Multimodal optical analysis of meningioma and comparison with histopathology

Meningioma is the most frequent primary central nervous system tumor. The risk of recurrence and the prognosis are correlated with the extent of the resection that ideally encompasses the infiltrated dura mater and, if required, the infiltrated bone. No device can deliver real‐time intraoperative histopathological information on the tumor environment to help the neurosurgeon to achieve a gross total removal. This study assessed the abilities of nonlinear microscopy to provide relevant and real‐time data to help resection of meningiomas. Nine human meningioma samples (four World Health Organization Grade I, five Grade II) were analyzed using different optical modalities: spectral analysis and imaging, lifetime measurements, fluorescence lifetime imaging microscopy, fluorescence emitted under one‐ and two‐photon excitation and the second‐harmonic generation signal imaging using a multimodal setup. Nonlinear microscopy produced images close to histopathology as a gold standard. The second‐harmonic generation signal delineated the collagen background and two‐photon fluorescence underlined cell cytoplasm. The matching between fluorescence images and Hematoxylin and Eosin staining was possible in all cases. Grade I meningioma emitted less autofluorescence than Grade II meningioma and Grade II meningioma exhibited a distinct lifetime value. Autofluorescence was correlated with the proliferation rates and seemed to explain the observed differences between Grade I and II meningiomas. This preliminary multimodal study focused on human meningioma samples confirms the potential of tissue autofluorescence analysis and nonlinear microscopy in helping intraoperatively neurosurgeons to reach the actual boundaries of the tumor infiltration.

Correspondence between H&E staining (top pictures) and the two‐photon fluorescence imaging (bottom pictures)     

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

Background  

Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far.     

Insulin Receptor Dysfunction Impairs Cellular Clearance of Neurotoxic Oligomeric A��

Accumulation of amyloid β (Aβ) oligomers in the brain is toxic to synapses and may play an important role in memory loss in Alzheimer disease. However, how these toxins are built up in the brain is not understood. In this study we investigate whether impairments of insulin and insulin-like growth factor-1 (IGF-1) receptors play a role in aggregation of Aβ. Using primary neuronal culture and immortal cell line models, we show that expression of normal insulin or IGF-1 receptors confers cells with abilities to reduce exogenously applied Aβ oligomers (also known as ADDLs) to monomers. In contrast, transfection of malfunctioning human insulin receptor mutants, identified originally from patient with insulin resistance syndrome, or inhibition of insulin and IGF-1 receptors via pharmacological reagents increases ADDL levels by exacerbating their aggregation. In healthy cells, activation of insulin and IGF-1 receptor reduces the extracellular ADDLs applied to cells via seemingly the insulin-degrading enzyme activity. Although insulin triggers ADDL internalization, IGF-1 appears to keep ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDL-induced surface insulin receptor loss. Our results suggest that dysfunctions of brain insulin and IGF-1 receptors contribute to Aβ aggregation and subsequent synaptic loss.Abnormal protein misfolding and aggregation are common features in neurodegenerative diseases such as Alzheimer (AD),² Parkinson, Huntington, and prion diseases (1–3). In the AD brain, intracellular accumulation of hyperphosphorylated Tau aggregates and extracellular amyloid deposits comprise the two major pathological hallmarks of the disease (1, 4). Aβ aggregation has been shown to initiate from Aβ1–42, a peptide normally cleaved from the amyloid precursor protein (APP) via activities of α- and γ-secretases (5, 6). A large body of evidence in the past decade has indicated that accumulated soluble oligomers of Aβ1–42, likely the earliest or intermediate forms of Aβ deposition, are potently toxic to neurons. The toxic effects of Aβ oligomers include synaptic structural deterioration (7, 8) and functional deficits such as inhibition of synaptic transmission (9) and synaptic plasticity (10–13), as well as memory loss (11, 14, 15). Accumulation of high levels of these oligomers may also trigger inflammatory processes and oxidative stress in the brain probably due to activation of astrocytes and microglia (16, 17). Thus, to understand how a physiologically produced peptide becomes a misfolded toxin has been one of the key issues in uncovering the molecular pathogenesis of the disease.Aβ accumulation and aggregation could derive from overproduction or impaired clearance. Mutations of APP or presenilins 1 and 2, for example, are shown to cause overproduction of Aβ1–42 and amyloid deposits in the brain of early onset AD (18, 19). Because early onset AD accounts for less than 5% of entire AD population, APP and presenilin mutations cannot represent a universal mechanism for accumulation/aggregation of Aβ in the majority of AD cases. With respect to clearance, Aβ is normally removed by both global and local mechanisms, with the former requiring vascular transport across the blood-brain barrier (20, 21) and the latter via local enzymatic digestions by several metalloproteases, including neprilysin, insulin-degrading enzyme (IDE), and endothelin converting enzymes 1 and 2 (22–24).The fact that insulin is a common substrate for most of the identified Aβ-degrading enzymes has drawn attention of investigators to roles of insulin signaling in Aβ clearance. Increases in insulin levels frequently seen in insulin resistance may compete for these enzymes and thus contribute to Aβ accumulation. Indeed, insulin signaling has been shown to regulate expression of metalloproteases such as IDE (25, 26), and influence aspects of Aβ metabolism and catabolism (27). In the endothelium of the brain-blood barrier and glial cells, insulin signaling is reported to regulate protein-protein interactions in an uptake cascade involving low density lipoprotein receptor-related protein and its ligands ApoE and α2-macroglobulin, a system known to bind and clear Aβ via endocytosis and/or vascular transport (28, 29). Similarly, circulating IGF-1 has been reported to play a role in Aβ clearance probably via facilitating brain-blood barrier transportation (30, 31).In the brain, insulin signaling plays a role in learning and memory (32–34), potentially linking insulin resistance to AD dementia. Recently we and others have shown that Aβ oligomers interact with neuronal insulin receptors to cause impairments of the receptor expression and function (35–37). These impairments mimic the Aβ oligomer-induced synaptic long term potentiation inhibition and can be overcome by insulin treatment (35, 38). Consistently, impairments of both IR and IGF-1R have been reported in the AD brain (39–41).Based on these results, we ask whether impairment of insulin and IGF-1 signaling contribute to Aβ oligomer build-up in brain cells. To address this question, we set out to test roles of IR and IGF-1R in cellular clearance and transport of Aβ oligomers (ADDLs) applied to primary neuronal cultures and cell lines overexpressing IR and IGF-1R. Our results show that insulin and IGF-1 receptors function to reduce Aβ oligomers to monomers, and prevent Aβ oligomer-induced synaptic toxicity both at the level of synapse composition and structure. By contrast, receptor impairments resulting from “kinase-dead” insulin receptor mutations, a tyrosine kinase inhibitor of the insulin and IGF-1 receptor, or an inhibitory IGF-1 receptor antibody increase ADDL aggregation in the extracellular medium. Our results provide cellular evidence linking insulin and IGF-1 signaling to amyloidogenesis.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

Role of Prostate Apoptosis Response 4 in Translocation of GRP78 from the Endoplasmic Reticulum to the Cell Surface of Trophoblastic Cells

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB) and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4), a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.     

Early endothelial progenitor cells in bone marrow are a biomarker of cell therapy success in patients with critical limb ischemia

Background aimsEndothelial progenitor cells (EPC) have been proposed for autologous angiogenic therapy. The objectives of this study were to quantify EPC in the peripheral blood and bone marrow mononuclear cells (BM-MNC) of patients with critical limb ischemia that had received BM-MNC as a cell therapy product, and to study the putative relationship between the presence of EPC and the process of neovascularization in toe or transmetatarsal amputation specimens.MethodsEarly and late endothelial progenitor cells (CFU-EC and ECFC) were cultivated and quantified according to published methods in peripheral blood and BM-MNC from patients with critical limb ischemia (CLI; n = 11) enrolled in the OPTIPEC trial (http://clinicaltrials.gov/ct2/show/NCT00377897) to receive BM-MNC as a cell therapy product.ResultsEight out of the 11 patients had undergone amputations. Three of the patients displayed a neoangiogenic process that was associated with a higher number of CFU-EC in BM-MNC, while CD3⁺, CFU-GM and CD34⁺ in BM-MNC, and EPC in peripheral blood, did not correlate with the appearance of newly formed vessels. As expected, circulating CFU-EC and ECFC counts were significantly lower in CLI patients compared with age-matched controls.ConclusionsIn patients with critical limb ischemia, EPC in peripheral blood were decreased compared with healthy individuals. However, in BM-MNC we found that relative numbers of CFU-EC could be used as an indicator to discriminate patients with neoangiogenic processes. These results need to be confirmed in a randomized study.