miR-409-3p靶向CXCL9调控滋养层细胞的增殖、迁移和侵袭

目的探讨微小RNA-409-3p(miR-409-3p)对滋养层细胞增殖、迁移和侵袭影响及作用机制。方法实时荧光定量PCR(RT-qPCR)检测正常妊娠胎盘组织和子痫前期胎盘组织中miR-409-3p和干扰素伽玛诱导的单核细胞因子(CXCL9)mRNA表达水平,蛋白印迹(Western blot)法检测CXCL9蛋白表达水平。转染miR-409-3p模拟物或CXCL9小干扰RNA至滋养层细胞HTR8/SVneo,构建miR-409-3p过表达或CXCL9表达抑制的HTR8/SVneo细胞,四甲基噻唑蓝染色法(MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,Western blot检测细胞周期蛋白D1(CyclinD1)、p21、基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达水平。双荧光素酶报告基因实验验证miR-409-3p与CXCL9调控关系。结果与正常妊娠胎盘组织比较,子痫前期胎盘组织中miR-409-3p水平升高(P<0.05),CXCL9 mRNA和蛋白水平降低(P<0.05)。miR-409-3p过表达或干扰CXCL9表达后,HTR8/SVneo细胞培养48 h和72 h后OD值、迁移和侵袭数、CyclinD1、MMP-2和MMP-9蛋白表达水平降低(P<0.05),p21蛋白表达水平升高(P<0.05)。miR-409-3p在HTR8/SVneo细胞中靶向负调控CXCL9表达。CXCL9过表达逆转了miR-409-3p过表达对HTR8/SVneo细胞增殖、迁移和侵袭的影响。结论 miR-409-3p抑制HTR8/SVneo细胞的增殖、迁移和侵袭与下调CXCL9表达有关。     

STAT3 and ERK Signaling Pathways Are Implicated in the Invasion Activity by Oncostatin M through Induction of Matrix Metalloproteinases 2 and 9

PurposeOur previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo.ResultsOSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition.ConclusionBoth STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.     

Epithelioid trophoblastic tumor: morphological and immunohistochemical study of three lung lesions

Epithelioid trophoblastic tumor (ETT) is a term proposed for an unusual variant of trophoblastic tumor that is closely related to choriocarcinoma but shows monomorphic growth of highly atypical trophoblastic cells instead of the typical dimorphic pattern of choriocarcinoma. We report here 3 cases of ETT, all of which were lung lesions probably originating from uterine trophoblastic disease. The antecedent pregnancies of the 3 cases were hydatidiform mole, invasive mole, and term pregnancy, respectively. The tumors were composed of highly atypical mononucleate cells, which mainly involved alveolar spaces, forming nests with central eosinophilic necrosis. Multinucleate giant cells were found within the nests, but they were fewer in number than in typical choriocarcinoma. The tumors were not associated with extensive hemorrhage or necrosis, except for 1 case, in which the ETT was combined with typical dimorphic choriocarcinoma. Immunohistochemically, multinucleate giant cells and occasional mononucleate tumor cells showed positivity for human chorionic gonadotropin. Staining for human placental lactogen was positive in rare multinucleate giant cells, and in 1 case, tumor cells showed diffuse positivity for placental alkaline phosphatase. Because ETT has a remarkably epithelioid appearance in cytological and architectural features, differentiation from the epithelial malignancies is problematic. Trophoblastic markers are frequently expressed in nontrophoblastic tumors, and reactivity for those markers alone is not sufficient for exclusion of other tumors. Rather, evidence of ETT comes from a combination of morphological features, immunohistochemical study, and clinical history.     

