下文參考用於說明的實例應用來描述本發明的若干態樣。應瞭解，闡述眾多特定細節、關係及方法以提供對本發明的充分理解。然而，一般熟習此項技術者將易於認識到，可在無一或多個特定細節的情況下或通過其他方法來實踐本發明。本發明不受動作或事件的所說明次序限制，這是因為一些動作可按不同次序發生及/或可與其他動作或事件同時發生。 本文中所使用的術語僅用於達成描述特定實施例的目的，且並不意欲限制本發明。除非上下文以其他方式清楚地指示，否則如本文中所使用，單數形式「一(a)」、「一(an)」及「所述」意欲包括複數形式。 如本文中所使用，如本文所用的術語「表達」定義為特定核苷酸序列由其啟動子驅動的轉錄及/或轉譯。 如本文中所使用，如本文所用的術語「啟動子」定義為起始聚核苷酸序列的特異性轉錄所需的由細胞的合成機制或引入的合成機制識別的DNA序列。 如本文中所使用，如本文所用的術語「抗原」或「Ag」定義為引起免疫反應的分子。此免疫反應可涉及抗體產生或特異性免疫勝任細胞的活化或兩者。熟習此項技術者應瞭解，包括幾乎所有蛋白質或肽的任何大分子均可充當抗原。 如本文中所使用，「Tn抗原」表示GalNAca-O-Ser/Thr，亦即其中GalNAc殘基直接經α連接至在細胞內或在細胞表面上表達的多肽鏈的絲胺酸或蘇胺酸殘基的羥基的抗原。 如本文中所使用，如本文所用的術語「抗體」是指特異性結合抗原的免疫球蛋白分子。抗體可為來源於天然來源或來源於重組來源的完整免疫球蛋白且可為完整免疫球蛋白的免疫反應部分。抗體通常為免疫球蛋白分子的四聚體。本發明中的抗體可呈多種形式存在，包括例如多株抗體、單株抗體、Fv、Fab及F(ab)2
以及單鏈抗體、人類抗體及人類化抗體。 如本文中所使用，術語「多株抗體」是指包括針對一種抗原或多種抗原內相同及/或不同抗原決定基的多種不同抗體的抗體群體。 如本文中所使用，術語「單株抗體」是指自均質或基本上均質抗體的群體獲得的抗體。術語「單株」不限於任何用於製造抗體的特定方法。一般而言，單株抗體的群體可由細胞、細胞群體或細胞株產生。 如本文所使用，術語「患者」、「個體(subject)」、「個體(individual)」及其類似術語在本文中可互換使用，且是指可進行本文所述的方法的任何動物或其細胞，無論活體外或是原位。在某些非限制性實施例中，患者、個體(subject)或個體(individual)為人類。 如本文中所使用，如本文所用的術語「免疫球蛋白」或「Ig」定義為充當抗體的一類蛋白質。由B細胞表達的抗體有時稱作BCR (B細胞受體)或抗原受體。此類蛋白質中包括的五個成員為IgA、IgG、IgM、IgD及IgE。IgA是存在於諸如唾液、淚液、母乳、胃腸道分泌物及呼吸道及泌尿生殖道的黏液分泌物的身體分泌物中的初級抗體。IgG是最常見的循環抗體。IgM為在大部分個體中主要免疫反應中產生的主要免疫球蛋白。其為凝集、補體結合及其他抗體反應中最有效的免疫球蛋白，且對於防禦細菌及病毒具有重要意義。IgD雖為無已知的抗體功能的免疫球蛋白，但可充當抗原受體。IgE是通過在暴露於過敏原時引起介體自肥大細胞及嗜鹼細胞釋放來介導速發超敏反應的免疫球蛋白。 如本文中所使用，「疫苗」為包含抗原的免疫原組合物，當投藥至個體，誘導或刺激或引發細胞或體液對疫苗抗原的免疫反應。疫苗可含有對個體產生較加強免疫反應的佐劑。 如本文中所使用，「佐劑」為用於與抗原或抗原組合結合的物質以產生較抗原或抗原組合單獨為強的免疫反應。 如本文中所使用，「刺激免疫反應」、「誘導免疫反應」及「引發免疫反應」除非另有所述，在本文中可交替使用，其包括，但不限於，誘導、刺激或引發由個體免疫反應介導的治療或預防效果。 如本文中所使用，「有效量」意謂提供治療或預防益處的量。 Tn抗原進行疫苗接種抑制NF-κB活性，且經由Tn免疫接種誘導的抗Tn抗體的作用抑制發炎。本發明疫苗組合物及方法可有效治療或預防發炎疾病。Tn免疫接種增加血清抗Tn抗體效價，同時其減少灌洗的蛋白質及細胞因子。本發明因此提出Tn免疫接種可減弱發炎相關的疾病及器官損傷。本發明發展一種抗發炎的疫苗組合物以及發炎與肺損傷(特別是高氧所致的肺損傷)以及牙周病進展的治療或預防。Tn免疫接種也減小肺損傷的平均線性截距及肺損傷評分。此外，肺損傷的改善伴隨NF-κB活性的降低。 在一個態樣中，本發明提供一種單劑疫苗，每劑包含約0.02 mg (優選為約0.1 mg)至約2 mg的Tn免疫原及佐劑溶液，所述Tn免疫原及佐劑溶液的比例為約0.5至約2 (v/v) : 約0.5至約2 (v/v)。在一具體實施例，Tn免疫原及佐劑溶液的比例為約1 (v/v) : 約1 (v/v)。 在一些具體實施例，Tn免疫原的劑量範圍自約0.02 mg至約0.1 mg、約0.02 mg至約0.05 mg、約0.1 mg至約1.5 mg、約0.1 mg至約1.2 mg、約 0.1 mg至約1 mg、約0.1 mg至約0.8 mg、約0.1 mg至約0.5 mg、約0.2 mg至約1.5 mg、約0.2 mg至約1.2 mg、約0.2 mg至約1 mg、約0.2 mg 至約0.8 mg、約0.2 mg至約0.5 mg、約0.5 mg至約2 mg、約0.5 mg 至約1.5 mg、約0.5 mg至約1.2 mg、約0.5 mg至約1 mg、約0.5 mg 至約0.8 mg、約1.0 mg 至約2 mg。佐劑溶液的體積範圍自約 0.2 ml至約1 ml、約0.2 ml至約0.8 ml或約 0.2 ml 至約0.6 ml。 在另一個態樣，本發明提供一種單劑疫苗於個體誘導免疫反應以治療或預防發炎疾病的方法，其中所述單劑疫苗包含約0.1 mg至約2 mg的Tn免疫原。 在一個態樣中，本發明提供一種單劑疫苗於個體誘導免疫反應以治療或預防發炎疾病的方法，其包含投與包含每劑約0.1 mg至約2 mg的Tn免疫原單劑疫苗至個體，以兩週一次的時間間隔投藥至少四次。在一個實施例中，所述方法進一步包含在第四次免疫接種後的一週後進行額外免疫接種約0.1 mg至約2 mg的Tn免疫原。在一具體實施例中，該額外免疫接種可在最後免疫接種後進行一或多次。 根據本發明方法的Tn免疫原投藥可產生較控制組高的血清效價的抗-Tn抗體。在一具體實施例中，所產生的抗-Tn抗體的血清效價較控制組高至少2、2.5、3、3.5、4、4.5、5、5.5或6倍。本文中所使用的「控制組」為載體多肽。 在一具體實施例中，Tn免疫接種降低細胞或個體中白細胞介素-6 (IL-6)及TNF-α的量或降低NF-κB的活性。 在一具體實施例中，細胞或個體具有升高的Tn表達。在另一個實施例中，Tn表達由TNF-α及IL-6上調。在另一其他實施例中，升高的Tn含量通常由細胞因子-Cosmc信號傳導軸調控。 在一具體實施例中，所述發炎疾病是齒根骨膜炎(牙周病)的進展、器官損傷或器官纖維化。在另一實施例中，器官損傷是肺損傷、腎損傷或肝損傷。在另一實施例中，肺損傷是高氧誘發的肺損傷。在另一實施例中，器官纖維化是肺纖維化、肝纖維化或腎纖維化。 在一具體實施例中，所述方法進一步包含在第四次免疫接種後的一週後進行額外免疫接種的步驟。 在一些具體實施例中，Tn免疫原可與載體多肽結合。該Tn免疫原及載體多肽是處於約3至約8:約1的重量比下；優選地，重量比為約5:約1。在一些實施例中，該多肽包括(但不限於)抗原呈現細胞(APC)結合域及富含半胱胺酸的結構域。在一些實施例中，APC結合域為免疫球蛋白(Ig) Fc片段或毒素的受體結合域。在另一實施例中，APC結合域為綠膿桿菌外毒素A (Pseudomona
s exotoxin A)、破傷風毒素或霍亂毒素的受體結合域。在另一實施例中，APC結合域為人類Ig的Fc片段。在其他一些實施例中，該富含半胱胺酸的結構域含有10個胺基酸殘基的片段，其中至少3個為半胱胺酸殘基。在另一其他實施例中，富含半胱胺酸的結構域含有6個半胱胺酸殘基。優選地，富含半胱胺酸的結構域具有Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys的胺基酸序列。在另一其他實施例中，該富含半胱胺酸的結構域含有所述胺基酸序列的2至30個重複序列。優選地，該富含半胱胺酸的結構域含有所述胺基酸序列的7個重複序列。在另一實施例中，Tn經由連接子(如含有順丁烯二亞醯胺官能基的連接子；例如，N-順丁烯二亞醯胺(N-maleimide)或6-順丁烯二亞醯胺基己酸N-琥珀醯亞胺酯(N-succinimidyl-6-maleimidocaproate))連接於半胱胺酸殘基。優選地，Tn免疫原與載體多肽結合為Fc片段-7重複的Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-N-順丁烯二亞醯胺-Tn或Fc片段-7重複的Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-6-順丁烯二亞醯胺基己酸N-琥珀醯亞胺酯-Tn。 在一個實施例中，Tn免疫原為經O-連接至絲胺酸或蘇胺酸的N-乙醯基半乳糖胺，其具有以下結構：；R為絲胺酸或蘇胺酸。 在一具體實施例中，Tn免疫原在0.2 ml至2 ml佐劑存在下投與。在一些具體實施例中，該佐劑為氫氧化鋁、磷酸鋁、羥磷灰石、Bordetella pertussis
死菌、Mycobacterium bovis
死菌、類毒素、鯊烯、Quil A、皂素、IL-1、IL-2、IL-12、佛朗氏完全佐劑或佛朗氏不完全佐劑。在另一體實施例治中，佐劑為磷酸鋁。 包含本發明的Tn免疫原的醫藥組合物可投與已受發炎所苦的個體。在治療應用中，將組合物以足以引發針對所存在抗原的有效免疫反應及治癒或至少部分阻滯症狀及/或併發症的量投與患者。足以實現此量定義為「治療有效量」。對此用途有效量將視例如肽組成、投與方式、所治療的疾病的階段及嚴重程度、患者體重及整體健康狀態以及處方醫師的判斷而定。但初始免疫接種(用於治療性或預防性投與)的範圍一般為約0.1 mg至約2 mg的Tn免疫原，接著依照本文所描述之加強療程，為約0.1 mg至約2 mg的加強劑量的Tn免疫原。 所述用於治療的疫苗組合物意欲以非經腸、局部、經鼻、經口或局部投與。優選地，該醫藥組合物以非經腸投與，例如靜脈內、皮下、皮內或肌肉內投與。優選地，疫苗經肌肉內投與。本發明提供以非經腸投與的組合物，其包含該疫苗組合物之溶液，該疫苗組合物溶解或懸浮於可接受的載劑(優選為水性載劑)。此等組合物可通過傳統習知的滅菌技術滅菌或可經無菌過濾。所得水溶液可封裝以按原樣使用，或凍乾，該經凍乾的製劑在投與之前與無菌溶液組合。所述組合物可含有為接近生理條件而需要的醫藥學上可接受的輔助物質，諸如pH調節劑及緩衝劑、張力調節劑、濕潤劑及其類似物，例如乙酸鈉、乳酸鈉、氯化鈉、氯化鉀、氯化鈣、脫水山梨糖醇單月桂酸酯、三乙醇胺油酸酯等。 本文所提供本發明的實例，乃藉由實例例示，而非限制。實例 材料與方法 動物
