Tuya Shilagard

Visualizing Hepatitis C Virus Infections in Human Liver by Two-Photon Microscopy

BACKGROUND & AIMS Although hepatitis C virus (HCV) is a common cause of cirrhosis and liver cancer, efforts to understand the pathogenesis of HCV infection have been limited by the low abundance of viral proteins expressed within the liver, which hinders the detection of infected cells in situ. This study evaluated the ability of advanced optical imaging techniques to determine the extent and distribution of HCV-infected cells within the liver. METHODS We combined 2-photon microscopy with virus-specific, fluorescent, semiconductor quantum dot probes to determine the proportion of hepatocytes that were infected with virus in frozen sections of liver tissue obtained from patients with chronic HCV infection. RESULTS Viral core and nonstructural protein 3 antigens were detected readily in liver tissues from patients with chronic infection without confounding tissue autofluorescence. Specificity was confirmed by blocking with specific antibodies and by tissue colocalization of distinct viral antigens. Between 7% and 20% of hepatocytes were infected in patients with plasma viral RNA loads of 10(5) IU/mL or greater. Infected cells were in clusters, which suggested spread of the virus from cell to cell. Double-stranded RNA, a product of viral replication, was abundant within cells at the center of such clusters, but often scarce in cells at the periphery, consistent with more recent infection of cells at the periphery. CONCLUSIONS Two-photon microscopy provides unprecedented sensitivity for the detection of HCV proteins and double-stranded RNA. Studies using this technology indicate that HCV infection is a dynamic process that involves a limited number of hepatocytes. HCV spread between cells is likely to be constrained by host responses.

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

In vivo multimodal nonlinear optical imaging of mucosal tissue

We present a multimodal nonlinear imaging approach to elucidate microstructures and spectroscopic features of oral mucosa and submucosa in vivo. The hamster buccal pouch was imaged using 3-D high resolution multiphoton and second harmonic generation microscopy. The multimodal imaging approach enables colocalization and differentiation of prominent known spectroscopic and structural features such as keratin, epithelial cells, and submucosal collagen at various depths in tissue. Visualization of cellular morphology and epithelial thickness are in excellent agreement with histological observations. These results suggest that multimodal nonlinear optical microscopy can be an effective tool for studying the physiology and pathology of mucosal tissue.

C6 knock-out mice are protected from thrombophilia mediated by antiphospholipid antibodies

Background: Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6−/−) mice. Methods: C6−/− mice or the wild-type C3H/HeJ (C6+/+) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. Results: Thrombus sizes were significantly larger in C6+/+ treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6+/+ mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6−/− mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6+/+ counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6−/− mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6+/+) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β2glycoprotein I (aβ2GPI) antibodies. Conclusions: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

IL-6 Regulates Extracellular Matrix Remodeling Associated With Aortic Dilation in a Fibrillin-1 Hypomorphic mgR/mgR Mouse Model of Severe Marfan Syndrome

Background Development of thoracic aortic aneurysms is the most significant clinical phenotype in patients with Marfan syndrome. An inflammatory response has been described in advanced stages of the disease. Because the hallmark of vascular inflammation is local interleukin‐6 (IL‐6) secretion, we explored the role of this proinflammatory cytokine in the formation of aortic aneurysms and rupture in hypomorphic fibrillin‐deficient mice (mgR/mgR). Methods and Results MgR/mgR mice developed ascending aortic aneurysms with significant dilation of the ascending aorta by 12 weeks (2.7±0.1 and 1.3±0.1 for mgR/mgR versus wild‐type mice, respectively; P<0.001). IL‐6 signaling was increased in mgR/mgR aortas measured by increases in IL‐6 and SOCS3 mRNA transcripts (P<0.05) and in cytokine secretion of IL‐6, MCP‐1, and GM‐CSF (P<0.05). To investigate the role of IL‐6 signaling, we generated mgR homozygous mice with IL‐6 deficiency (DKO). The extracellular matrix of mgR/mgR mice showed significant disruption of elastin and the presence of dysregulated collagen deposition in the medial‐adventitial border by second harmonic generation multiphoton autofluorescence microscopy. DKO mice exhibited less elastin and collagen degeneration than mgR/mgR mice, which was associated with decreased activity of matrix metalloproteinase‐9 and had significantly reduced aortic dilation (1.0±0.1 versus 1.6±0.2 mm change from baseline, DKO versus mgR/mgR, P<0.05) that did not affect rupture and survival. Conclusion Activation of IL‐6‐STAT3 signaling contributes to aneurysmal dilation in mgR/mgR mice through increased MMP‐9 activity, aggravating extracellular matrix degradation.

Use of high-resolution confocal imaging of the vaginal epithelial microstructure to detect microbicide toxicity.

OBJECTIVE High-resolution optical imaging by confocal reflectance microscopy (CRM) was investigated for its ability to delineate the epithelial microstructure of the vaginal tract and detect alterations that may result from the use of vaginal microbicides. METHODS The vaginal tracts of Swiss Webster mice treated with medroxyprogesterone acetate were exposed in vivo to a 4% nonoxynol-9 (N-9)-containing gel or saline. The vaginal tract was removed 4 h, 16 h, or 48 h after treatment and imaged by CRM without staining, and biopsy specimens were obtained from the imaged regions and processed for histological analysis. RESULTS In control mice, CRM revealed a columnar epithelium and lamina propria with features resembling those observed via histological analysis. CRM revealed an exfoliated epithelium 4 h and 16 h after N-9 treatment, and quantitative measurement of epithelial thickness revealed a mean thickness (+/- standard error of the mean) of approximately ~41.7 +/- 1.7 mum in control specimens, compared with 14.9 +/- 4.5 mum for specimens obtained 4 h after treatment and 24.4 +/- 2.1 mum for specimens obtained 16 h after treatment. Inflammation 4 h after treatment was indicated through detection of inflammatory infiltrates. In samples collected 48 h after treatment, the epithelium was regenerating. The time line of changes in the morphological structure and epithelial thickness detected by CRM closely resembled that of changes revealed by histological analysis. CONCLUSIONS This study demonstrates that CRM can delineate the epithelial structure and detect indicators of inflammation after treatment with N-9 and that it may be a useful imaging tool for evaluating the effects of vaginal microbicides.

Gold nanorods for intravital vascular imaging of preneoplastic oral mucosa

We explore the feasibility of using gold nanorods with efficient two-photon luminescence properties as contrast agents for intravital imaging of neoplasia. This investigation spanned ex vivo characterization in cells/tissue to in vivo implementation in an oral carcinogenesis model. GNRs were >40 times brighter than surrounding tissue. Intravital imaging revealed 3D microvasculature, and in dysplasia, abnormal vessels (dense and tortuous) compared to normal. GNRs were diffusely distributed in lesions after 24 hours. No known previous study has revealed abnormal vessel structure in dysplasia by imaging. Results suggest GNRs can function as high-contrast agents for in vivo visualization of carcinogenesis features.

Multiphoton autofluorescence microscopy and second harmonic generation microscopy of oral epithelial neoplasms

The objective of this study was to evaluate the nonlinear optical microscopy techniques of multiphoton autofluorescence microscopy (MPAM) and second harmonic generation microscopy (SHGM) for noninvasive imaging of oral epithelial carcinogenesis. In vivo imaging was performed in a hamster model for oral carcinogenesis to characterize optical and morphometric alterations during neoplastic transformation. Data is presented showing alterations in morphometry and collagen density during the precancerous phase of neoplastic transformation.

Temporal characterization of lymphatic metastasis in an orthotopic mouse model of oral cancer

The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models.

In vivo multiphoton and second harmonic generation microscopy of epithelial carcinogenesis

Multiphoton microscopy and second harmonic generation microscopy were used to image epithelial changes in a hamster model for oral malignant transformation. In vivo imaging was performed to characterize morphometric alterations in normal and precancerous regions. Morphometric measurements such as cell nucleus area and epithelial thicknesses obtained from MPM-SHGM were in excellent agreement with histology obtained after in vivo imaging. MPM-SHGM was highly sensitive to spectroscopic and architectural alterations throughout carcinogenesis, showing statistically significant changes in morphology. MPM revealed hyperkeratosis, nuclear enlargement/crowding in dysplasia, and immune cell infiltration. SHGM revealed alterations in submucosal architecture, with a decrease in SHG density evident during early stages of precancer. By combining MPM with SHGM, the basement membrane could be identified in normal, hyperplasia, and dysplasia samples and in some cases of early invasion. The combined technique of MPM-SHGM has the potential to serve as an adjunct to biopsy for assessing precancerous changes and will be investigated further for that purpose. Additionally, the method can provide spatiotemporal assessment of early neoplastic changes in order to elucidate the stages of transformation in vivo and could be used to assess therapeutic efficacy of agents being tested for the treatment of epithelial precancers/cancer.