Coxiella burnetii Induces Apoptosis during Early Stage Infection via a Caspase-Independent Pathway in Human Monocytic THP-1 Cells

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection

Classification of Human Retinal Microaneurysms Using Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography

Purpose. Microaneurysms (MAs) are considered a hallmark of retinal vascular disease, yet what little is known about them is mostly based upon histology, not clinical observation. Here, we use the recently developed adaptive optics scanning light ophthalmoscope (AOSLO) fluorescein angiography (FA) to image human MAs in vivo and to expand on previously described MA morphologic classification schemes. Methods. Patients with vascular retinopathies (diabetic, hypertensive, and branch and central retinal vein occlusion) were imaged with reflectance AOSLO and AOSLO FA. Ninety-three MAs, from 14 eyes, were imaged and classified according to appearance into six morphologic groups: focal bulge, saccular, fusiform, mixed, pedunculated, and irregular. The MA perimeter, area, and feret maximum and minimum were correlated to morphology and retinal pathology. Select MAs were imaged longitudinally in two eyes. Results. Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging revealed microscopic features of MAs not appreciated on conventional images. Saccular MAs were most prevalent (47%). No association was found between the type of retinal pathology and MA morphology (P = 0.44). Pedunculated and irregular MAs were among the largest MAs with average areas of 4188 and 4116 μm2, respectively. Focal hypofluorescent regions were noted in 30% of MAs and were more likely to be associated with larger MAs (3086 vs. 1448 μm2, P = 0.0001). Conclusions. Retinal MAs can be classified in vivo into six different morphologic types, according to the geometry of their two-dimensional (2D) en face view. Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging of MAs offers the possibility of studying microvascular change on a histologic scale, which may help our understanding of disease progression and treatment response

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals

Twist expression promotes migration and invasion in hepatocellular carcinoma

Background: Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC). Methods: We examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion. Results: We detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively. Conclusion: Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition

The Lost South Gobi Microcontinent: Protolith Studies of Metamorphic Tectonites and Implications for the Evolution of Continental Crust in Southeastern Mongolia

The Central Asian Orogenic Belt, or Altaids, is an amalgamation of volcanic arcs and microcontinent blocks that records a complex late Precambrian–Mesozoic accretionary history. Although microcontinents cored by Precambrian basement are proposed to play an integral role in the accretion process, a lack of isotopic data hampers volume estimates of newly produced arc-derived versus old-cratonic crust in southeastern Mongolia. This study investigates metamorphic tectonites in southern Mongolia that have been mapped as Precambrian in age, largely on the basis of their high metamorphic grade and high strain. Here we present results from microstructural analyses and U-Pb zircon geochronology on samples from Tavan Har (44.05° N, 109.55° E) and the Yagan-Onch Hayrhan metamorphic core complex (41.89° N, 104.24° E). Our results show no compelling evidence for Precambrian basement in southeastern Mongolia. Rather, the protoliths to all tectonites examined are Paleozoic–Mesozoic age rocks, formed during Devonian–Carboniferous arc magmatism and subsequent Permian–Triassic orogenesis during collision of the South Mongolia arc with the northern margin of China. These results yield important insights into the Paleozoic accretionary history of southern Mongolia, including the genesis of metamorphic and igneous basement during the Paleozoic, as well as implications for subsequent intracontinental reactivation

The caspase inhibitor ZVAD-fmk failed to inhibit NMII induced cell death.

<p>Panel 4A, FACS analysis of Annexin-V staining in ZVAD-fmk treated NMII infected THP-1 cells. Control cells were treated with staurosporine (1 µm) or staurosporine with ZVAD-fmk (50 µm) for 4 h. NMII infected THP-1 cells were treated with ZVAD-fmk (50 µm) and refreshed daily up to 48 h post infection. Fluorescence was detected using a fluorescence-activated cell sorter to analyze necrotic (PI+), non-apoptotic (negative for both dyes), early apoptotic (Annexin+/PI−), and late apoptotic cells (Annexin+/PI+). Panel 4B, percentage of apoptotic cells in Z-VAD-fmk treated NMII infected THP-1 cells. Data shown are the Mean±SE from at least three independent experiments. ^*denotes significant differences (*<i>p</i><0.05). Panel 4C, Western blot of Caspase-3 and PARP activities in ZVAD-fmk treated NMII infected cells. Lane 1, normal THP-1 cells; lane 2, staurosporine treated THP-1 cells; lane 3, staurosporine with ZVAD-fmk treated THP-1 cells; lane 4, NMII infected THP-1 cells; lane 5, NMII infected THP-1 cells treated with ZVAD-fmk.</p

FACS Analysis of Annexin-V staining of NMII infected THP-1 cells.

<p>Approximately 1×10⁶ NMII infected or uninfected cells were double stained with Annexin-V-FITC and PI. Panel 3A, representative FACS scatter plots of THP-1 cells. Fluorescence was detected using a fluorescence-activated cell sorter to analyze necrotic (PI+), non-apoptotic (negative for both dyes), early apoptotic (Annexin+/PI−), and late apoptotic cells (Annexin+/PI+). Panel 3B, percentages of apoptotic cells in NMII infected THP-1 cells. Data shown represents the Mean±SE from at least three independent experiments. ^*denotes significant differences (***<i>p</i><0.001) between infected and uninfected cells at each time point post infection. Panel 3C, double staining of intracellular <i>C. burnetii</i> and apoptotic cell DNA. Intracellular <i>C. burnetii</i> was stained by IFA with rabbit anti-<i>Coxiella</i> polyclonal antibodies and apoptotic host nuclei were stained with TUNEL staining. Upper panel: Cells stained with anti-<i>Coxiella</i> antibodies; Middle panel: TUNEL stating of apoptotic host nuclei; Lower panel: Merge. From left to right 1) Normal cell control; 2) Staurosporine (1 µm) treated apoptotic control cell; 3) NMII infected cells at 24 h post infection; 4) NMII infected cells at 48 h post infection; 5) NMII infected cells at 72 h post infection.</p