Effects of mycophenolate mofetil on cutaneous lupus erythematosus in (NZB × NZW) F1 mice

AbstractBackgroundFew studies have evaluated the effects and precise molecular mechanism of mycophenolate mofetil (MMF) in the treatment of human cutaneous lupus erythematosus (CLE). Our findings shed light on the therapeutic effects of MMF in a UVB-induced NZB × NZW (NZBW) F1 CLE mouse model.MethodsContinuous MMF treatment (60 mg/kg/day) was administered up to Day 50 from the beginning of UVB induction (Day 0; 20 weeks old), as the pathologic features of CLE are present after 50 days. The therapeutic effects of MMF treatment in NZBW lupus mice were examined by comparing histopathological changes, lupus band test (deposition of immune complexes at the dermal–epidermal junction) and colocalization of autoantibodies with a dermal autoantigen Dsg3, and by evaluating the associations of local matrix metalloprotease activities.ResultsMMF improved survival in the NZBW lupus mice from 35.7% to 81.8%. The proteinuria, blood urea nitrogen, and interleukin 6 levels were significantly reduced after MMF treatment. The dermal lymphocytic infiltration, deposition of immune complexes at the dermal–epidermal junction, colocalized autoantibodies with Dsg3, and epidermal matrix metalloprotease activity were also attenuated in MMF-treated NZBW F1 mice.ConclusionThe results confirmed that MMF could substantially attenuate skin damage due to CLE in the NZBW F1 mouse model

Bronchiolitis Obliterans Organizing Pneumonia in Swine Associated with Porcine Circovirus Type 2 Infection

Bronchiolitis obliterans organizing pneumonia (BOOP) is a chronic respiratory disease. Although the pathogenesis of BOOP is still incompletely understood, BOOP is responsive to steroids and has a good prognosis. In our five pigs with chronic postweaning multisystemic wasting syndrome (PMWS), typical BOOP lesions were revealed. All five porcine lungs showed typical intraluminal plugs, and porcine circovirus type 2 (PCV2) was identified. They also exhibited similar pathologic findings such as proliferation of type II pneumocytes and myofibroblasts (MFBs), extracellular collagen matrix (ECM) deposition, and fragmentation of elastic fibers. MFBs migration correlative molecules, for instance, gelatinase A, B and osteopontin, appeared strongly in the progressing marginal area of polypoid intraluminal plugs of fibrotic lesion. These molecules colocalized with the active MFBs. Both gelatinase activity and intercellular level of active MFBs were significantly increased (P < .05). Porcine chronic bronchopneumonia leads to BOOP and it is associated with PCV2 persistent infection. Swine BOOP demonstrates similar cellular constituents with human BOOP. Perhaps their molecular mechanisms of pathogenesis operate in a similar way. Thus we infer that the swine BOOP can be considered as a potential animal model for human BOOP associated with natural viral infection. Moreover, it is more convenient to obtain samples

Activin B Promotes Epithelial Wound Healing In Vivo through RhoA-JNK Signaling Pathway

Background: Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated. Principal Findings: In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19) and RhoA (L63) were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 59-bromodeoxyuridine (BrdU) incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing. Conclusion: These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock

Acute cardiac damage and early activation of myocardial matrix metalloproteinases after subarachnoid hemorrhage.