五週大雌性C57BL/6NCrlBltw小鼠得自BioLASCO Taiwan Co., Ltd並置於無菌室。動物維持在約25o
C並整個實驗隨意供應顆粒食物與水。實驗流程已由臺北醫學大學實驗動物照護及使用委員會核可(LAC-2016-0047)。疫苗製備
疫苗的製備是將Tn接合至如先前研究所述(H.L. Chiang, C.Y. Lin, F.D. Jan, Y.S. Lin, C.T. Hsu, J. Whang-Peng, L.F. Liu, S. Nieh, C.C. Lin, J. Hwang, A novel synthetic bipartite carrier protein for developing glycotope-based vaccines. Vaccine 30 (2012) 7573–7581
)的載體。Tn在glycotope/載體蛋白重量比為5比1，接合至ratFc(Cys42)Histag2 or GST(Cys6)Histag2。在含有20 mM 磷酸鈉、pH 7.9、8 M尿素、500 mM咪唑及0.2 mM TCEP的緩衝液中進行接合。48小時後，接合物於含0.2 mM TCEP的磷酸緩衝液(PBS)中再折疊。GST(Cys6)在含0.2 mM TCEP的PBS中透析。不同的glycotope與連接子(順丁烯二亞醯胺基己酸N-琥珀醯亞胺酯)在4o
C下接合至GST(Cys6)48小時。小鼠實驗組
以20μg劑量的Tn疫苗或載體蛋白質(10 μg of mFc(Cys42-Tn)Histag2)在100 μl佐劑的存在下，以雙週的時間兼隔皮下免疫5週大雌性C57BL/6NCrlBltw小鼠4次，並在第4次免疫後1週額外追加1次免疫接種。從面靜脈抽血，使用酵素免疫分析法(ELISA)在第0、42及49天測定抗-Tn抗體的效價。最後免疫接種後4天，將小鼠暴露於室內空氣(room air；RA)或富含氧的大氣(100% O2)至多96小時。氧曝露的進行以4公升/分鐘連續遞送於透明60 × 50 × 40公分Plexiglas室，且氧量以ProOx Model 110監測器(NexBiOxy, Hsinchu, Taiwan)監測，每日檢查濕度，值為60–80%。取得下列4個組：載體蛋白 + RA (n = 6)、Tn疫苗 + RA (n = 6)、載體蛋白 + O2
(n = 6)及Tn疫苗 + O2 (n = 5)。小鼠在O2
處理96小時後以異氟醚深度麻醉。肺以0.6 ml、0.9%生理食鹽水在4o
C灌洗，灌洗肺內外3次，接著復原。針對每一動物重複清洗程序超過2次，收集3次的洗液並記錄總體積。支氣管肺泡刷洗檢查後，將右肺結紮，左肺在25公分H2
O的壓力下，以4%緩衝的三聚甲醛以氣管內滴注法固定。通過 ELISA 分析血清抗 Tn- 抗體的量
將GST(Cys6-Tn)以1.5μg/ml的濃度塗覆於96孔槽平底盤上(Falcon Labware, Lincoln Park, NJ, USA)。將經各種不同稀釋的血清加入各經塗覆的孔槽中。於37°C反應2小時後，以PBS清洗這些孔槽三次。接著，將與過氧化酶共軛的抗-人類免疫球蛋白加入，並將所述盤於37°C反應1小時。受質溶液含有0.54 mg/ml的2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)(2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)及0.01% H2
O2
及0.1M檸檬酸(pH 4.2)。以410 nm讀取吸收度值。支氣管肺泡灌洗液蛋白及細胞因子分析
使用二辛可寧酸(bicinchoninic acid)試驗(Pierce Chemical, Rockford, IL, USA)量測支氣管肺泡灌洗液(BALF)中的總蛋白質濃度。所述BALF中的IL-6及TNF-α的量是使用ELISA套組(Cloud-Clone Corp., Houston, TX, USA)測定。數據分別以mg/ml及pg/ml的方式表達。NF-kB 的西方墨點分析
次細胞劃分(subcellular protein fractionation)是以用於組織的次細胞蛋白劃分套組(Thermo Scientific, Melbourne, VIC, Australia, cat# 87790)來完成。核蛋白提取物被用於偵測NF-κB p65(SC-372, Santa Cruz Biotechnologies, Santa Cruz, CA, USA)次單元及PCNA (SC-7907)；細胞質蛋白提取物被用於偵測IκB-α(SC-1643)及β-肌動蛋白(SC-47778)。蛋白質濃度是使用二辛可寧酸(bicinchoninic acid)試驗套組測定。使用12%的十二烷基硫酸鈉聚丙烯酰胺凝膠將蛋白質分開並轉印至聚偏二氟乙烯(polyvinylidene difluoride)膜上，並將所述膜於5%脫脂牛奶中於室溫進行阻斷1小時。將所述膜與抗體於4°C反應隔夜。隨後，將所述膜及與HRP共軛的二級抗體於室溫反應1小時。依照製造商的指南，通過增強化學發光(enhanced chemiluminescence)試劑將訊號視覺化。抗β-肌動蛋白及PCNA的抗體分別被用作核及細胞質蛋白裝載量的內部控制組。所有點墨實驗皆使用不同小鼠至少進行三次。肺部型態測定
為將分析標準化，切片是來自右肺的右中葉。將5-μm肺組織切片以蘇木精及曙紅染色並測定型態。在不重疊的10個視野中測定平均線性截距(MLI)，其為肺泡平均直徑的指標。組織學
將肺組織於含4%三聚甲醛(paraformaldehyde)的磷酸緩衝液中固定，以石蠟包埋，以蘇木精及曙紅染色，且由對實驗流程及組別為盲的病理學家檢測。肺損傷是以以下四個標準來評分：1)肺泡充血、2)出血、3)嗜中性球在空氣腔或血管壁的浸潤及4)肺泡壁的厚度。每一項目是依據如下述的五分等級來分級：0為最小的(少的)損傷、1為輕微的損傷、2為中等的損傷、3為嚴重的損傷及4為最大的損傷。NF-kB 的 免疫組織化學染色
在例行的去石蠟步驟後，通過將載玻片浸於0.01 mol/L的檸檬酸鈉緩衝液(pH6.0)中來進行熱誘導的抗原決定基修復(retrieval)。為阻斷內生性過氧化酶的活性及非特異性抗體的結合，在與作為一級抗體的兔多株抗-NF-κB P65抗體(1:50稀釋; Abcam Inc., Cambridge, MA, USA)於4°C反應20小時前，先將切片於含有10%正常山羊血清及0.3%H2
O2
的0.1 mol/L PBS中於室溫預反應1小時。接著將這些切片以生物素化的(biotinylated)山羊抗-兔IgG(1:200稀釋，Vector, CA, USA)於室溫處理1小時。接著依照製造商的建議與來自ABC套組(抗生物素蛋白-生物素複合物(Avidin-Biotin Complex), Vector, CA, USA)的試劑進行反應，且透過二氨基聯苯胺(diaminobenzidine)受質套組(Vector, CA, USA)來視覺化所述反應的產物。所有經免疫染色的切片是通過Olympus BX 43來觀察及拍照。統計分析
所有數據是以平均值±SD呈現。統計分析是使用單因子變異數分析，並以杜凱氏事後檢定用於多重組別比較。當P＜ 0.05時，差異被認為是統計上顯著的。實例 1 抗 Tn 抗體的血清效價
在免疫接種之前所有小鼠中抗Tn抗體的含量為低的，且將其計為背景(圖1)。接受載體蛋白(亦即，Fc片段-7重複的Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-6-順丁烯二亞醯胺基己酸N-琥珀醯亞胺酯)且圈養在室內空氣或高氧中的小鼠顯示背景血清抗Tn抗體含量(圖1A)，而接受Tn疫苗接種的小鼠在Tn疫苗接種後發展高血清抗Tn抗體效價，且在疫苗接種若干個月後抗Tn抗體保持高含量(圖1B)。實例 2 存活及體重
在整個研究期間，暴露於室內空氣或高氧的小鼠均存活。暴露於高氧的小鼠顯示犧牲時的體重顯著低於在RA中飼養的小鼠(圖2)。實例 3 支氣管肺泡灌洗液蛋白及細胞因子分析
將小鼠用載體蛋白處理接著暴露於高氧，在BALF中，這些小鼠顯示比暴露於RA的小鼠顯著更高的總蛋白質及IL-6含量(圖3A及3B)。另一方面，用Tn疫苗處理且暴露於高氧的小鼠顯示BALF中顯著低於用載體蛋白處理的小鼠的IL-6含量(圖3B)。在BALF中，用載體蛋白處理且暴露於高氧的小鼠顯示TNF-α含量顯著高於暴露於RA的小鼠(圖3C)。在BALF中，用Tn疫苗處理且暴露於高氧的小鼠顯示TNF-α含量較低。然而，差異未達到顯著性。實例 4 組織學結果
圖4A中呈現來自暴露於RA及高氧的小鼠的經蘇木精及曙紅染色的代表性肺切片。如更大的線性截距所指示，高氧導致發炎細胞浸潤且肺實質簡化。與用載體蛋白或Tn疫苗處理且暴露於RA的小鼠相比，用載體蛋白處理且暴露於高氧的小鼠顯示顯著更高的肺損傷評分及MLI(圖4B及4C)。用Tn疫苗處理顯著減少高氧誘發的肺損傷評分及MLI增加。實例 5NF-κB 的免疫組織化學
雖然主要在肺泡巨噬細胞的細胞質中發現NFκB的免疫組織化學染色，但肺泡巨噬細胞的細胞核及少數肺泡上皮細胞中亦顯示免疫反應性(圖5A)。經載體蛋白免疫接種的高氧組的肺展示比對照及經Tn處理的高氧組更強的NFκB免疫反應性。實例 6 NF-kB 及 IkB α 的西方墨點分析
與用載體蛋白或Tn疫苗處理且暴露於RA的小鼠相比，用載體蛋白處理且暴露於高氧的小鼠展示顯著更高的核NF-κB p65及胞溶質磷酸化-IκBα含量(圖5B及5C)。即使在經高氧處理的大鼠中，用Tn疫苗處理的大鼠組亦顯著減少NFκB p65及胞溶質磷酸化-IκBα的含量。實例 7 ： 發炎組織及細胞中升高的 Tn 含量
為檢查升高的Tn含量是否與發炎相關，使用免疫組織化學(IHC)在發炎組織中量測Tn含量。在動脈粥樣硬化、支氣管炎及齒根骨膜炎的組織中觀測到Tn含量顯著增加，但在其對應正常組織中未觀測到此增加(圖6A)。為研究發炎性細胞因子對Tn含量的可能調控，使用來自經LPS刺激的單核細胞U937細胞的條件培養基。觀測到補充來自經LPS刺激的U937細胞的一天條件培養基的人類齒齦纖維母細胞(HGF)中Tn含量以LPS劑量依賴性方式增加(圖6B)。與來自無LPS下培養的U937細胞的培養基相比，在來自經LPS (10、30或100 ng/ml)處理24小時的U937細胞的條件培養基中發炎性細胞因子(例如TNF-α、IL-6及IL-1β)的分泌顯著更高(圖6C)。實例 8 ： HGF 中 TNF- α 及 IL-6 上調 Tn 表達
