Tumor-Associated Macrophage-Induced Invasion and Angiogenesis of Human Basal Cell Carcinoma Cells by Cyclooxygenase-2 Induction

Tumor-associated macrophages (TAMs) and cyclooxygenase-2 (COX-2) are associated with invasion, angiogenesis, and poor prognosis in many human cancers. However, the role of TAMs in human basal cell carcinoma (BCC) remains elusive. We found that the number of TAMs infiltrating the tumor is correlated with the depth of invasion, microvessel density, and COX-2 expression in human BCC cells. TAMs also aggregate near COX-2 expressing BCC tumor nests. We hypothesize that TAMs might activate COX-2 in BCC cells and subsequently increase their invasion and angiogenesis. TAMs are a kind of M2 macrophage derived from macrophages exposed to Th2 cytokines. M2-polarized macrophages derived from peripheral blood monocytes were cocultured with BCC cells without direct contact. Coculture with the M2 macrophages induced COX-2-dependent invasion and angiogenesis of BCC cells. Human THP-1 cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages with M2 functional profiles. Coculture with PMA-treated THP-1 macrophages induced COX-2-dependent release of matrix metalloproteinase-9 and subsequent increased invasion of BCC cells. Macrophages also induced COX-2-dependent secretion of basic fibroblast growth factor and vascular endothelial growth factor-A, and increased angiogenesis in BCC cells

Taiwanese Dermatological Association consensus for the management of atopic dermatitis

AbstractBackground/ObjectiveThis report describes the 2014 consensus of the Taiwanese Dermatological Association (TDA) regarding the treatment of atopic dermatitis (AD). The TDA consensus is distributed to practices throughout Taiwan to provide recommendations for therapeutic approaches for AD patients to improve their quality of life.MethodsThe information in the consensus was agreed upon by a panel of national experts at TDA AD consensus meetings held on March 16, May 4, and June 29, 2014. The consensus was in part based on the 2013 Asia–Pacific AD guidelines and the guidelines of the American Academy of Dermatology, with modification to reflect the clinical practice in Taiwan.ResultsThe amendments were drafted after scientific discussions focused on the quality of evidence, risk, and benefits; all the consensus contents were voted on by the participating dermatologists, with approval by at least 75% for inclusion.ConclusionThe consensus provides a comprehensive overview of treatment for AD, with some local and cultural considerations for practitioners in Taiwan, especially the use of wet dressings/wraps, systemic immunomodulatory agents, and complementary therapies

Incorporating gene co-expression network in identification of cancer prognosis markers

<p>Abstract</p> <p>Background</p> <p>Extensive biomedical studies have shown that clinical and environmental risk factors may not have sufficient predictive power for cancer prognosis. The development of high-throughput profiling technologies makes it possible to survey the whole genome and search for genomic markers with predictive power. Many existing studies assume the interchangeability of gene effects and ignore the coordination among them.</p> <p>Results</p> <p>We adopt the weighted co-expression network to describe the interplay among genes. Although there are several different ways of defining gene networks, the weighted co-expression network may be preferred because of its computational simplicity, satisfactory empirical performance, and because it does not demand additional biological experiments. For cancer prognosis studies with gene expression measurements, we propose a new marker selection method that can properly incorporate the network connectivity of genes. We analyze six prognosis studies on breast cancer and lymphoma. We find that the proposed approach can identify genes that are significantly different from those using alternatives. We search published literature and find that genes identified using the proposed approach are biologically meaningful. In addition, they have better prediction performance and reproducibility than genes identified using alternatives.</p> <p>Conclusions</p> <p>The network contains important information on the functionality of genes. Incorporating the network structure can improve cancer marker identification.</p

