C组给予多次光动力疗法和D组给予顺铂腹腔注射。对比治疗前和治疗2周后肿瘤体积,治疗前后裸鼠体质量、瘤体积、瘤质量变化及抑瘤率,并分别采用western-blot和免疫组化法对各组裸鼠肿瘤组织MIF表达情况进行检测,行对比分析。结果:B组、C组和D组体质量、瘤体积和瘤质量变化和A组比较差异均有统计学意义(P<0.05),且C组和D组上述指标及抑瘤率和B组比较差异均有统计学意义(P<0.05),而C组和D组间各项指标差异均无统计学意义(P>0.05);电镜分析结果显示各组肿瘤胞浆可见明显空泡,团间有明显坏死灶,并且随着光动力疗法剂量增高,坏死区域增大越明显。B组、C组和D组MIF蛋白IOD值均较A组显著降低(P<0.05),且C组和D组MIF蛋白相对表达量和IOD值均较B组显著降低(P<0.05),而C组和D组MIF蛋白相对表达量和IOD值比较差异均无统计学意义(P>0.05)。结论:采用多次光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤效果和顺铂相近,且可显著降低MIF表达水平。     

载芬维A铵脂质体抑制A375黑素瘤细胞裸鼠皮下移植瘤的初步研究

目的 探讨载芬维A铵脂质体（4-HPR-L）对裸鼠皮下人恶性黑素瘤的抑制作用。方法 采用薄膜-超声分散法制备4-HPR-L。通过皮下接种黑素瘤A375细胞至BALB/c裸鼠右侧腋窝建立黑素瘤荷瘤裸鼠模型。取10只荷瘤裸鼠模型,随机等分为两组,分别给予尾静脉注射同浓度细胞膜近红外荧光探针（DiR）溶液和DiR脂质体（DiR-L）,应用小动物活体成像仪观察给药后6、12、24 h药物在体内分布情况。取30只荷瘤裸鼠,随机等分为3组,即对照组、4-HPR组和4-HPR-L组,分别经尾静脉每次注射5%（质量分数）葡萄糖溶液0.2 ml、25 mg/kg 4-HPR和4-HPR-L溶液,于接种A375细胞后第8、10、12、14、16、18、20、22天给药,动态监测给药后各组裸鼠的体重和肿瘤体积,观察生存情况。于末次给药后第2天处死裸鼠,取心、肝、脾、肺、肾及肿瘤组织,HE染色和免疫组化染色观察黑素瘤体内转移情况,并用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测肿瘤细胞凋亡情况。计量资料采用单因素方差分析和独立样本t检验进行分析。结果 小动物活体成像仪显示,DiR-L能较长时间滞留于肿瘤组织,给药24 h后在肿瘤部位仍可观察到较强荧光;定量分析显示,肿瘤组织中DiR-L荧光强度（22.85 ± 1.66）显著高于DiR（8.45 ± 0.97,t = 12.957,P < 0.01）。与对照组和4-HPR组相比,4-HPR-L组末次给药后第2天离体瘤重明显降低（F = 27.055,t值分别为4.768、6.640,均P < 0.05）。HE染色显示,4-HPR-L组2只裸鼠发生肝脏转移,而对照组、4-HPR组全部裸鼠发生肝脏转移。荷瘤鼠的生存期观察显示,4-HPR-L组裸鼠于接种后76 d内全部死亡,对照组和4-HPR组分别于接种后56 d和59 d内全部死亡。对照组凋亡指数为（12.14 ± 1.33）‰,4-HPR组为（67.17 ± 15.18）‰,4-HPR-L组为（152.73 ± 11.27）‰,3组间差异有统计学意义（F = 167.588,P < 0.05）,4-HPR-L组与对照组和4-HPR组相比,t值分别为18.162、11.075,均P < 0.05。结论 4-HPR-L能够有效抑制裸鼠皮下黑素瘤体积增长和黑素瘤细胞转移,并延长裸鼠生存期。     

Tumor apoptosis by indole-3-acetic acid/light in B16F10 melanoma-implanted nude mice

Recently, we reported that UVB-activated indole-3-acetic acid (IAA) induces the apoptosis of G361 human melanoma cells. In the present study, we used IAA and visible light combinations to treat B16F10 melanoma-implanted nude mice using an experimental intense pulsed light (IPL) therapy model. We first investigated whether activated IAA by horseradish peroxidase (HRP) or UVB causes apoptosis of B16F10 melanoma cells. IAA/HRP or IAA/UVB combination lead to apoptosis of B16F10 cells, as reported in other cell lines. Interestingly, IAA alone was not cytotoxic. These findings suggested the potential use of IAA in the treatment of melanoma. For the future clinical use, we also tested whether visible light has the same effects like UVB and found that visible light also activates IAA to produce free radicals and that IAA/visible light decreased cell viability significantly. Based on these results, IAA/IPL combination was tried whether it can induce apoptosis in vivo status. TUNEL staining showed that IAA/IPL treatment induced apoptosis of tumor cells. In addition, the expressions of p53, Fas, and PARP were upregulated in the IAA/IPL-treated group than in untreated control, demonstrating that IAA/IPL treatment caused apoptosis in melanoma-implanted nude mice. In conclusion, we showed that IAA/IPL induces melanoma regression in B16F10 melanoma-implanted nude mice. These results suggest the potential use of IAA/IPL in the treatment of malignant melanoma.     

大蒜素对小鼠黑素瘤细胞B16-F1增殖和凋亡的影响

目的探讨大蒜素对小鼠黑素瘤细胞B16-F1增殖及凋亡的影响。方法传代培养小鼠黑素瘤B16-F1细胞,使细胞浓度为5×104个/m l,分别加入不同浓度(3,6,9,12,15μg/m l)的大蒜素细胞培养液。培养12,24,36,48 h后,噻唑蓝(MTT)法检测细胞生存活性,原位末端标记(TUNEL)法检测细胞凋亡,定量RT-PCR法检测ras基因的表达水平。结果3~15μg/m l的大蒜素对B16-F1细胞均有杀伤作用,当大蒜素的浓度为3~6μg/m l,诱导肿瘤细胞凋亡,且呈浓度时间相关性。当大蒜素的浓度较高时,细胞呈中毒反应,凋亡作用不明显。3~15μg/m l的大蒜素下调ras基因的表达,并且呈显著的浓度相关性。结论大蒜素对小鼠黑素瘤B16-F1细胞有杀伤作用,可以明显诱导肿瘤细胞的凋亡,并且对肿瘤细胞ras基因的表达均有不同程度的下调作用。     

人瘢痕疙瘩成纤维细胞裸鼠荷瘤模型的建立

目的 建立一种简单、成功率高的人瘢痕疙瘩裸鼠荷瘤模型。 方法 27只雌性BALB/c裸鼠随机分为5组,A、B、C组每组5只,在每只裸鼠腋窝下接种人瘢痕疙瘩成纤维细胞和Matrigel胶混悬液0.1 ml,细胞浓度分别为1.0 × 104个/μl Matrigel胶（A组）、3.0 × 104个/μl Matrigel胶（B组）、5.0 × 104个/μl Matrigel胶（C组）。取C组成形瘤块修剪成若干个5 mm × 5 mm × 5 mm大小的组织块移植于D组裸鼠（8只）的腋窝皮下;E组裸鼠（4只）皮下注射100 μl Matrigel胶作为对照组。A、B、C组出瘤后30 d、D组瘤块移植后30 d肉眼观察瘤体的形成过程及变化,并在第31天处死裸鼠进行组织病理学检查,分析其移植后成形的瘤体组织学变化及裸鼠的心、肝、脾、肺、肾组织的变化。 结果 A、B、C组裸鼠成瘤率为100%,出瘤时间分别为（90.20 ± 3.96） d、（61.00 ± 2.92） d、（39.60 ± 3.20） d。出瘤30 d时3组瘤体体积分别为（288.34 ± 25.29） mm3、（1 370.63 ± 105.24） mm3、（1 940.98 ± 184.37） mm3,3组差异有统计学意义（F = 138.74,P < 0.05）。D组裸鼠成形瘤块移植后瘤块体积先略增大后逐渐减小,移植第14天始持续增大,8只中7只成瘤。E组裸鼠未见瘤体形成。组织病理学检查,各组瘤体的组织形态在镜下一致,与人瘢痕疙瘩组织相似,未见其他脏器组织学改变及转移瘤灶。结论 裸鼠皮下接种人瘢痕疙瘩成纤维细胞可建立瘢痕疙瘩裸鼠荷瘤模型,而且已成瘤组织修剪成一定体积再次移植于裸鼠皮下也可以建立瘢痕疙瘩裸鼠荷瘤模型。     

Response of epidermolysis bullosa fibroblasts to factors derived from macrophages and polymorphonuclear leukocytes in terms of collagenase production

In order to investigate the role of inflammatory cells in altering the collagenase production by epidermolysis bullosa (EB) fibroblasts, macrophage and polymorphonuclear leukocyte (PMN) factors obtained from mouse peritoneal fluids were added to the fibroblast culture system, and collagenase activity was assayed after a 48-h incubation. Data obtained here revealed that the response of collagenase production by fibroblasts was quite different, depending on the type of EB. Namely, EB dystrophica recessiva (EBDR) (n = 2) fibroblasts produced significant amounts of collagenase in the range of 5.07 (U/ml) to 6.04 in response to macrophage-conditioned medium, macrophage lysate, and PMN lysate, compared with 0.13 in the absence of these. On the other hand, EB dystrophica dominans (EBDD) (n = 1) fibroblasts showed little or no overt increase in enzyme production in the presence of macrophage lysate and PMN lysate, which resulted in a moderate increase to 3.82 in response to macrophage-conditioned medium. Furthermore, EB simplex (EBS) (n = 1) fibroblasts produced collagenase up to 3.84 in response to these three factors. These factors can be inactivated by treating with trypsin, pronase, and phenylglyoxal. Our data clearly indicated that, in the comparisons of EBDD and EBS fibroblasts, EBDR fibroblasts showed quite high response to factors derived from macrophages and PMNs in terms of collagenase production. This fact may raise a clue that accounts for the high levels of tissue collagenase activity, which plays a potentially major role in blister formation in EBDR.     

L929成纤维细胞在皮肤B16黑素瘤细胞成瘤中的作用

目的研究成纤维细胞在皮肤黑素瘤成瘤中的作用。方法以小鼠皮肤成纤维细胞系L929与小鼠皮肤黑素瘤细胞系B16为研究对象,细胞共培养体外诱导L929成纤维细胞分化为肌成纤维细胞,免疫细胞学检测a-SMA的表达,Western blot证实表型的变化;建立C57小鼠皮肤黑素瘤移植瘤动物模型,研究L929成纤维细胞对B16黑素瘤移植成瘤率的影响。结果B16黑素瘤细胞可以诱导部分L929成纤维细胞发生表型变化;L929成纤维细胞可提高B16黑素瘤细胞移植成瘤率。结论肿瘤细胞外基质中的成纤维细胞与皮肤黑素瘤细胞的成瘤性有关。     