為確定細胞因子是否可使Tn含量升高，HGF用各種量的純化細胞因子處理。如圖7A中所示，HGF中的Tn含量對TNF-α的反應最大，對IL-6的反應中等，且對IL-1β無反應，即使在實驗條件下100 ng/ml的濃度下。TNF-α (30 ng/ml)使Tn升高顯示為時間依賴性的。在TNF-α處理4小時後，HGF中的Tn含量基本上不變。在8至12小時間觀測到Tn含量逐漸增加。Tn含量在TNF-α處理24小時後逐漸減少，且在TNF-α處理48小時後顯著減少(圖7B)。實例 9 ： TNF- α 經由下調 COSMC 基因來上調 Tn 表達
為探索在細胞因子介導的Tn含量上調下面的可能分子機制，研究TNF-α對COSMC
基因的mRNA含量的作用。如圖8A中所示，TNF-α (100 ng/ml，處理24小時)顯著下調HGF中的COSMC
mRNA。相比之下，TNF-α未顯著改變T-合成酶mRNA含量。在TNF-α處理後，觀測到HGF中Cosmc及T-合成酶的蛋白質含量的結果類似(圖8B、8C及8D)。TNF-α對COSMC
基因下調的作用可能涉及其啟動子中CpG島的高甲基化。使用亞硫酸氫鹽焦磷酸測序定量COSMC
基因的啟動子中的甲基化變化，TNF-α處理使四個CpG
位點顯著高甲基化(圖9A)。用去甲基化劑預處理HGF以劑量依賴性方式減少COSMC
啟動子中四個CpG
位點的甲基化(圖9A)，且相對應地，增加COSMC
mRNA的表達且減少Tn的含量(圖9B)。總而言之，吾人的結果表明細胞因子介導的Tn含量上調是由涉及COSMC
基因啟動子的高甲基化的COSMC
下調引起。Several aspects of the invention are described below with reference to example applications for illustration. It will be appreciated that numerous specific details, relationships, and methods are described to provide a thorough understanding of the invention. However, it will be readily apparent to those skilled in the art that the present invention may be practiced without one or more specific details or by other methods. The present invention is not limited by the illustrated order of acts or events, as some acts may occur in different orders and/or may occur concurrently with other acts or events. The terminology used herein is for the purpose of the description of the embodiments, The singular forms "a", "an" and "the" are intended to include the plural. As used herein, the term "expression" as used herein, is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. As used herein, the term "promoter" as used herein, is defined as the DNA sequence recognized by the synthetic machinery of the cell or the introduced synthetic machinery required for the specific transcription of the starting polynucleotide sequence. As used herein, the term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response can involve antibody production or specific immunity to competent cell activation or both. Those skilled in the art will appreciate that any macromolecule comprising almost all proteins or peptides can serve as an antigen. As used herein, "Tn antigen" means GalNAca-O-Ser/Thr, that is, a serine or sulphite in which a GalNAc residue is directly linked to a polypeptide chain expressed in a cell or on a cell surface via an alpha. The antigen of the hydroxyl group of the residue. As used herein, the term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds an antigen. The antibody may be an intact immunoglobulin derived from a natural source or derived from a recombinant source and may be an immunoreactive portion of an intact immunoglobulin. The antibody is typically a tetramer of immunoglobulin molecules. The antibodies of the present invention may exist in various forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab) 2 as well as single chain antibodies, human antibodies, and humanized antibodies. As used herein, the term "multi-species antibody" refers to a population of antibodies comprising a plurality of different antibodies directed against the same and/or different epitopes within an antigen or antigens. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of homogeneous or substantially homogeneous antibodies. The term "single plant" is not limited to any particular method used to make antibodies. In general, a population of monoclonal antibodies can be produced by a cell, a population of cells, or a population of cells. As used herein, the terms "patient,""subject,""individual," and the like are used interchangeably herein and refer to any animal or cell thereof that is capable of performing the methods described herein. , whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human. As used herein, the term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. An antibody expressed by a B cell is sometimes referred to as a BCR (B cell receptor) or an antigen receptor. The five members included in such proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tract. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most individuals. It is the most effective immunoglobulin in agglutination, complement binding and other antibody reactions, and is important for defense against bacteria and viruses. Although IgD is an immunoglobulin having no known antibody function, it can serve as an antigen receptor. IgE is an immunoglobulin that mediates rapid hypersensitivity by causing the release of mediators from mast cells and basophils when exposed to allergens. As used herein, a "vaccine" is an immunogen composition comprising an antigen that, when administered to an individual, induces or stimulates or elicits an immune response of the cell or body fluid to the vaccine antigen. The vaccine may contain an adjuvant that produces a more potent immune response to the individual. As used herein, an "adjuvant" is a substance that is used in combination with an antigen or antigen combination to produce a stronger immune response than an antigen or antigen combination alone. As used herein, "stimulated immune response", "induced immune response" and "priming immune response" are used interchangeably herein, unless otherwise indicated, including, but not limited to, induction, stimulation or initiation by an individual. The immune response mediated therapeutic or prophylactic effect. As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit. Vaccination of the Tn antigen inhibits NF-κB activity, and the action of anti-Tn antibodies induced by Tn immunization inhibits inflammation. The vaccine compositions and methods of the invention are effective in treating or preventing inflammatory diseases. Tn immunization increases serum anti-Tn antibody titers while reducing lavaged proteins and cytokines. The present invention therefore suggests that Tn immunization can attenuate inflammation-related diseases and organ damage. The present invention develops an anti-inflammatory vaccine composition as well as treatment or prevention of inflammation and lung damage (especially lung damage caused by hyperoxia) and progression of periodontal disease. Tn immunization also reduced the mean linear intercept and lung injury scores for lung injury. In addition, improvement in lung injury is accompanied by a decrease in NF-κB activity. In one aspect, the invention provides a single dose vaccine comprising from about 0.02 mg (preferably from about 0.1 mg) to about 2 mg of a Tn immunogen and an adjuvant solution, the Tn immunogen and an adjuvant solution The ratio is from about 0.5 to about 2 (v/v): from about 0.5 to about 2 (v/v). In a specific embodiment, the ratio of Tn immunogen to adjuvant solution is about 1 (v/v): about 1 (v/v). In some embodiments, the dose of the Tn immunogen ranges from about 0.02 mg to about 0.1 mg, from about 0.02 mg to about 0.05 mg, from about 0.1 mg to about 1.5 mg, from about 0.1 mg to about 1.2 mg, from about 0.1 mg to about 1 mg, from about 0.1 mg to about 0.8 mg, from about 0.1 mg to about 0.5 mg, from about 0.2 mg to about 1.5 mg, from about 0.2 mg to about 1.2 mg, from about 0.2 mg to about 1 mg, from about 0.2 mg to about 0.8 mg From about 0.2 mg to about 0.5 mg, from about 0.5 mg to about 2 mg, from about 0.5 mg to about 1.5 mg, from about 0.5 mg to about 1.2 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 0.8 mg, about From 1.0 mg to about 2 mg. The volume of the adjuvant solution ranges from about 0.2 ml to about 1 ml, from about 0.2 ml to about 0.8 ml, or from about 0.2 ml to about 0.6 ml. In another aspect, the invention provides a method of inducing an immune response in a subject to treat or prevent an inflammatory disease in a single dose vaccine, wherein the single dose vaccine comprises from about 0.1 mg to about 2 mg of a Tn immunogen. In one aspect, the invention provides a method of inducing an immune response in an individual to treat or prevent an inflammatory disease comprising administering a single dose of a Tn immunogen vaccine comprising from about 0.1 mg to about 2 mg per dose to the individual, Dosing at least four times at biweekly intervals. In one embodiment, the method further comprises performing an additional immunization of from about 0.1 mg to about 2 mg of the Tn immunogen one week after the fourth immunization. In a specific embodiment, the additional immunization can be performed one or more times after the last immunization. Administration of a Tn immunogen according to the methods of the invention produces an anti-Tn antibody that is higher in serum titer than the control group. In a specific embodiment, the serum titer of the produced anti-Tn antibody is at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 times higher than the control group. As used herein, a "control group" is a carrier polypeptide. In a specific embodiment, Tn immunization reduces the amount of interleukin-6 (IL-6) and TNF-[alpha] in a cell or an individual or decreases the activity of NF-[kappa]B. In a specific embodiment, the cell or individual has elevated Tn expression. In another embodiment, Tn expression is up-regulated by TNF-[alpha] and IL-6. In yet other embodiments, elevated Tn levels are typically regulated by