Allergens in Atopic Dermatitis

Allergens play an essential role in atopic dermatitis, either intrinsic or extrinsic. They provoke cutaneous inflammation via IgE-dependent and cell-mediated immune reactions. Food allergens have a well-known contribution to disease activity of atopic dermatitis, especially in infants and young children. However, the importance of inhaled allergens is still under investigation. For clinical implication, identification of individualized allergens is an ideal strategy for better control of atopic dermatitis and avoidance of atopic march. The aim of this article is to discuss the common allergens in atopic dermatitis (AD), the specificity and sensitivity of laboratory tests for allergens, and the clinical effect of various preventions

The Regression of Hemangioma of Infancy is Regulated by Sympathetic Nerve

【研究背景及目的】 嬰兒型血管瘤（hemangioma of infancy, HOI）是嬰兒期最常見的腫瘤，在足月兒其盛行率約為1％-2.6％。此血管瘤與其他皮膚血管腫瘤的最大差異為其病程包含「增生期、退化期、休止期」。就病理學而言，「嬰兒型血管瘤」屬於微血管增生所形成之血管瘤(capillary hemangioma)，可以位於皮膚的真皮淺層或深層，亦可能侵犯內臟。 「嬰兒型血管瘤」通常在三至九個月時迅速增大(增生期)，然後在九至十二個月時開始萎縮(退化期)，最後在2-5歲時只剩下脂肪與纖維組織(休止期)。基於臨床經驗，大約百分之六十的「嬰兒型血管瘤」最後會萎縮成外觀尚可接受的疤痕組織，因此，「嬰兒型血管瘤」的治療策略向來以保守的「等待與觀察｣為主要原則，除非有特殊的考量或併發症，才會考慮積極的治療方式。 對於「嬰兒型血管瘤」如此神秘而有趣的自然消失過程，如果能破解其奧秘，應該能成為一種新穎的血管退化療法。因此，過去有許多學者致力於「嬰兒型血管瘤」的各個層面之研究。就病理生理學(pathophysiology)而言，「嬰兒型血管瘤」的微血管與腦部/胎盤血管同樣含有glucose transporter I (GLUT-1)，屬於具有血液與組織間的障蔽功能的微血管。因為其他種類的良性皮膚血管腫瘤都不含GLUT-1，所以GLUT-1可以當作「嬰兒型血管瘤」在病理學鑑別診斷上具有高度專一性的標記。 除了GLUT-1，FcγRII, merosin, Lewis Y antigen (Le Y)等三種胎盤血管的標記也存在於「嬰兒型血管瘤」所以胎盤血管可能與「嬰兒型血管瘤」有相關性。此外，「嬰兒型血管瘤」與胎盤內皮細胞的轉錄體(transcriptome)相似性極高。所以，胎盤血管可能與「嬰兒型血管瘤」有重大相關性。 從另一個角度而言，也有一些文獻證明「嬰兒型血管瘤」內皮細胞有某些胎兒血管的特徵，暗示它可能是一種具有胎兒血管特色的腫瘤或是血管發育過程早期中斷的產物。例如:新生兒的「嬰兒型血管瘤」內皮細胞的第一型血小板-內皮細胞之 接合分子(platelet-endothelial cell adhesion molecule, PECAM-1)與 von Willebrand氏因子(von Willebrand factor, vWf)含量遠少於正常新生兒血管，但比較接近正常胎兒血管。新生兒的「嬰兒型血管瘤」與正常胎兒血管都富含第一型膠原蛋白(type I collagen) ，然而正常新生兒血管卻富含第四型膠原蛋白(type IV collagen)。增生期「嬰兒型血管瘤」的內皮細胞表現出CD 34, CD31, lymphatic endothelial hyaluronan receptor-1 (LYVE-1)，是不成熟的血管。 就血管生成促進因子(angiogenesis stimulating factors)而言，「嬰兒型血管瘤」組織在增生期富含鹼性纖維母細胞生長因子(basic fibroblast growth factor, bFGF)與血管內皮細胞生長因子(vascular endothelial growth factor, VEGF)；在退化初期，「嬰兒型血管瘤」組織的VEGF含量趨於正常，bFGF則在退化晚期趨於正常。也有許多證據顯示，「嬰兒型血管瘤」附近的異常組織對「嬰兒型血管瘤」的增生具有外在的貢獻:基質細胞(stromal cells)能分泌VEGF;增生期的表皮細胞富含bFGF與VEGF。此外，也有零星證據顯示其他的生長因子與血管新生調控因子與「嬰兒型血管瘤」各階段的關係，例如: 第一型金屬蛋白酵素的組織抑制因子(tissue inhibitor of metalloproteinase 1, TIMP-1)，第二型胰島素生長因子(insulin-like growth factor 2) ，組織缺氧誘生因子α等等 (hypoxia inducible factor-1 alpha, HIF-1α)。 亦有許多證據顯示「嬰兒型血管瘤」具有內在性質的改變，例如: 內皮細胞呈現單株性質(monoclonality)，內皮細胞對血管生成抑制因子endostatin有異常的反應，內皮細胞具有突變的VEGF接受器，angiopoietin-2的失調，以及對angiopoietin-1的過度反應，類固醇接受器亞型(glucose receptor isotypes)的比例變化，胺2,3-雙加氧酵素(indoleamine 2,3- dioxygenase )的含量變化等等。 從解剖學的角度研究「嬰兒型血管瘤」，是一個新方向。Waner年提出「臉部的血管瘤位置不是隨機分佈的」－所有局部型臉部的嬰兒型血管瘤都集中分佈在胚胎時期中胚層與中胚層或中胚層與外胚層的融合處、所有的廣泛型血管瘤也都屬於節段式分佈。我門因此認為此一發現暗示著「嬰兒型血管瘤」的生成可能受到局部神經的誘導，而且「嬰兒型血管瘤」的組織中也可能含有異常的神經組織。我們過去的實驗曾利用雷射都卜勒血流儀 (laser Doppler fluxmetry, LDF)測量18名3-8個月嬰兒的增生期血管瘤表面皮膚的血流，實驗結果顯示它對44℃的熱刺激缺乏血流調控的反應(血管擴張)，原因可能是「嬰兒型血管瘤」組織的交感神經缺乏或功能異常所引起。 綜合上述兩項證據，我們假設神經組織對HOI細胞的演化，可能具有調控的作用。本研究將測量不同時期「嬰兒型血管瘤」的神經密度與血管瘤細胞凋亡數目，提供神經與「嬰兒型血管瘤」演化相關性的初級證據。 【研究方法與材料】 皮膚血管瘤標本來自北台灣三家醫學中心20年內的病理切片庫藏，切除時年齡為0-24個月大的嬰兒。先以免疫組織化學染色法篩選出GLUT-1 呈陽性的「嬰兒型血管瘤」。