Endogenous μ-opioid peptides modulate immune response towards malignant melanoma

Abstract: Opioids exert major effects not only in the central nervous system but also in immune responses. We investigated the effects of μ‐opioid peptides, secreted by tumor cells, on anti‐tumor immune responses. For this purpose, tumor growth was studied in wild‐type and μ‐opioid receptor–deficient (MOR?/?) mice injected with B16 melanoma cells. The ability of these cells to produce opioids was studied by Western blots in vitro. Finally, biopsy material from human melanomas was investigated by immunohistochemistry for ß endorphin expression. Injection of B16 melanoma cells, producing endogenous ß endorphin, in the flank of MOR?/? mice revealed a profound reduction in tumor growth, paralleled by a significantly higher infiltration of immune cells into the tumors, when compared to tumor growth after injection of B16 melanoma cells into wild‐type mice. Opioids present in B16 cell supernatant significantly reduced the proliferation of normal but not MOR?/? leucocytes. Immunohistochemical analyses of biopsies from human melanoma tissues showed a positive correlation between expression of ß endorphin and tumor progression. Our data provide evidence that μ‐opioid peptides may play a major role in cancer progression by modulating immune response. This finding may have implications for the future optimization of immunointerventions for cancer.     

T-钙黏蛋白对小鼠皮下氮烯咪胺耐药黑素瘤B16F10细胞株的影响

目的观察T-钙黏蛋白(cadherin)对小鼠皮下氮烯咪胺(dacarbazine,DTIC)耐药黑素瘤B16F10细胞株的影响。方法通过体外分步诱导法诱导B16F10氮烯咪胺耐药细胞株(DTIC-R B16F10)。采用CCK-8法检测DTIC-R B16F10细胞的增殖情况。将T-cadherin基因转染至DTIC-R B16F10细胞。免疫组织化学检测转染后T-cadherin的表达情况。培养DTIC-R B16F10细胞,分为6组:对照组、pEGFP-N1组、T-cadherin组、DTIC组、pEGFP-N1联合DTIC组和T-cadherin联合DTIC组。通过CCK-8法和Transwell小室实验检测T-cadherin与氮烯咪胺联合对DTIC-R B16F10细胞活性和迁移的影响。建立荷瘤小鼠模型,观察T-cadherin对小鼠皮下注射DTIC-R B16F10细胞后肿瘤生长的影响。采用析因分析法评价T-cadherin联合氮烯咪胺对细胞活性、迁移和小鼠皮下瘤生长的影响。结果DTIC-R B16F10组、B16F10组与DTIC-R B16F10+DTIC组A值差异无统计学意义(P>0.05)。免疫组织化学检测表明细胞表达T-cadherin。T-cadherin联合DTIC组细胞活性、迁移细胞数及肿瘤重量均显著低于其他组(P<0.05)。析因分析显示,T-cadherin联合氮烯咪胺对DTIC-R B16F10的细胞活性、迁移和小鼠皮下瘤生长的抑制有协同作用。结论T-cadherin可抑制黑素瘤小鼠皮下瘤的生长,增加黑素瘤对氮烯咪胺的敏感性。     

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.     

腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

目的　探讨腺病毒载体介导的内皮抑素基因（Ad-mES）在体外和体内的生物学活性。方法　不同感染复度（MOI）的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响。观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达。电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况。结果　腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%。靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强。瘤细胞接种后第8天,各组成瘤率100%。开始出现小鼠死亡的最早日：PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天。结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长（P < 0.05）,肿瘤体积增长减慢（P < 0.05）。     

腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

目的 探讨腺病毒载体介导的内皮抑素基因(Ad-mES)在体外和体内的生物学活性.方法 不同感染复度(MOI)的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响.观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达.电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况.结果 腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%.靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强.瘤细胞接种后第8天,各组成瘤率100%.开始出现小鼠死亡的最早日:PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天.结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长(P<0.05),肿瘤体积增长减慢(P<0.05).     

RANK is expressed in metastatic melanoma and highly upregulated on melanoma-initiating cells

Melanoma accounts for ～ 79% of skin cancer-related deaths, and the receptor activator of NF-κB (RANK)-receptor activator of NF-κB ligand (RANKL) pathway has been shown to be involved in the migration and metastasis of epithelial tumor cells. In this study, we demonstrate that RANK was significantly increased in peripheral circulating melanoma cells, primary melanomas, and metastases from stage IV melanoma patients compared with tumor cells from stage I melanoma patients. However, upregulated RANK expression was not found in stage IV melanoma patients with bone metastases compared with stage IV melanoma patients without bone metastases, providing a possible explanation for the clinical observation that melanoma cells do not preferentially metastasize to bone tissue. Strikingly, RANK-expressing melanoma cells from peripheral blood, primary tumors, or metastases of stage IV patients coexpressed ATP-binding cassette (ABC) B5 and CD133, both markers characteristic of melanoma-initiating cells, suggesting a tumor stem cell-like phenotype. In support of this hypothesis, RANK-expressing melanoma cells showed a reduced Ki67 proliferation index compared with RANK(-) melanoma cells from the same patient and are able to induce tumor growth in immunodeficient mice. Together, our data demonstrate that RANK expression is increased in metastatic melanoma and highly upregulated on melanoma-initiating cells, suggesting that RANK might be involved in the development and maintenance of melanoma-initiating cells and possibly in metastatic spreading.     

Transduced monocyte/macrophages targeted to murine skin by UV light

We have selectively targeted monocyte/macrophages overexpressing an immunomodulatory molecule, latency-associated peptide (LAP), a naturally occurring antagonist for transforming growth factor-beta1, to murine skin utilizing UV light to produce a cutaneous influx of transduced monocyte/macrophages. Bone marrow (BM) cells from BALB/c mice were transduced in vitro with a retroviral construct containing green fluorescent protein (GFP) for detection and human LAP (hLAP) as a test molecule. The transduced BM cells were then cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) to produce differentiation to monocyte/macrophages. More than 80% of transduced BM cells were CD11b-positive and MOMA-positive by fluorescence-activated cell-sorter analysis and secreted LAP by ELISA after 10 days of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF). Transduced monocyte/macrophages containing either GFP or hLAP-GFP were then injected intravenously into BALB/c mice. One-half of recipients in each group were exposed to UVB (72 mJ) to induce monocyte/macrophage infiltration into skin. Recipients were sacrificed 60 h after UV irradiation. We found transduced cutaneous macrophages expressing GFP by examining with fluorescence microscopy frozen skin sections of recipient mice immunostained with antibodies to GFP and to macrophage marker F4/80. We identified hLAP sequences by polymerase chain reaction (PCR) of total DNA in recipient blood and UV-irradiated skin but not in unirradiated skin. LAP sequences were also detected at much lower levels in other organs (lung, spleen, and liver) by PCR. Therefore, we have shown that genetically altered monocytic cells can be injected intravenously and targeted to mouse skin by UV exposure. This macrophage-based gene-transfer method may be a potentially useful immunotherapeutic approach for delivering monocyte/macrophage-derived products to skin.     

T-cell-immunity-based inhibitory effects of orally administered herbal medicine juzen-taiho-to on the growth of primarily developed melanocytic tumors in RET-transgenic mice

We examined the effect of oral administration of juzen-taiho-to, one of the most popular herbal medicines in Japan, on primary melanocytic tumor growth in RET-transgenic mice. There was virtually no difference between the lengths of tumor-free stages in the juzen-taiho-to-treated mice and the untreated littermate control mice. The rate of tumor growth in the juzen-taiho-to-treated mice, however, was greatly suppressed during the entire period after the initial tumor development. Correspondingly, the life span of juzen-taiho-to-treated transgenic mice was longer (over 6 mo in mean value) than that of control mice. We partially elucidated the mechanism of the antitumor effect of juzen-taiho-to. The addition of juzen-taiho-to at any of a wide range (50-1600 microg per ml) of concentrations to in vitro cultures of Mel-Ret cells, a malignant melanoma cell line derived from a RET-transgenic mouse, caused neither cell death nor cell cycle arrest directly. The addition of 50-400 microg per ml of juzen-taiho-to to cultures of murine spleen cells, however, promoted their DNA synthesis. More importantly, peritoneal exudate cells from the juzen-taiho-to-treated transgenic mice, in which the ratio and number of T cells were increased, displayed an antitumor immunity against Mel-Ret cells in vitro. Interestingly, the peritoneal-exudate-cell-associated antitumor immunity was further augmented by the addition of 200-400 microg per ml of juzen-taiho-to in vitro. This immunity, which was primarily conveyed by Thy-1+ T cells, was antigen (RET/melanoma) specific and cytotoxic. Amongst various chemical ingredients of juzen-taiho-to examined in this study, glycirrhizin displayed an action, partially replacing that of juzen-taiho-to, in promoting anti-Mel-Ret immunity when supplementarily added in vitro. These results suggest that juzen-taiho-to suppresses once-developed primary melanocytic tumors through potentiation of T-cell-mediated antitumor cytotoxic immunity in vivo.     

Imiquimod enhances the systemic immunity attained by local cryosurgery destruction of melanoma lesions

Melanoma lesions can be frozen in vivo, resulting in necrotic death of malignant cells and in tumor antigen release suitable for cross-presentation by professional antigen-presenting cells. Imiquimod is a small molecule with adjuvant pro-inflammatory effects that can be topically delivered as a cream. Local cryosurgery of B16/ovalbumin (OVA)-derived subcutaneous tumor nodules leads to curative destruction of the lesions. If imiquimod is repeatedly applied on the cryo-treated lesion, a conspicuous, leukocyte-rich inflammatory infiltrate appears during the days following treatment. Mice treated by cryosurgery plus imiquimod rejected rechallenges of B16/OVA in 90% of the cases, whereas cryosurgery alone failed to prevent tumor grafting in 70% of the cases. The combination treatment of B16/OVA tumors was also able to protect 60% of the mice against outgrowth of a lethal dose of non-transfected B16 tumor cells. Addition of imiquimod to cryosurgery results in increases of the cellular immune response against tumor antigens as measured by in vitro IFN-gamma production and T-cell proliferation in response to OVA. The potent memory response is not only directed against the OVA epitope, but also toward a broader range of B16 antigens. Our data indicate that these combined treatments turn the treated tumor lesion into an autologous tumor vaccine, which is even able to cause vitiligo in several cases. These preclinical data and the simplicity of the procedures warrant the design of a pilot clinical trial.     

Effect of cepharanthin on superoxide anion (O2-) production by macrophages

Our previous report showed the inhibitory action of cepharanthin on oxygen-derived free radical production by polymorphonuclear leukocytes (PMN). In the present study, we examined the effects of cepharanthin on production of superoxide anion (O₂^–), one oxygen radical, by macrophages in vitro and in vivo. Phorbol myristate acetate-induced O₂^– production by mouse peritoneal exudate cells (PEC), whose constituent cell types were identified as macrophages, lymphocytes, and PMN (75, 20, and less than 5%, respectively), was measured by ferricytochrome C reduction assay. Superoxide anion production by PEC, which depended mainly on the macrophage component, was inhibited 34% by 5 μg/ml cepharanthin and 85% by 50 μg/ml. These results indicate that cepharanthin suppresses O₂^– production by macrophages as well as by PMN. The fact that no inhibition of O₂^– by a xanthine-xanthine oxidase system was observed indicates that cepharanthin is not a scavenger of O₂^–. Carrageenan-induced paw edema is due to the activity of macrophages. Participation of O₂^– and other oxygen radicals is implicated in this edema, because the swelling is inhibited by administration of superoxide dismutase. The effects of cepharanthin on O₂^– production by macrophages was examined using this experimental system in mice. However, no significant inhibition of the edema by cepharanthin was observed.