臨床上，蛛網膜下腔出血（subarachnoid hemorrhage；SAH）的病人經常有急性心肌損傷及心室功能不全的現象。其致病原因最主要是因腦出血後，顱內壓升高導致交感神經過度興奮而來。這種交感神經風暴會促使心肌過度收縮而造成心肌損傷。但是，對於蛛網膜下腔出血引發心臟收縮功能不全的分子機轉研究則尚未有人提出。因此，建立一種簡單、穩定、可靠的動物模型，在蛛網膜下腔出血引起早期心肌損傷及心室功能障礙的病理生理機制研究上極為重要。肌鈣蛋白 I（cardiac troponin I；cTnI）已知是心肌細胞調控心臟收縮的一個重要結構蛋白，如果心肌中的肌鈣蛋白 I 被分解，則心臟的收縮會不完全而使心肌受損。基質金屬蛋白酶（matrix metalloproteinases；MMPs）在心肌細胞所扮演的角色不僅可以分解細胞外的細胞間質（extracellular matrix；ECM），亦可在很短的時間內（幾分鐘）分解細胞內的肌鈣蛋白 I，致使心臟收縮不完全。因此本實驗則提出蜘蛛膜下腔出血後，基質金屬蛋白酶可在心肌組織中被活化而增加心肌肌鈣蛋白 I 的分解，導致急性心臟收縮功能不全。我們利用 3/0 nylon 線穿刺大白鼠大腦動脈，模擬蛛網膜下腔出血。在出血後極短時間內（0-180分鐘）有類似臨床常見顱內壓、平均動脈壓及心跳顯著性的升高，並伴隨著心律的異常、左心室舒張功能指數（−LV dP/dtmax）的下降以及交感神經傳遞物正腎上腺素的上升。180分鐘後以肉眼觀察心室有明顯擴張、塌陷及鬱血的現象，並有類似蛛網膜下腔出血病患心肌 myocytolysis 和橫紋消失的病理病變產生。由血液肌鈣蛋白 I 濃度的上升及心肌肌鈣蛋白 I 免疫組織化學染色的流失，可進一步診斷為早期心肌損傷。因此，本動物模式可作為模擬人類蛛網膜下腔出血誘發急性心臟損傷及心肌功能障礙的實驗模式。接著進行導致心臟收縮功能不全之分子機轉研究。首先研究心臟受損後血液及心肌基質金屬蛋白酶（MMP-2，MMP-9）表現量的變化，以及基質金屬蛋白酶對於負責調節心肌收縮的心肌肌鈣蛋白 I 裂解作用的影響。出血後左心室功能明顯下降，血液中 pro-MMP-9 及肌鈣蛋白 I 濃度明顯上升。心肌內很快的（甚至於提早至30分鐘）activated MMP-2、pro-MMP-2 及 pro-MMP-9 活性增強，伴隨著心肌肌鈣蛋白 I 完整性的降低及裂解比例的增高。在相關性的分析上，心肌基質金屬蛋白酶活性的增強、心肌肌鈣蛋白 I 蛋白含量的減少與心室舒張能力的維持有密切的關係。這些結果顯示早期蛛網膜下腔出血可誘發活化基質金屬蛋白酶及水解心肌肌鈣蛋白 I，而導致心室功能不全。因此，本實驗結果可作為臨床上蛛網膜下腔出血引發早期心臟損傷與心功能不全預防和治療之方針。Early cardiac damage and reversible ventricular dysfunction (stunning) have been reported in humans with subarachnoid hemorrhage (SAH). It has been considered that sympathetic activity with catecholamine-mediated injury is the most likely cause of cardiac injury after SAH. However, the molecular pathogenesis of ventricular dysfunction in SAH hemorrhage remains unknown. Investigations of early cardiac damage and ventricular dysfunction occurring immediately after SAH are important, but would be impossible to conduct without a good animal model. Cardiac troponin I (cTnI) is a key regulatory protein in cardiac muscle contraction and relaxation. Selective proteolysis of cTnI has recently been proposed to play a key role in the contractile dysfunction observed in stunned myocardium. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases which are synthesized as zymogens. MMPs not only extracellularly remodel the ECM but also act intracellularly in contractile dysfunction hearts by degrading troponin I. Therefore, we wish to test the hypothesis that activation of MMPs (MMP-2 or/and MMP-9) and subsequent cleavage of troponin I in ventricular may cause acute myocardial damage and contractile dysfunction after SAH. SAH was induced by the endovascular suture method in rats. SAH induced increases in mean blood pressure, heart rate, intracranial pressure and plasma norepinephrine accompanied with electrocardiogram abnormalities and ventricular dysfunction. The heart was dilated. Myocardial damage was evident from the increased plasma cTnI and marked loss of myocardial tissue cTnI in myocytolytic areas at 180 minutes after SAH. Thus, we create a rat SAH model that can be applied in studying mechanisms of the early myocardial damage and ventricular dysfunction following SAH and for evaluating therapeutic strategies in SAH patients. To study the molecular mechanisms leading to contractile dysfunction in the acute stage of SAH, we examined the temporal profiles of MMPs and cTnI in blood and heart tissue and to determine whether changes in these profiles are associated with myocardial dysfunction and cTnI degradation. Following SAH, there was an early increase (as early as 30 minutes) in activated MMP-2 expression, pro-MMP-2 and pro-MMP-9 activities in myocardium that was accompanied by a decrease in tissue cTnI integrity. The enhanced myocardial MMP-2 and -9 activities and the loss of tissue cTnI content correlated with the –LV dP/dtmax (index of left ventricular diastolic function). The degradation of tissue cTnI and the release of plasma cTnI were also increased following SAH. These data showed that SAH could induce the activation of MMPs and proteolysis of cTnI that lead to cardiac dysfunction.目次 中文摘要 ……………………………………………………………..…….…. i Abstract ………………………………………………..…………................ iii 目次 .………………………………………………………………………..… v 圖表目次 .……………………………………………………………..……... vi 第一章 緒論 .…………………………………………………………..…….. 1 第一節 前言 ……………………………………………………………..... 1 第二節 文獻探討 .……………………………………………………..….. 1 一、蛛網膜下腔出血與心肌損傷的的關係 ……………………..…. 1 二、致病機轉-兒茶酚胺的角色 ………………………………….… 3 三、基質金屬蛋白酶（MMPs）參與急性心肌損傷的機制 …….. 5 第三節 研究目的與假說 .……………………………………………….... 8 第二章 動物模式建構 蜘蛛網膜下腔出血誘發急性心肌受損之動物模式建構 .……… 10 第一節 前言.…………………………………………………………….... 10 第二節 材料與方法 .………………………………………………….…. 11 第三節 結果 .…………………………………………….……………..... 14 第四節 討論 .………………………………………………………….…. 16 第三章 致病機轉研究 蛛網膜下腔出血誘發早期心肌基質金屬蛋白酶活化及肌鈣 蛋白 I 降解的研究 .…………………………………………….. 28 第一節 前言 .…………………………………………………………..… 28 第二節 材料與方法 …………………………………………………...… 29 第三節 結果 ………………………………………………..………….… 32 第四節 討論 …………………………………………………................... 34 第四章 總結論 ………….………………………………………………….. 50 第五章 參考文獻 .………………………………………………………….. 51 第六章 個人相關研究發表清單 ….……………………………………….