再依據H&E染色病理切片的型態差異將「嬰兒型血管瘤」分為增生期，退化初期與退化晚期三種族群。然後個別再執行「蛋白質基因產物9.5」(protein gene product 9.5，PGP9.5)，「酪胺酸羥化酶」(tyrosine hydroxylase，TH) 與 「活化凋亡蛋白3」(activated caspase 3，AC3) 免疫組織染色。在可見光顯微鏡下將上述染色結果以數位化相機拍攝200倍放大影像，再以影像分析軟體Image Pro Plus&reg;分析圖形。 最後，我們以ANOVA與Wilcoxon rank sum test分析不同時期嬰兒型血管瘤的神經密度、交感神經密度與血管瘤細胞凋亡數目的差異，各別定義p 值小於0.001與0.01為顯著統計差異; 再以Pearson correlation analysis 分析神經密度、交感神經密度與血管瘤細胞凋亡數目的相關性，定義p 值小於0.01為顯著統計差異。 【結果】 總共得到103個血管瘤標本，其中42個為GLUT-1 positive， 確立診斷為「嬰兒型血管瘤」。12個是增生期，19個是退化初期，11個是退化晚期。「蛋白質基因產物9.5」免疫組織染色結果顯示「嬰兒型血管瘤」 的神經密度( 平均值± 標準差，單位: μm2/μm2):增生期-(5.2±2.9)x10-5，退化初期- (1016.7±173.6 )x 10-5，退化晚期- (0.5±0.8) x 10-5。「酪胺酸羥化酶」免疫組織染色結果顯示「嬰兒型血管瘤」的交感神經密度(平均值±標準差，單位: μm2/μm2): 增生期- (3.2± 2.8) x 10-5，退化初期-(1043.8±212.7) x 10-5，退化晚期-(0.3 ±0.5) x 10-5。「活化凋亡蛋白3」免疫組織染色結果顯示「嬰兒型血管瘤」的凋亡細胞密度(平均值±標準差，單位: cells/μm2): 增生期-(0.0± 0.0) x10-5，退化初期- (112.7± 25.0) x 10-5，退化晚期-(1.0 ±1.8 )x10-5。上述結果以ANOVA與Wilcoxon rank sum test分析，各別為p&lt;0.001與p&lt;0.01，具有統計上的顯著差異; 以Pearson correlation analysis 分析神經密度、交感神經密度與血管瘤細胞凋亡數目的相關性，p 值小於0.001，具有統計上的顯著差異。 【結論】「嬰兒型血管瘤」在退化初期有明顯的細胞凋亡與不尋常的神經纖維增生現象，而且主要是交感神經。 值得注意的是，正常的皮膚微血管並無交感神經相伴行，但是本實驗發現「嬰兒型血管瘤」在退化初期有異常的交感神經纖維穿梭在微血管間，代表著交感神經纖維可能扮演「嬰兒型血管瘤」退化的調控者。交感神經對於微血管或其它種類血管的負向調節則尚無文獻論及，所以本實驗的發現具有獨特的意義。【Background and Purpose】 Hemangioma of infancy ( HOI）is the most common tumor in infancy, with an estimated prevalence of 1-2.6％ in neonate [Hlmdahl K et al., 1955]. It is characteristic by its typical involution of nascent, proliferating, involuting, and involuted stages. About one third of the patients are born with visible lesions, and the others are invisible until several weeks old. HOI is not a hereditary disease, and its cause is unknown. However, some risk factors have been reported, such as Caucasian, female, prematurity with birth body weight less than 1500 g, and a history of chorionic villi sampling. [Bowers RE et al., 1960; Droler BA et al., 1999 ; Mulliken JB et al., 1988]. HOI is one of capillary hemangiomas, usually located in superficial or deep dermis. It often appears on the head and neck area, and sometimes may invade the viscera。Usually, it is proliferating during the first three to nine months of age, and then becomes involuting between the age of nine and twelve months. Finally, HOI entered the involuted stage at the age of two to five years old. Approximately, 10% of the HOI patients completely resolved per year. For differential diagnosis, non-involuting congenital hemangioma (NICH) and rapidly-involuting congenital hemangioma (RICH) should be excluded by lacking of typical evolution of HOI: NICH usually does not involute and grow proportionally with increasing age [Enjolras O,et al., 2001]; RICH is often completely involuted in one year [Lopez-Gutierrez et al., 2005]. According to clinical experience, about 60% of HOI will finally resolve in a cosmetically acceptable scar tissue. Thus, the policy of treating HOI has long been “wait and see” or so-called “benign neglect”. For destructive HOI or severe complications, early intervention is necessary. Damage to the face, compression to important organs (eyes, noses, throat), and ulceration are the major indications for early treatment. The well-established therapies are corticosteroid ( topical, oral, and injection), topical imiquimod (Aldara&reg;), pulsed dye laser, chemotherapy (cyclophosphamide or vincristine), interferon α-2a or 2b, and surgery [Garzon MC et al., 2005; Zvulunov A et al., 2002; Sidbury R et al.,2003; Welsh O et al., 2004; Hazen PG., et al. 2005; Barrio VR et al., 2005; Turner et al., 1994; Boehm DK et al., 1995; Perez PJ et al., 1995; Garmendia G et al., 2001; Rampini E et al., 2000; Tamayo L et al., 1997; Illum N et al., 1995; Chan YC et al., 2005]。Although corticosteroid is the first choice of treatment for proliferating HOI, the response rate is about 30%. Imiquimod cream enhance the local production of interferon, has been proved to be effective in recent 3 years. The second-line treatment for proliferating HOI is chemotherapy or interferon injection, however, their side effects are immunosuppression and neurological deficit, respectively [Dubois J et al., 2002; Barlow CF et al., 1998]. For laser therapy, pulsed dye laser (595nm) is most effective. But, the depth of treatment is 1-2 mm only. Early laser treatment may leave more scars or pigment change [Batta K et al., 2002]. Thus, dye laser therapy is usually recommended for children older than one year. If we can find out the reason for mysterious involution of HOI, it would contribute a lot to novel therapy for angiogenesis inhibition. So far, there have been many studies on various aspects of HOI. From the view point of pathophysiology, HOI is capillary hemangioma. North first reported that HOI, like brain and placenta, has glucose transporter I (GLUT-1) located on the endothelial membrane of capillaries [North PE et al., 2000]. Other common benign cutaneous vascular tumor does not have GLUT-1, so GLUT-1 is a specific marker for differential diagnosis. More importantly, NICH and RICH also do not contain GLUT-1 [Leon-villapalos J et al., 2005]. Since capillaries of placenta contain GLUT-1, the association between placenta and HOI was highly suspected. North found another three placental capillaries’ antigens were also expressed in HOI- FcγRII, merosin, and Lewis Y antigen (Le Y). Thus, they proposed two hypothesis on “placenta as an origin of HOI”-one was “aberrant angioblast entered the skin vessels, another was “placental thrombotic cells developed in the skin” [North PE et al., 2001]. Barnes reported that the transcriptomes of the endothelial cells of HOI and placenta have high degree of similarity [Barnes et al., 2005]. Therefore, the close resemblance of HOI and placental vessels may explain the regression of HOI. Besides, some studies showed that the endothelial cells of HOI had characteristics similar to fetal capillaries. It implies that HOI may be a capillary tumor with the nature of fetal vessel or a product of early interruption of vessel development. Dosanjhr reported noted the level of platelet-endothelial cell adhesion molecule (PECAM-1) and von Willebrand factor (vWf) in HOI of the newborn was far less than that of normal newborn, but was similar to that of normal fetus. He also found that type I collagen was rich in newborn’s HOI and normal fetus, while type IV collagen was rich in normal newborn. [Dosanjh A et al., 2000]. Dadras found the endothelium of proliferating HOI expressed CD 34, CD31, and lymphatic endothelial hyaluronan receptor-1 (LYVE-1), while involuting HOI did not. They concluded that proliferating HOI was consisted of immature vessels, because it expressed both vascular and lymphatic markers. [Dadras SS et al., 2004]. The role of growth factors and angiogenesis stimulating factors in the pathogenesis for HOI has been noted since 1994. Proliferating HOI secrete high level of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). At involuting stage, VEGF is normal, but bFGF is still increased at early involuting stage [Takahashi K et al., 1994]. Level of urinary bFGF was high in infants with HOI. However, it was normal in patients with arteriovenous malformation [Dosquet C et al., 1998]. Besides, some evidences showed that abnormal tissues around HOI produced angiogenesis stimulating factors-Berard found the stromal cells of HOI could secret VEGF [Berard M et al., 1997]; Bielenberg reported that proliferating HOI had hyperplastic epidermis containing abundant bFGF and VEGF [Bielenberg et al., 1999]. Other factors such as tissue inhibitor of metalloproteinase 1(TIMP-1), insulin-like growth factor 2, hypoxia inducible factor-1 alpha (HIF-1α) had been reported to be related to different stages of HOI [Takahashi K et al., 1994; Ritter MR et al., 2002; Chen D et al., 2005]. Although angiogenesis stimulating factors may explain endothelial hyperplasia of proliferating HOI, there are some further evidences about intrinsic abnormalities of HOI, for example, monoclonality of endothelial cells [Boye E et al., 2001] and loss of heterozygosity on chromosome 5q [Berg JN et al., 2001]. Boye found that endothelial cells of HOI had abnormal response to endostatin [Boye E et al., 2001]. Walter noted that HOI had mutant VEGF receptor [Walter JW et al., 2002]. Yu reported endothelial cells of HOI had increased expression of Tie2, dysregulation of angiopoietin-2, and hyperreactivity to angiopoietin-1 [Yu Y et al., 2001]. Jiang found that endothelium of various stages of HOI had different density of glucose receptor isotypes [Jiang XW et al., 2001]. Yu also found that proliferative HOI contained endothelial progenitor cells (EPCs) [Yu Y et al., 2004]. Ritter first reported the relationship between proliferative HOI and immune cells- proliferative HOI had abundant CD8+ cytotoxic T cells. Indoleamine 2, 3- dioxygenase, an enzyme associated with regulation of T cell function, is more abundant in proliferative HOI than involuting HOI. It may suggest that the regression of HOI is associated with the inhibition of T cell function [Ritter et al., 2003 et al., 1986]. A new approach to study HOI is via the viewpoint of anatomy. Waner reported “the distribution of facial hemangiomas is nonrandom”- the focal hemangiomas on the face were all located near lines of mesenchymal or mesenchymal-ectodermal embryonic fusion, while the diffuse hemangiomas showed a segmental distribution [Waner M et al., 2003]. Thus, it may imply that HOI was influenced by local abnormal nerve tissues. Moreover, HOI may contain abnormal nerve tissues. Recently we demonstrated that regulation of blood flow in proliferating HOI is impaired. Laser Doppler fluxmetry with heating provocation could hardly induce vasodilatation in proliferating HOI of 18 infants, aged 0-8 months old. It may be caused by abnormal function of local sympathetic nerves. For further investigation about the relationship between HOI and nerve tissue, we hypothesize that nerve tissue may regulate the involution of HOI. To compare the change of nerve density at different stages of HOI, we will measure the density of nerve and apoptotic cells of HOI at various stages. Thus, it may provide primary evidence about the role of nerve tissue in HOI. 【Materials and Methods】 specimen collection From 3 medical centers in Northern Taiwan, paraffin-embedded specimens of hemangiomas were obtained. The age at excision was 0-24 months old. histopathology examination 5 um paraffin sections were deparaffinized in xylene for 30 minutes. After hydration with 100%, 95%, 85% alcohol, and pure water for several minutes, they were stained with Hematoxylin & Eosin (H & E). Under light microscopy, they were classified as proliferating, early involuting, and late involuting hemangiomas by morphological criteria: (1) endothelial hyperplasia, plump and multi-layered; with narrow lumens (2) single-layered endothelium, flat; with obvious, not tortuous lumens (3) single-layered endothelium, flat; with dilated and tortuous. Pyogenic granulomas were excluded. immunohistochemistry GLUT-1 immunohistochemical staining was done first to confirm the diagnosis of HOI. And then PGP9.5 immunohistochemical staining was performed on all HOI specimens to show nerve density. Tyrosine hydroxylase and activated caspase 3 immunohistochemistry staining were also done for further demonstration of sympathetic nerve and apoptotic cells in HOI. image processing and data analysis All results were photographed by digital camera under light microscope at magnification of 200 fold. The conditions for taking photographs were: (1) avoided the neighboring areas of venules, arterioles, and eccrine glands (2) focus on the area of highest nerve/apoptotic cell density, and then took four successive pictures. Photographs were then analyzed with Image Pro Plus&reg;. The density of nerve was defined as “summation of individual areas of nerves divided by the area of the picture (unit: μm2/μm2). The density of apoptotic cells was defined as “numbers of apoptotic cells divided by the area of the picture (unit: cells/μm2). data analysis and statistics ANOVA and Wilcoxon rank sum test were used to analyze the difference of nerve density and apoptosis events at different stages of HOI. P&lt;0.01 was defined as statistically significant. We also analyzed the correlation of nerve density, sympathetic nerve density, and apoptotic cell density by Pearson correlation analysis. P&lt;0.01 was defined as statistically significant. 【Results】 specimen collection and staging result of HOI There were103 cutaneous hemangiomas. Among them, 42 were GLUT-1 positive (Fig. 1A), diagnosing as HOI: 12 were proliferating HOI, 19 were early involuting HOI, 11 were late involuting HOI (Fig. 2). Among 61 GLUT-1 negative hemangiomas (Fig. 1B), there were 3 pyogenic granulomas (Table 1). early involuting HOI had highest density of nerve Nerve density at different stages of 42 HOI demonstrated by PGP9.5 immunohistochemistry showed (mean ± SD, unit: μm2/μm2): proliferating- (5.2±2.9) x 10-5, early involuting-(1016.7±173.6) x10-5, late involuting-(0.5±0.8) x10-5 (Fig. 3, Fig. 4). The difference was statistically significant by ANOVA and Wilcoxon rank sum test (Table 2). early involuting HOI had highest density of sympathetic nerve Tyrosine hydroxylase immunohistochemistry showed the sympathetic nerve density at different stages of HOI (mean ± SD, μm2/μm2) was: proliferating-(3.2±2.8) x10-5, early involuting-(1043.8±212.7) x 10-5, late involuting-(0.3±0.5) x 10-5 (Fig. 5, Fig. 6). The difference was statistically significant by ANOVA and Wilcoxon rank sum test (Table 3). the majority of nerves in early involuting HOI were sympathetic nerves Compared with density of nerves, nearly each of the sympathetic nerve density of early involuting HOI had an approximate value (Fig. 7). early involuting HOI had highest density of apoptotic cells Activated caspase 3 immunohistochemistry showed the apoptotic cell density at different stages of HOI (mean ±SD, cells/μm2): proliferative- (0.0±0.0) x 10-5，early involuting-(112.7±25.0) x 10-5，late involuting-(1.0±1.8) x 10-5(Fig. 8, Fig. 9). The difference was statistically significant by ANOVA test (Table 4). density of nerve, sympathetic nerve, and apoptotic cells at different stages of HOI had strong correlation It showed that early involuting HOI showed an extraordinary high density of nerves, sympathetic nerves, and apoptotic cells. By Pearson’s correlation test, the coefficient of nerve density and sympathetic nerve was 0.97094, of nerve density and apoptotic cell density was 0.95329, and of apoptotic cell density and sympathetic nerve density was 0.95252. They were all statistically significant by Pearson correlation analysis (Table 5). 【Discussion】 diagnosis and staging of HOI Although GLUT-1 has been a well-known specific marker for pathological diagnosis of HOI [North PE et al., 2000], However, most of the studies on HOI did not confirm the diagnosis of HOI by GLUT-1 immunohistochemical staining. They usually made the diagnosis by clinical observation. In this study, we used GLUT-1 immunohistochemistry to confirm the diagnosis of HOI. We found that only 41% of capillary hemangiomas were HOI (42%, if pyogenic granulomas were excluded). It was far less than the percentage reported in previous study [North PE et al., 2000], in which the percentage of HOI was 97%. Thus, the importance of GLUT-1 in diagnosis and histological study of HOI should be kept in mind. The controversy of clinical staging of HOI is mainly on the early stage of involution. The regression of HOI was evidenced by reduction of the size and change of the color (became dark red or grayish white). However, the initiation of regression may originate from deeply central part of the tumor, which is invisible. Thus, it may need quantification of urinary bFGF or immuno- histochemical staining of tissue markers to identify the early change of HOI. We may expect that molecules related with cell proliferation/death or angiogenesis regulation may be useful markers [Chen D et al., 2005; Ritter MR et al., 2002; Takahashi K et al., 1994]. Previous studies did find several tissue biomarkers at various stages of HOI, however their staging system was based on clinical observation and age of the patients. Thus, cellular markers for staging of HOI still need further investigation. Moreover, our study created strict morphological criteria for pathological staging of HOI. We also define the difference between early and late involuting HOI, in order to find the change that may be related to the regression of HOI. early involuting HOI had highest density of nerves, sympathetic nerves, and apoptotic cells It has been well known that PGP9.5 is a pan-neuronal marker, the best target for immunohistochemical staining of nerve fibers [Reilly DM et al., 1997]. However, among previous studies on nerve of HOI, only one used PGP9.5 immunohistochemistry [Adegboyega PA et al., 2005; Jang YC et al., 2000]. They neither used GLUT-1 immunohistochemistry to confirm the diagnosis of HOI, nor did precise staging for HOI. Thus, the power of their conclusion about the nerve density at various stages of HOI was not strong enough. The reason that PGP9.5 immunohistochemistry was rarely used in studies on nerves of HOI is technical difficulty. PGP9.5 can be easily stained with fresh frozen tissues; however, it is poorly stained with paraffin sections. Our specimens were old paraffin sections, which needed time-consuming repeated testing procedures to define the optimal conditions. Our success at demonstrating PGP9.5 immunohistochemistry in this collection of HOI paraffin-embedded specimen has a great contribution to the reliability of nerve density of HOI. Our study showed that early involuting HOI had highest density of nerves, which was far more than that of proliferating and late involuting HOI. The nerve density of proliferating and late involuting HOI was extremely low. In previous studies that used S-100 protein or PGP9.5 immunohistochemistry to evaluate the nerve density of HOI, they found no significant difference between proliferating/ involuting HOI and normal dermis [Adegboyega PA et al., 2005; Jang YC et al., 2000]. Our results were quite different from previous studies. It may be due to different criteria of staging. On the other hand, the proliferating HOI of previous studies might include “early involuting” HOI, so they had similar nerve density to normal dermis. Because our study strictly defined the entity of early involuting HOI, we discovered a new finding that had never been noted. The constitution of nerves of HOI had not been further investigated in previous reports; however, we found that early involuting HOI had large population of sympathetic nerves. This is novel and unexpected, because there is no sympathetic nerve innervation beyond terminal arteriole in normal dermis. It may imply that abnormal sympathetic nerves appeared in early involuting HOI have a role in inducing regression of HOI. density of nerves, sympathetic nerves, and apoptotic cells at different stages of HOI had strong correlation Our results showed that density of nerves, sympathetic ner