Interleukin-12 inhibits cell invasion in choriocarcinoma

Gestational trophoblastic disease (GTD) is a unique disease that arises from allografting of the conceptus, and has a characteristic morphology and biological behavior. It encompasses a spectrum of interrelated diseases, including hydatidiform mole, invasive mole, choriocarcinoma and placental-site trophoblastic tumor, but its pathogenesis remains unrevealed. Particularly, choriocarcinoma is a highly malignant tumor with poor prognosis. In this study, we cultured the human choriocarcinoma cell line JEG-3 in vitro. After treating the cells with different doses of interleukin (IL)-12, the cell invasion was observed. We also detected the expression of matrix metalloproteinases (MMP)-9 and tissue inhibitors of metalloproteinases (TIMP)-1 and the cell cycle of JEG-3 cells. Our data indicated that IL-12 inhibits cell invasion in a dose- and time-dependent manner through regulating the expression of MMP-9 and TIMP-1. In addition, treatment with IL-12 redistributes the phases of the cell cycle in JEG-3 cells. These findings suggest an antitumor role of IL-12 in choriocarcinoma, with far reaching possibilities for understanding the mechanisms of IL-12.     

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.     

Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells

We recently identified two novel aminopeptidases, placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP). Enzymatically, P-LAP degrades oxytocin, vasopressin, and angiotensin III, while A-LAP degrades angiotensin II and kallidin. In this study we investigated the expression and localization of P-LAP and A-LAP in human trophoblastic cells in the normal placenta (n = 26), gestational choriocarcinoma (n = 8), and placental site trophoblastic tumor (n = 3). On immunoblot analysis both P-LAP and A-LAP proteins were detected in normal placenta and five choriocarcinoma tissues, as well as in two choriocarcinoma cell lines. Immunohistochemical staining of normal placental tissues demonstrated that P-LAP was not only localized in villous syncytiotrophoblasts but also highly expressed in extravillous trophoblasts (EVTs) invading the decidua or maternal spiral arteries. The expression level of P-LAP on these invasive EVTs reached a maximum during the late first to second trimesters of pregnancy, and it decreased in the third trimester. Similarly, A-LAP was strongly expressed in EVTs invading the decidua or spiral arteries in the second trimester of pregnancy, while it was weakly or moderately expressed in villous cytotrophoblasts or EVTs located in the cell columns. These two aminopeptidases were more strongly expressed in all eight choriocarcinomas and three placental site trophoblastic tumors and mainly localized to the intermediate-type trophoblastic tumor cells invading the uterine myometrium or stromal vessels. In summary P-LAP and A-LAP were predominantly expressed in the invasive phenotype of EVTs during placentation, as well as in the invasive tumor cells of trophoblastic neoplasms. These results suggest the involvement of these aminopeptidases in invasiveness of both normal and malignant intermediate-type trophoblasts possibly through degradation of specific peptide substrates.     

Expression pattern alterations of syndecans and glypican-1 in normal and pathological trophoblast

Syndecans (syn-1, -2, -3, -4) and glypican-1 are proteoglycans expressed during development in association with changes in tissue organization and differentiation. They participate in the modulation of growth factor actions and in cell-cell and cell-matrix adhesion. The expression of syn-1, -2, -3, -4, and glypican-1 has been studied in normal human placenta and in gestational trophoblastic disease such as hydatidiform mole, invasive mole, and choriocarcinoma, using immunohistochemistry and western blots. Syndecan-3 was not expressed in normal or pathological tissues. During normal gestation, the other proteoglycans showed a specific staining pattern, which for some was modified during pregnancy. For instance, syn-1 was only expressed in syncytiotrophoblast; syn-4 was mainly localized in the villous and extravillous cytotrophoblast in the first trimester, whereas at term it was expressed in the syncytiotrophoblast. The most striking results are the altered expression patterns of syndecans and glypican-1 in pathological tissues. These proteoglycans showed a progressive decrease of immunostaining related to the increase of severity of trophoblastic disease, in particular in invasive mole and choriocarcinoma. In addition, dysregulation in the localization of the expression patterns was observed for syn-2 and -4. Because changes in syndecan expression enable cells to become more or less responsive to their micro-environment, the down-regulation and/or dysregulation of syndecans in relation to the degree of severity of trophoblastic diseases provides new insights into the progression of these pathologies.     

Promyelocytic leukaemia (PML) protein expression in human placenta and choriocarcinoma

Promyelocytic leukaemia (PML) protein, the product of the pml gene, is heterogeneously expressed in various normal and neoplastic tissues, and the fusion of the pml gene with retinoic acid receptor-alpha is believed to be a central mechanism in acute PML tumourigenesis. As PML is important for controlling major cellular processes, such as growth and differentiation, it is believed that it plays an important role during human gestation. The human placenta is a critical organ for the maintenance of gestation, but the expression pattern and functional significance of PML in the placenta have not been documented. The present study has therefore investigated the expression of PML in the human placenta and in choriocarcinoma, and has observed the biological effects following the overexpression of PML in choriocarcinoma cell lines (BeWo and JEG-3). In the human placenta, PML expression was readily found in villous stromal fibroblasts, capillary endothelial cells, Hofbauer cells, and occasionally in amnion cells. Moreover, immunoblotting of placental lysates demonstrated increased PML expression with increasing gestation. Interestingly, PML expression was confined to intermediate trophoblasts and syncytiotrophoblastic giant cells at the placental site (placental site giant cells) in the trophoblastic cell population. Intermediate trophoblasts at non-placental sites, and villous cytotrophoblasts and syncytiotrophoblasts consistently did not express PML. Further screening of PML expression in hydatidiform moles (n = 4) and choriocarcinomas (n = 7) also revealed selective PML expression in intermediate trophoblastic cells and syncytiotrophoblastic cells, but not in the cytotrophoblastic populations, which corresponds well with observations in the placental bed. Adenoviral transduction of PML resulted in a marked reduction in cell growth in both choriocarcinoma cell lines, which was associated with increased apoptosis. The findings of the present study strongly suggest that PML plays an important role in human placental development and growth, and in the pathobiology of trophoblasts and trophoblastic neoplasia.     

Origin of histiocyte-like cells and multinucleated giant cells in malignant fibrous histiocytoma: Neoplastic or reactive?

The origin of histiocyte-like cells in malignant fibrous histiocytoma (MFH) remains controversial. To determine whether histiocyte-like cells and multinucleated giant cells show reactive or neoplastic proliferation, we transplanted human storiform-pleomorphic MFH to nude mice and investigated the origin of histiocyte-like cells using the DNA in situ hybridization (ISH) system. In addition, we analyzed the mRNA expression of mouse c-fms and human colony stimulating factor-1 (CSF-1); immunohistochemical expression of markers detectable in cells of monocyte/macrophage lineage. The DNA ISH revealed neoplastic proliferation of fibroblastic cells and bizarre multinucleated giant cells of human origin. Monocyte/macrophage lineage cells were seen in parental tumors, whereas they did not participate in neoplastic proliferation in transplanted tumors. The parental tumors expressed human CSF-1 mRNA and the histiocyte-like cells in transplanted tumors expressed 'mouse' c-fms mRNA. These results suggest that MFH induce infiltration of monocyte/macrophage and CSF-1 is one of the mediators involved in this phenomenon, because the human CSF-1 can act as a ligand to the mouse c-fms. Histiocyte-like cells in MFH should be considered as a reactive monocyte/macrophage lineage rather than as an element of neoplasm.     

Induction of prostaglandin E2 production by leukemia inhibitory factor promotes migration of first trimester extravillous trophoblast cell line, HTR-8/SVneo

BACKGROUND: The invasion of first trimester extravillous trophoblast (EVT) to decidua is an important event in placentation. Leukemia inhibitory factor (LIF) is an essential factor for mouse implantation, and it is reported that LIF may be involved in human first trimester EVT invasion. Prostaglandin E2 (PGE2) is also known as a critical factor for first trimester EVT invasion. In this study, we investigated the role of LIF in PGE2 production and EVT invasion using a human first trimester EVT cell line, HTR-8/SVneo. METHODS AND RESULTS: Co-stimulation with LIF and IL-1beta induced higher amounts of PGE2 production and further migration of HTR-8/SVneo cells compared with that by stimulation with LIF or IL-1beta alone. Enhanced PGE2 production was most probably due to the enhanced expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). PGE2 produced by HTR-8/SVneo cells promoted the migration of HTR-8/SVneo cells. A COX-2 inhibitor suppressed PGE2 production and the migration of HTR-8/SVneo cells. Agonists to PGE2 receptors, EP1, EP2 and EP4, promoted the migration of HTR-8/SVneo cells. Moreover, stimulation with LIF up-regulated EP1, EP2 and EP4 expression in HTR-8/SVneo cells. CONCLUSIONS: It is suggested that LIF participates in placentation through EVT invasion by up-regulating PGE2 production and PGE2 receptor expression in first trimester EVT.     