Effects of mycophenolate mofetil on cutaneous lupus erythematosus in (NZB × NZW) F1 mice

Background: Few studies have evaluated the effects and precise molecular mechanism of mycophenolate mofetil (MMF) in the treatment of human cutaneous lupus erythematosus (CLE). Our findings shed light on the therapeutic effects of MMF in a UVB-induced NZB × NZW (NZBW) F1 CLE mouse model. Methods: Continuous MMF treatment (60 mg/kg/day) was administered up to Day 50 from the beginning of UVB induction (Day 0; 20 weeks old), as the pathologic features of CLE are present after 50 days. The therapeutic effects of MMF treatment in NZBW lupus mice were examined by comparing histopathological changes, lupus band test (deposition of immune complexes at the dermal–epidermal junction) and colocalization of autoantibodies with a dermal autoantigen Dsg3, and by evaluating the associations of local matrix metalloprotease activities. Results: MMF improved survival in the NZBW lupus mice from 35.7% to 81.8%. The proteinuria, blood urea nitrogen, and interleukin 6 levels were significantly reduced after MMF treatment. The dermal lymphocytic infiltration, deposition of immune complexes at the dermal–epidermal junction, colocalized autoantibodies with Dsg3, and epidermal matrix metalloprotease activity were also attenuated in MMF-treated NZBW F1 mice. Conclusion: The results confirmed that MMF could substantially attenuate skin damage due to CLE in the NZBW F1 mouse model

High-dose Norepinephrine Induces Disruption of Myocardial Extracellular Matrix and Left Ventricular Dilatation and Dysfunction in a Novel Feline Model

Intravenous norepinephrine (NE) at a dose of 1-6 μg/kg/minute can induce increased extracellular matrix (ECM) and hypertrophic cardiomyopathy. This study aimed to investigate the effects of a higher dose of NE on cardiac remodeling. Methods: After intraperitoneal urethane-chloralose anesthesia, 7 cats (3.03 ± 0.58 kg) received intravenous infusion of NE 30 (ig/kg/minute for 3 hours. Aortic blood pressure and heart rate (HR) were measured by polygraphy at 0, 5,15, 30, 60, 90, 120, and 180 minutes. Left ventricular size and ejection fraction (EF) were measured by M-mode echocardio-graphy before and after NE administration. Histopathology was performed by hematoxylin-eosin, silver impregnation, and Sirius red staining. Activity of matrix metalloproteinases (MMP) in the left ventricle was measured by zymography. Results: Mean blood pressure (mmHg) increased from 139 ± 20 to 198 ± 19,187 ± 23, and 166 ± 16 at 15, 30, and 60 minutes, respectively, during NE infusion. HR (beats/minute) decreased from 214 ± 10 to 158 ± 28 at 15 minutes and then recovered gradually. The left ventricles showed significant dilatation (end-diastolic diameter: from 1.20 ± 0.18 to 1.58 ± 0.23cm, p = 0.003; end-systolic diameter: from 0.62 ± 0.23 to 1.35 ± 0.29cm, p = 0.002) and hypokinesia (EF: from 86.2 ± 5.2 to 33.1 ± 16.5%, p< 0.001). Histopathology revealed that left ventricular myocytes were elongated, wavy, and fragmented, while collagen fibers were overstretched, straightened, and disrupted. MMP-9 activity was significantly elevated (p = 0.003 vs. control), while MMP-2 activity was unchanged. Conclusion: High-dose NE increases MMP-9 activity and causes ECM disruption, left ventricular dilatation and dysfunction

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca2+ transients and TGF- -signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation