Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell-wall polysaccharides of Arabidopsis petals.

A procedure for the simultaneous analysis of cell-wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long-chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell-wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.

A novel Th cell epitope of Candida albicans mediates protection from fungal infection.

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.

Diagnostic performance of &supl;⁸F-fluorodeoxyglucose positron emission tomography in giant cell arteritis: a systematic review and meta-analysis.

PURPOSE: The aim of this study was to conduct a systematic review and perform a meta-analysis on the diagnostic performances of (18)F-fluorodeoxyglucose positron emission tomography (FDG PET) for giant cell arteritis (GCA), with or without polymyalgia rheumatica (PMR). METHODS: MEDLINE, Embase and the Cochrane Library were searched for articles in English that evaluated FDG PET in GCA or PMR. All complete studies were reviewed and qualitatively analysed. