Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL

BACKGROUND: Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied. OBJECTIVES: To investigate the effect of viable and nonviable cells of Propionibacterium acnes in both exponential and stationary growth phases, and of P. acnes GroEL on cytokine production by human primary keratinocytes. METHODS: Actively proliferating or contact-inhibited keratinocytes were cocultured with viable or formaldehyde-killed P. acnes cells in either the exponential or stationary phase of growth. Culture supernatants were assayed by enzyme-linked immunosorbent assay for the cytokines interleukin (IL)-1alpha, tumour necrosis factor (TNF)-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF). Keratinocytes were also stimulated with different concentrations of P. acnes GroEL and supernatants assayed for cytokines. RESULTS: Viable P. acnes in the stationary phase of growth stimulated keratinocyte monolayers to produce significantly higher amounts of IL-1alpha, TNF-alpha and GM-CSF than unstimulated keratinocytes. Viable exponential-phase bacteria stimulated production of significantly higher amounts of TNF-alpha and GM-CSF but these levels were significantly lower than those for stimulation with stationary-phase bacteria. Nonviable P. acnes from either growth phase was not able to stimulate cytokine production. P. acnes GroEL at concentrations in the range 0.05-1.0 micro g mL(-1) was able to induce increased production of cytokines by keratinocytes in a dose-dependent manner. This was analogous to stimulation with Escherichia coli GroEL. CONCLUSIONS: Stimulation of cytokine production by P. acnes and P. acnes GroEL may be important in the pathogenesis of inflammatory acne vulgaris and may have wider implications for the immunomodulation of the human immune system by commensal skin microorganisms.     

黑素瘤细胞生长因子表达及微血管密度研究

目的:探讨血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)在人黑素瘤中的表达和微血管密度(MVD)的关系。方法:采用免疫组化方法检测42例黑素瘤组织中的VEGF、bFGF的表达;利用第Ⅷ因子相关抗原(FⅧ-RAg)血管内皮细胞特异性染色,对肿瘤MVD进行计数和分析。结果:42例黑素瘤组织中,表达VEGF32例,bFGF29例,两指标均表达25例,VEGF与bFGF的表达呈正相关,VEGF、bFGF表达与淋巴结转移有关;色素痣组织不表达VEGF、bFGF;黑素瘤MVD明显高于色素痣。结论:MVD随着VEGF、bFGF表达的增强而增加,VEGF和bFGF在促进黑素瘤血管形成和促进肿瘤转移方面起协同作用。     

In Vivo human melanoma cytokine production: Inverse correlation of GM-CSF production with tumor depth

Abstract: Melanomas produce multiple cytokines which may influence their growth in vivo . Experimental evidence suggests that granulocyte macrophage-colony stimulating factor (GM-CSF) can induce a potent anti-melanoma response, whereas interleukin-8 (IL-8) may act as a growth factor in human melanoma. Little is currently known regarding the production of these cytokines by human melanoma in vivo . In this study we tested the hypothesis that endogenous production of GM-CSF and IL-8 can be correlated with the depth of human malignant melanoma surgical specimens. We examined 45 melanocytic human tissue samples consisting of 27 primary cutaneous melanomas, 9 metastatic melanomas, and 9 dysplastic nevi for in vivo GM-CSF and IL-8 production using immunohistochemistry. The majority of thin melanomas (≤0.76 mm) stained highly positive for GM-CSF with little or no staining for IL-8 whereas the medium (≤0.76 –≤4.0 mm) and thick (>4.0 mm) melanoma specimens showed little or no staining for GM-CSF and significant amounts of IL-8 staining. Metastatic melanoma as well as dysplastic nevi specimens had little or no GM-CSF and IL-8 staining. These results support the hypothesis that endogenous melanoma cytokines such as GM-CSF and IL-8 with opposing effects on tumor progression play an important role in melanoma growth and regulation.     

Antiproliferative effect of hyperthermia and ACNU on cultured human malignant melanoma cells

Human malignant melanoma cultured cells were treated either with ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chlorethyl)-3-nitro sourea hydrochloride), hyperthermia, or the combination of ACNU and hyperthermia. The combination treatment inhibited the cell growth to a slightly synergistic degree compared to the respective single treatments. The present in vitro experimental results support in part the finding of our previous report that the combination treatment with ACNU and hyperthermia have a significantly synergistic antitumor effect to human melanoma transplanted to nude mice. However, the synergistic effect was much less intense in the present in vitro experiment. The difference may have resulted from the environmental differences between in vitro and in vivo experimental systems.     

Expression of growth factor receptors on human melanoma cells: Comparison of modulating effects of interferons and retinoids

Abstract Amocrine and paracrine growth factors are important mediators in malignant transformation. Interferons (IFN) and retinoids (RX) are well-known differentiative and immunomodulating agents with effects on subsets of different human tumors including malignant melanoma. In this study, we examined the modulating effects of three IFN and seven different RX on human melanoma cell lines regarding growth factor receptor expression. Growth factor receptor expression, including PDGF-R, NGF-R, EGF-R, IR, IGF-l-R, TFR and c-kil. was studied by immunhislochemistry and FACSsean analysis. Both groups of sub-stances modulated the expression of some growth factor receptors. Upre-gulation of PDGF-R was seen after treatment with IFN as well as with RX. In contrast, EGF-R was found to be downregulated in two EGF-R-positive cell lines by IFN and. on the other hand, induced by RX in two EGF-R-negalive cell lines. The expression of NGF-R was modulated am-biguously by these substances but demonstrated a cell line specificity in the different melanoma cell lines tested. Additionally, some of the tested growth factor receptors were not markedly changed regarding their ex-pression by treatment with IFN and RX (IR. 1GF-I-R, e-kit, TFR).     

血管内皮生长因子过度表达促进恶性黑素瘤细胞增殖机制研究

目的:研究血管内皮生长因子(VEGF)过度表达促进恶性黑素瘤(MM)细胞增殖的机制。方法:通过电穿孔法将VEGF165cDNA转染至MM细胞系A375中,细胞计数法、MTT法和酶联免疫吸附实验(ELISA)法分别检测A375细胞体外增殖效应和其分泌的VEGF蛋白水平,化学比色法和蛋白印迹法(Western Blot)分别检测转染前后的A375细胞合成的诱导型、内皮型、神经型一氧化氮合酶(iNOS、eNOS、nNOS)的活性和蛋白表达。结果:转染VEGF165cDNA后,A375细胞明显增殖,其分泌的VEGF在转染后72 h、96 h明显增加(P<0.01),其合成的iNOS的活性和蛋白表达在转染后48 h、72 h、96 h明显升高(P≤0.05),但在转染前后未检测到eNOS和nNOS的活性及蛋白表达,一氧化氮合酶抑制剂L-NAME呈剂量依赖性地抑制转染72 h后的A375细胞增殖活性。结论:iNOS可能在VEGF过度表达促进MM细胞增殖中发挥重要作用。     

Radioimaging of malignant melanoma xenografts in nude mice using 111In-labeled monoclonal antibody against human malignant melanoma

By using ¹¹¹In-labeled monoclonal antibody (ZME-018) against human malignant melanoma, we examined its usefulness in radioimmunodetection of human melanoma xenografts in nude mice. Two human malignant melanoma cell lines were used in this study, KHm-1/4; and KHm-3/ps. KHm-1/4 cells express melanoma-associated antigen which is reactive with ZME-018, whereas KHm-3/ps cells do not. ZME-018 was conjugated with DTPA (diethylene triamine pentaacetic acid) first; then the conjugate was labeled with ¹¹¹In. After these procedures, labeled ZME-018 retained its binding activity against KHm-1/4 cells in vitro. Nude mice, bearing melenoma xenografts started from injections into the right hind legs, were i.p. or i.v. injected with ∽300 μCi (60 μg) of labeled ZME-018. Tumor images were taken with gamma camera at 24 hr intervals. Clear tumor images were obtained by 24 hrs after injection. The best imaging was obtained at 72 hrs with no background of internal organs. Specific localization was confirmed by the absence of imaging in the mice which received free ¹¹¹In only and also by using control mice bearing antigen-negative melanoma cells (KHm-3/PS). An example of tissue distribution of the labeled antibody in terms of tumor to tissue ratio at day 3 is as follows: tumor/intestine; 20/1, tumor/liver; 10/1, tumor/blood; 8/1, tumor/muscle; 8/1, tumor/heart; 3/1, tumor/kidney; 2/1. This study demonstrates the future applicability of ¹¹¹In-labeled monoclonal anti-melanoma antibody for radioimmunodetection of metastatic lesions in melanoma patients.     

黑素瘤抑制性活性因子在黑素瘤不同阶段及基底细胞癌中的表达

目的：探讨黑素瘤抑制性活性因子（MIA）在黑素瘤及基底细胞癌中的表达及其作用。方法：应用sP免疫组化技术检测38份黑素瘤石蜡标本、35份基底细胞癌石蜡标本以及32份色素痣石蜡标本中MIA的表达水平。结果：MIA在所有色素痣以及基底细胞癌中均呈阴性表达，而在原位黑素瘤、侵袭性黑素瘤、有淋巴结转移的黑素瘤、无淋巴结转移的黑素瘤阳性表达率分别为21．4％、91．6％、94．1％和42．8％。结论：MIA在黑素瘤的发生发展中起重要作用，MIA有可能成为临床诊断、治疗黑素瘤的靶点。     

Clinicopathologic and prognostic significance of SATB1 in cutaneous malignant melanoma

Background

Special AT-rich sequence-binding protein-1 (SATB1), a new type of gene regulator, has been reported to be expressed in several human cancers and may have malignant potential. However, no data on SATB1 expression and its relationship to tumor progression in cutaneous malignant melanoma (CMM) has yet been reported.

Objective

We examined the immunohistochemical expression of SATB1 in CMM to determine whether it could serve as a prognostic marker.

Methods

A total of 97 samples of primary CMM and controls were immunostained for SATB1. The following clinicopathologic variables were evaluated: age, gender, subtype, SATB1 expression, Breslow thickness, Clark level, presence of ulceration, lymph node metastasis, distant metastasis, and survival. Statistical analyses were performed to assess for associations. Several parameters were analyzed for survival using the Kaplan-Meier method and Cox proportional-hazards model.

Results

Forty cases (85.1%) of CMM showed positive staining for SATB1 by immunohistochemistry. The intensity of SATB1 staining was significantly higher in CMM than in nevus NV and normal skin (NS) (P < 0.01). High SATB1 expression was significantly correlated with Breslow thickness, Clark level, mortality, presence of ulceration, and lymph node metastasis (P < 0.01). Moreover, Kaplan-Meier analysis revealed that SATB1 overexpression was significantly associated with worse survival (P < 0.01). Further univariate analysis and multivariate regression analysis indicated that SATB1 expression was an independent prognostic marker for CMM (P = 0.03).

Conclusions

The overexpression of SATB1 correlated with metastatic potential of CMM and is a novel independent prognostic marker for predicting outcome.     

腺病毒介导p27mt基因转移诱导恶性黑素瘤细胞凋亡的实验研究

目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。     

A case of pedunculated malignant melanoma of the vaginal mucosa.

We report an 84-year-old Japanese woman who presented with a pedunculated malignant melanoma of the vaginal mucosa. Mucosal melanoma is believed to be more common in Japan than other countries, but such tumors of the vulvovaginal region are quite unusual. In our patient, three tumors were connected by a narrow pedicle. Three black tumors measuring 5-10 mm in diameter with a common pedicle were seen on the vaginal mucosa at five o'clock from the cervix. The tumors were hanging from the narrow pedicle. On histologic examination, they were diagnosed as malignant melanoma. Resection was done with a distal margin of 3 cm from the tumors and a margin of 1 cm from the cervix. The patient has had no evidence of local recurrence or distant metastasis. In our patient, the three main tumors had a common pedicle, which seems to be a unique finding. Since pedunculated malignant melanomas are rare, making a clinical diagnosis is difficult. Although pedunculated melanomas are recognized as having a high malignant potential because these lesions are generally thick, a relatively good outcome is sometimes reported. In our patient, there was no tumor infiltration into the dermis of the pedicle, and this may be one reason for the good outcome at present. There has been no previous report of a mucosal melanoma consisting of three tumors like those in the present patient.     

Synergistic effects of hyperthermia and peplomycin against human malignant melanoma xenografts

Two types of human malignant melanoma were treated either with peplomycin alone, hyperthermia alone, or a combination of peplomycin and hyperthermia. The antitumor effect of combination therapy was evaluated by tumor growth curves, maximal suppression rate, and histopathology. A marked antitumor effect of combination therapy with peplomycin (5 mg/kg) and hyperthermia (43°C, 30 min) was obtained for both types of melanoma. Histopathologic findings revealed advanced central necrosis and pyknotic nuclei in the tumor cells. Although peplomycin alone (20 mg/kg, 6 times) had no antitumor effect on C24 melanoma, the peplomycin (5 mg/kg, 6 times) + heat group showed complete inhibition of the tumor growth. These results suggest that hyperthermia enhances the antitumor effect of peplomycin even against peplomycin-resistant tumor cells.     

HLA—I类分子在黑素瘤中的表达

目的：检测HLA—I（人类白细胞抗原一1）类分子在黑素瘤组织中的表达。方法：应用免疫组织化学方法检测77例黑素瘤组织中H1A—I类分子的表达,并对蛋白的表达进行半定量分析,以20例色素痣组织做对照。结果：在77例黑素瘤组织中,HLA—I类分子的阳性表达率为64％,而在色素痣组织中100％阳性表达。结论：在黑素瘤组织中HLA—I类分子的表达下降,可能与人黑素瘤细胞的免疫逃逸有关。     

Detection of inflammatory cytokines in psoriatic skin

Recent investigations have revealed the involvement of cytokines in the pathogenesis of psoriasis. This study examined the amount of inflammatory cytokines — interleukin-1 (IL-1), interleukin-6 (IL-6) and granulocyte macrophage colony-stimulating factor (GM-CSF) — released into the supernatants of organ cultures of involved and uninvolved skin from psoriatic patients and normal skin from healthy individuals. Bioassays were employed to detect the activities of IL-1 and IL-6. Enzyme-linked immunosorbent assay (ELISA) methods were used to quantitate immunoreactive IL-1, IL-1, IL-6 and GM-CSF. The activity of IL-1 in uninvolved psoriatic skin was found to be increased relative to that in involved and normal skin, while immunoreactive IL-1 was found only in involved skin. A neutralization experiment showed that bioactive IL-1 was mostly attributable to IL-1. Uninvolved psoriatic skin also secreted higher amounts of both bioactive and immunoreactive IL-6 compared with involved skin. Immunoreactive GM-CSF was detected in uninvolved skin only. These cytokines detected in uninvolved skin may have been released from epidermal or mesenchymal cells, since uninvolved skin contained fewer inflammatory infiltrates. Our results offer additional evidence that increased amounts of inflammatory cytokines in uninvolved skin may provide a preliminary condition and play important roles in the initial events in the evolution of psoriatic lesions.Part of this work was presented in abstract form at the Fifth International Psoriasis Symposium in San Fransisco, 10–14 July 1991     

Rapid growth of malignant melanoma in pregnancy

Malignant melanoma during pregnancy is a difficult problem as a variety of risks to both the mother and fetus must be weighed. We describe a rapidly progressive malignant melanoma diagnosed during pregnancy. There are no standarized guidelines for treatment; each case requires an individualized approach. We review the literature and present an algorithm to aid in approaching such patients.