a cytokine-Cosmc signaling axis. In a specific embodiment, the inflammatory disease is progression of gingival periostitis (periodontal disease), organ damage, or organ fibrosis. In another embodiment, the organ damage is a lung injury, a kidney injury, or a liver injury. In another embodiment, the lung injury is a hyperoxia-induced lung injury. In another embodiment, the organ fibrosis is pulmonary fibrosis, liver fibrosis, or renal fibrosis. In a specific embodiment, the method further comprises the step of performing an additional immunization one week after the fourth immunization. In some embodiments, a Tn immunogen can bind to a carrier polypeptide. The Tn immunogen and carrier polypeptide are at a weight ratio of from about 3 to about 8: about 1; preferably, the weight ratio is about 5: about 1. In some embodiments, the polypeptide includes, but is not limited to, an antigen presenting cell (APC) binding domain and a cysteine rich domain. In some embodiments, the APC binding domain is an immunoglobulin (Ig) Fc fragment or a receptor binding domain of a toxin. In another embodiment, the APC binding domain is a receptor binding domain of Pseudomonas exotoxin A, tetanus toxin or cholera toxin. In another embodiment, the APC binding domain is an Fc fragment of human Ig. In other embodiments, the cysteine-rich domain contains a fragment of 10 amino acid residues, at least 3 of which are cysteine residues. In yet other embodiments, the cysteine-rich domain contains six cysteine residues. Preferably, the cysteine-rich domain has an amino acid sequence of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys. In yet other embodiments, the cysteine-rich domain contains from 2 to 30 repeats of the amino acid sequence. Preferably, the cysteine-rich domain contains seven repeats of the amino acid sequence. In another embodiment, Tn is via a linker (eg, a linker containing a maleimide functional group; for example, N-maleimide or 6-butylene) N-succinimidyl-6-maleimidocaproate is attached to a cysteine residue. Preferably, the Tn immunogen binds to the carrier polypeptide as an Fc fragment-7 repeat of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-N-maleimide-Tn or Fc Fragment-7 repeated Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-6-m-butylene succinate-N-succinimide-Tn. In one embodiment, the Tn immunogen is N-ethylmercaptogalactosamine O-linked to a serine or threonine having the following structure: ; R is a serine or threonine. In a specific embodiment, the Tn immunogen is administered in the presence of 0.2 ml to 2 ml of adjuvant. In some embodiments, the adjuvant is aluminum hydroxide, aluminum phosphate, hydroxyapatite, Bordetella pertussis , Mycobacterium bovis , toxoid, squalene, Quil A, saponin, IL-1, IL - 2, IL-12, Fuller's adjuvant or Fraunhofer incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. A pharmaceutical composition comprising a Tn immunogen of the invention can be administered to an individual who has suffered from inflammation. In therapeutic applications, the composition is administered to the patient in an amount sufficient to elicit an effective immune response against the antigen present and to cure or at least partially arrest the symptoms and/or complications. Sufficient to achieve this amount is defined as "therapeutically effective amount." The effective amount for this use will depend, for example, on the composition of the peptide, the mode of administration, the stage and severity of the condition being treated, the weight and overall health of the patient, and the judgment of the prescribing physician. However, initial immunization (for therapeutic or prophylactic administration) generally ranges from about 0.1 mg to about 2 mg of the Tn immunogen, followed by an intensive course of treatment as described herein, which is from about 0.1 mg to about 2 mg. A dose of Tn immunogen. The vaccine composition for treatment is intended to be administered parenterally, topically, nasally, orally or topically. Preferably, the pharmaceutical composition is administered parenterally, for example intravenously, subcutaneously, intradermally or intramuscularly. Preferably, the vaccine is administered intramuscularly. The invention provides a composition for parenteral administration comprising a solution of the vaccine composition dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. These compositions may be sterilized by conventional conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, the lyophilized formulation being combined with a sterile solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting agents and buffers, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, which are required to be close to physiological conditions. , potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and the like. The examples of the invention are provided by way of example and not limitation. EXAMPLES Materials and Methods Animals Five-week-old female C57BL/6NCrlBltw mice were obtained from BioLASCO Taiwan Co., Ltd. and placed in a sterile room. Animals were maintained at approximately 25 o C and granulated food and water were supplied ad libitum throughout the experiment. The experimental procedure has been approved by the Laboratory Animal Care and Use Committee of Taipei Medical University (LAC-2016-0047). Vaccine Preparation Vaccines were prepared by conjugating Tn as described in previous studies ( HL Chiang, CY Lin, FD Jan, YS Lin, CT Hsu, J. Whang-Peng, LF Liu, S. Nieh, CC Lin, J. Hwang). , A novel synthetic bipartite carrier protein for developing glycotope-based vaccines. Vaccine 30 (2012) 7573–7581 ). Tn is 5 to 1 in glycotope/carrier protein weight ratio and ligated to ratFc (Cys42) Histag2 or GST (Cys6) Histatag2. The conjugation was carried out in a buffer containing 20 mM sodium phosphate, pH 7.9, 8 M urea, 500 mM imidazole, and 0.2 mM TCEP. After 48 hours, the conjugate was refolded in phosphate buffer (PBS) containing 0.2 mM TCEP. GST (Cys6) was dialyzed against PBS containing 0.2 mM TCEP. Glycotope with different linkers (alkylene maleic acyl group hexanoic acid N- succinimidyl ester (PEI)) joined to a GST (Cys6) 48 hours at 4 o C. The mouse experimental group was immunized subcutaneously with a 20 μg dose of Tn vaccine or carrier protein (10 μg of mFc (Cys42-Tn) Hista 2) in the presence of 100 μl of adjuvant for 5 weeks in a two-week period. Large female C57BL/6NCrlBltw The mice were administered 4 times and an additional immunization was performed 1 week after the 4th immunization. Blood was drawn from the facial vein, and the titer of the anti-Tn antibody was measured on days 0, 42, and 49 using an enzyme immunoassay (ELISA). Four days after the last immunization, the mice were exposed to room air (RA) or an oxygen-rich atmosphere (100% O2) for up to 96 hours. Oxygen exposure was continuously delivered to a transparent 60 x 50 x 40 cm Plexiglas chamber at 4 liters per minute with oxygen monitored by a ProOx Model 110 monitor (NexBiOxy, Hsinchu, Taiwan) with daily humidity readings of 60–80. %. The following four groups were obtained: carrier protein + RA (n = 6), Tn vaccine + RA (n = 6), carrier protein + O 2 (n = 6), and Tn vaccine + O2 (n = 5). Mice were deeply anesthetized with isoflurane after 96 hours of O 2 treatment. The lungs were lavaged in 0.6 ml, 0.9% saline solution at 4 o C, and the lungs were lavaged three times and then recovered. The washing procedure was repeated more than 2 times for each animal, 3 washes were collected and the total volume was recorded. After bronchoalveolar brushing examination, the right lung was ligated, and the left lung was fixed by intratracheal instillation with 4% buffered trioxane under the pressure of 25 cm H 2 O. Tn- amount of serum anti-antibody analysis by ELISA GST (Cys6-Tn) at 1.5μg / ml concentration of the coating in the groove 96 pans (Falcon Labware, Lincoln Park, NJ , USA). Serum from various dilutions was added to each coated well. After reacting at 37 ° C for 2 hours, the wells were washed three times with PBS. Next, anti-human immunoglobulin conjugated with peroxidase was added, and the plate was reacted at 37 ° C for 1 hour. The receptor solution contains 0.54 mg/ml of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) And 0.01% H 2 O 2 and 0.1 M citric acid (pH 4.2). Absorbance values were read at 410 nm. Bronchoalveolar lavage fluid protein and cytokine assays were tested using bicinchoninic acid (Pierce) Chemical, Rockford, IL, USA) The total protein concentration in bronchoalveolar lavage fluid (BALF) was measured. The amount of IL-6 and TNF-α in the BALF was determined using an ELISA kit (Cloud-Clone Corp., Houston, TX, USA) Data were expressed in mg/ml and pg/ml, respectively. Western blot analysis of NF-kB subcellular protein fractionation is a subcellular protein partitioning set for tissue. (Thermo Scientific, Melbourne, VIC, Australia, cat# 87790) was completed. Nuclear protein extracts were used to detect NF-κB p65 (SC-372, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) subunits and PCNA (SC-7907); cytoplasmic protein extract was used to detect IκB-α (SC-1643) and β-actin (SC-47778). The protein concentration was determined by using dicinocin. (bicinchoninic acid) test kit assay. Protein was separated and transferred to a polyvinylidene difluoride membrane using a 12% sodium dodecyl sulfate polyacrylamide gel, and the membrane was applied to 5 Blocking was performed for 1 hour at room temperature in % skim milk. The membrane was reacted with the antibody overnight at 4 ° C. Subsequently, the membrane and the secondary antibody conjugated with HRP were reacted at room temperature for 1 hour. The manufacturer's guideline visualizes the signal by enhanced chemiluminescence reagents. Anti-β-actin and PCNA antibodies are used as internal control groups for nuclear and cytoplasmic protein loading, respectively. Mice were at least three times. Pulmonary type was determined to normalize the analysis, and the sections were from the right middle lobe of the right lung. 5-μm lung tissue sections were stained with hematoxylin and eosin and assayed for morphology. The mean linear intercept (MLI), which is an indicator of the average diameter of the alveoli, was determined in one field of view. Histology was fixed in phosphate buffer containing 4% paraformaldehyde, embedded in paraffin, and Fine Red staining, and is detected by the process and the experimental group blind pathologist. Lung injury was scored by four criteria: 1) alveolar congestion, 2) hemorrhage, 3) infiltration of the neutrophil in the air cavity or vessel wall, and 4) thickness of the alveolar wall. Each item is graded according to a five-point scale as follows: 0 is the smallest (less) damage, 1 is the slight damage, 2 is the medium damage, 3 is the severe damage, and 4 is the largest damage. Of NF-kB Immunohistochemical staining of paraffin in the routine to step through the slides were immersed in 0.01 mol / L of sodium citrate buffer (pH 6.0) to the heat-induced epitope repair ( Retrieval). To block endogenous peroxidase activity and non-specific antibody binding, multiple anti-NF-κB P65 antibodies (1:50 dilution; Abcam Inc., Cambridge, MA, USA) were used as rabbit primary antibodies. The pellet was pre-reacted for 1 hour at room temperature in 0.1 mol/L PBS containing 10% normal goat serum and 0.3% H 2 O 2 before reacting at 4 ° C for 20 hours. These sections were then treated with biotinylated goat anti-rabbit IgG (1:200 dilution, Vector, CA, USA) for 1 hour at room temperature. The reaction was then carried out with reagents from the ABC kit (Avidin-Biotin Complex, Vector, CA, USA) according to the manufacturer's recommendations, and transmissive with diaminobenzidine. Kits (Vector, CA, USA) were used to visualize the products of the reaction. All immunostained sections were observed and photographed by Olympus BX 43. Statistical Analysis All data are presented as mean ± SD. Statistical analysis was performed using single factor variance analysis and was used for multiple recombination comparisons with Dukai's post hoc test. When P < 0.05, the difference was considered to be statistically significant. Example 1 titer of anti-Tn antibodies in all mice prior to vaccination anti-Tn antibody content low, its count and the background (FIG. 1). Accepting a carrier protein (ie, Fc fragment-7 repeats of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-6-m-butylene hydrazide hexanoic acid N-amber Mice with capsids in house air or hyperoxia showed background serum anti-Tn antibody content (Fig. 1A), whereas mice receiving Tn vaccination developed high serum anti-Tn antibody titers after Tn vaccination, and Anti-Tn antibodies remained high levels after several months of vaccination (Fig. 1B). Example 2 Survival and Body Weight Mice exposed to room air or hyperoxia survived throughout the study period. Mice exposed to hyperoxia showed significantly lower body weight at sacrifice than mice housed in RA (Figure 2). Example 3 Bronchoalveolar lavage fluid protein and cytokine analysis Mice were treated with carrier protein followed by exposure to hyperoxia. In BALF, these mice showed significantly higher total protein and IL-6 than mice exposed to RA. Content (Figures 3A and 3B). On the other hand, mice treated with Tn vaccine and exposed to hyperoxia showed significantly lower levels of IL-6 in BALF than mice treated with carrier protein (Fig. 3B). In BALF, mice treated with carrier protein and exposed to hyperoxia showed significantly higher TNF-[alpha] levels than mice exposed to RA (Fig. 3C). In BALF, mice treated with Tn vaccine and exposed to hyperoxia showed lower levels of TNF-[alpha]. However, the difference did not reach significance. Example 4 Histological Results Representative lung sections stained with hematoxylin and eosin from mice exposed to RA and hyperoxia are presented in Figure 4A. As indicated by the larger linear intercept, hyperoxia leads to infiltration of inflammatory cells and simplification of the lung parenchyma. Mice treated with vehicle protein and exposed to hyperoxia showed significantly higher lung injury scores and MLI compared to mice treated with carrier protein or Tn vaccine and exposed to RA (Figures 4B and 4C). Treatment with Tn vaccine significantly reduced hyperoxia-induced lung injury scores and increased MLI. Example 5 Immunohistochemistry of NF-κB Although immunohistochemical staining of NFκB was mainly found in the cytoplasm of alveolar macrophages, immunoreactivity was also observed in the nuclei of alveolar macrophages and a few alveolar epithelial cells (Fig. 5A). The lungs of the hyperoxia group immunized with the carrier protein exhibited stronger NFκB immunoreactivity than the control and the Tn-treated hyperoxia group. Example 6 Western blot analysis of NF-kB and IkB alpha Compared to mice treated with carrier protein or Tn vaccine and exposed to RA, mice treated with carrier protein and exposed to hyperoxia exhibited significantly higher nuclear NF - κB p65 and cytosolic phosphorylation-IκBα content (Fig. 5B and 5C). Even in the hyperoxia-treated rats, the rats treated with the Tn vaccine significantly reduced the levels of NFκB p65 and cytosolic phosphorylation-IκBα. Example 7 : Elevated Tn content in inflamed tissues and cells To examine whether elevated Tn levels are associated with inflammation, immunohistochemistry (IHC) was used to measure Tn levels in inflamed tissues. A significant increase in Tn content was observed in tissues of atherosclerosis, bronchitis, and root periosteum, but this increase was not observed in its corresponding normal tissues (Fig. 6A). To investigate the possible regulation of Tn levels by inflammatory cytokines, conditioned medium from LPS-stimulated monocyte U937 cells was used. The Tn content in human gingival fibroblasts (HGF) supplemented with one-day conditioned medium from LPS-stimulated U937 cells was observed to increase in a dose-dependent manner in LPS (Fig. 6B). Inflammatory cytokines (eg, TNF-α, IL- in conditioned medium from U937 cells treated with LPS (10, 30 or 100 ng/ml) for 24 hours compared to medium from U937 cells cultured without LPS The secretion of 6 and IL-1β) was significantly higher (Fig. 6C). Example 8 : Upregulation of Tn expression by TNF-[ alpha] and IL-6 in HGF To determine whether cytokines can increase Tn levels, HGF is treated with various amounts of purified cytokines. As shown in Figure 7A, the Tn content in HGF was the most responsive to TNF-[alpha], moderate to IL-6, and non-responsive to IL- l[beta], even at experimental concentrations of 100 ng/ml. TNF-α (30 ng/ml) showed an increase in Tn as a time-dependent. After 4 hours of TNF-α treatment, the Tn content in HGF was substantially unchanged. A gradual increase in Tn content was observed at 8 to 12 hours. The Tn content gradually decreased after 24 hours of TNF-α treatment and was significantly reduced after 48 hours of TNF-α treatment (Fig. 7B). Example 9 : TNF- α up-regulates Tn expression by down-regulating the COSMC gene To explore the possible molecular mechanisms underlying the upregulation of cytokine-mediated Tn levels, the effect of TNF-α on the mRNA content of the COSMC gene was investigated. As shown in Figure 8A, TNF-[alpha] (100 ng/ml, treatment for 24 hours) significantly down-regulated COSMC mRNA in HGF. In contrast, TNF-α did not significantly alter the T-synthase mRNA content. After TNF-α treatment, the results of protein content of Cosmc and T-synthase in HGF were similar (Fig. 8B, 8C and 8D). The effect of TNF-[alpha] on downregulation of the COSMC gene may involve hypermethylation of CpG islands in its promoter. Methylation changes in the promoter of the COSMC gene were quantified using bisulfite pyrophosphate sequencing, and TNF-[alpha] treatment resulted in significantly hypermethylation of the four CpG sites (Fig. 9A). Pretreatment of HGF with a demethylating agent reduced methylation of four CpG sites in the COSMC promoter in a dose-dependent manner (Fig. 9A) and, correspondingly, increased COSMC mRNA expression and decreased Tn content (Fig. 9) 9B). Taken together, our results indicate that cytokine-mediated up-regulation of Tn levels is caused by down-regulation of hypermethylated COSMCs involved in the COSMC gene promoter.