Caspase activity is downregulated in choriocarcinoma: a cDNA array differential expression study

BACKGROUND: Placental trophoblast can be considered to be pseudomalignant tissue and the pathogenesis of gestational trophoblastic diseases remains to be clarified.AIMS: To examine the role of caspases 8 and 10, identified by differential expression, on trophoblast tumorigenesis. METHODS: cDNA array hybridisation was used to compare gene expression profiles in choriocarcinoma cell lines (JAR, JEG, and BeWo) and normal first trimester human placentas, followed by confirmation with quantitative real time polymerase chain reaction and immunohistochemistry. Caspase 10 and its closely related family member caspase 8 were analysed. RESULTS: Downregulation of caspase 10 in choriocarcinoma was detected by both Atlastrade mark human cDNA expression array and Atlastrade mark human 1.2 array. Caspase 10 mRNA expression was significantly lower in hydatidiform mole (p = 0.035) and chorioarcinoma (p = 0.002) compared with normal placenta. The caspase 8 and 10 proteins were expressed predominantly in the cytotrophoblast and syncytiotrophoblast, respectively, with significantly lower expression in choriocarcinomas than other trophoblastic tissues (p < 0.05). Immunoreactivity for both caspase 8 and 10 correlated with the apoptotic index previously assessed by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (p = 0.02 and p = 0.04, respectively) and M30 (p < 0.001 and p = 0.003, respectively) approaches. CONCLUSIONS: These results suggest that the downregulation of capases 8 and 10 might contribute to the pathogenesis of choriocarcinoma.     

IL-22 secreted by decidual stromal cells and NK cells promotes the survival of human trophoblasts

Interleukin-22 (IL-22) has been implicated as an important immune regulator in many physiologic and pathological processes, but little is known about the IL-22 in the fetal-maternal interface. In this study, we demonstrated that co-culture of decidual stromal cells (DSCs) and decidual natural killer (dNK) cells resulted in increased secretion of IL-22, compared to culture of DSCs or dNK cells alone. The trophoblast cell line, HTR8/SVneo, expresses IL-22 receptor α1 (IL-22R1). Combinant human (rh) IL-22 significantly promoted the proliferation and viability, and inhibited the apoptosis of HTR8/SVneo cells. By Western blotting and immunohistochemistry, we confirmed that villi expressed IL-22R1, and the villi from unexplained spontaneous miscarriage patients expressed reduced levels of IL-22R1 than those from normal early pregnancy. These findings indicate that the IL-22 secreted by DSCs and dNK might promote the survival of trophoblasts and participate in the maintenance of pregnancy by binding to the IL-22R1. The reduced level of IL-22/IL-22R1 in villi might be involved in the occurrence of spontaneous miscarriage.     

胎盘粘连植入组织和人胎盘滋养层细胞中VCAM-1的表达及其与侵入性胎盘疾病的相关性

目的探讨胎盘粘连植入组织和人胎盘滋养层细胞中血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)的表达及其与侵入性胎盘疾病的相关性。方法采用酶联免疫吸附试验检测2017年9月至2018年9月期间我院收治的20例产妇静脉血中可溶性VCAM-1(sVCAM-1)的表达水平,根据sVCAM-1的平均值将另外随机选取的200例产妇分为高表达组和低表达组,计算两组侵入性胎盘发生率。采用免疫组化染色、实时定量PCR和Western blot检测40例侵入性胎盘组织和40例正常胎盘组织中的VCAM-1、TNF-α、NF-κB p65和IκBα表达。应用20 ng/mL的TNF-α处理人胎盘滋养层HTR8/SVneo细胞系后检测细胞中VCAM-1、TNF-α、NF-κB p65和IκBα的表达。结果20例健康产妇静脉血中的VCAM-1平均水平为36.32±3.25 ng/mL。高表达组的侵入性胎盘发生率(6.98%)显著高于低表达组(0.64%)(χ^2=0.922,P=0.009)。实时定量RT-PCR和Western blot结果表明,与对照组相比,侵入性胎盘组VCAM-1的mRNA和蛋白表达水平显著升高。免疫染色显示VCAM-1蛋白主要在滋养细胞的细胞质和膜中表达,侵入性胎盘组的VCAM-1阳性率(72.50%)显著高于对照组(17.50%)(Z=-5.063,P<0.001)。与对照组相比,侵入性胎盘组的TNF-α和NF-κB p65表达水平显著升高,而IκBα表达水平显著降低(P<0.05)。与未用TNF-α处理(0 ng/mL)的HTR8/SVneo细胞相比,应用20 ng/mL浓度的TNF-α处理细胞1周后,细胞中的NF-κB p65和VCAM-1表达水平显著升高,而IκBα表达水平显著降低(P<0.05)。结论VCAM-1在侵入性胎盘产妇血液和胎盘组织中均为高表达模式。VCAM-1的活化至少部分依赖于TNF-α、NF-κB信号通路的激活,从而诱导其在滋养细胞中高表达并引起过度侵袭,导致侵入性胎盘的发生。     

The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.     

Effects of Tumor Necrosis Factor-Alpha on Human Trophoblast Cell Adhesion and Motility

PROBLEM: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor-alpha (TNF-α) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF-α may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF-α on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion. METHODS: The effect of TNF-α on the motility of extended-lifespan first trimester trophoblasts (HTR) and JEG-3 choriocarcinoma cells was tested using the phagokinetic track motility assay. An in vitro adhesion assay was used to determine the effect of TNF-α on the adhesion of HTR and JEG-3 cells to laminin, a major basement membrane component. In addition, the effect of TNF-α on the surface expression of the laminin receptor β1 integrin subunit was examined using flow cytometry. RESULTS: HTR or JEG-3 cells were strongly adherent to laminin which was not significantly altered by TNF-α treatment. We also measured the effect of TNF-α on the surface expression of β1 integrin on HTR and JEG-3 cells; no difference was observed between control and treatment groups. Interestingly, the motility of both HTR and choriocarcinoma JEG-3 cells was significantly inhibited by TNF-α. CONCLUSIONS: The role of TNF-α in human embryo implantation is currently unknown. Our data demonstrate that TNF-α does not alter trophoblast cell adhesion to laminin, but significantly inhibits trophoblast cell motility in vitro, suggesting that TNF-α may play a regulatory role in trophoblast cell invasion.     

Folate ameliorates dexamethasone-induced fetal and placental growth restriction potentially via improvement of trophoblast migration

Overexposure to prenatal dexamethasone (Dex) leads to small placental and fetal size and the alteration of fetal programming. Folate plays important roles in processes associated with successful pregnancy, including angiogenesis and trophoblast invasion. Placental folate transport is altered with prenatal Dex administration. The purpose of this study was to investigate the protective role of maternal folate administration in placentas exposed to Dex. In vitro, four groups of C57BL/6J pregnant mice were utilized: 1) normal drinking water + Saline injection group (NN); 2) normal drinking water + Dex injection group (ND); 3) drinking water with folate + Saline injection group (FN); and 4) drinking water with folate + Dex injection group (FD). In vivo, four treatment groups of the human extravillous trophoblast HTR-8/SVneo cells were classified: 1) control (NN); 2) Dex treatment (ND); 3) folate treatment (FN); and 4) folate and Dex treatment (FD). The results showed the maternal folate increases the placental size, birth weight, and expression of matrix metalloproteinases 9 (MMP9) in a mice model of Dex overexposure. In human extravillous trophoblast HTR8/SVneo, folate ameliorated the Dex-induced supress of cell migration and improved the expression/activity of MMP2 and MMP9. In conclusion, folate might be a potential therapy intervention to reduce the adverse effects of prenatal Dex exposure partially via improved trophoblast migration.     

Trophoblasterkrankungen und Trophoblasttumoren

The clinico-pathologic characteristics and the histological/immunohistological differential diagnosis of villous trophoblastic lesions (complete, partial, and invasive hydatiform moles), gestational choriocarcinoma and trophoblastic tumors and pseudotumors of the placental site (placental-site trophoblastic tumor, exaggerated placental site reaction, placental site nodule) are reviewed in context with aspects of cytogenetics, epidemiology and molecular pathogenesis.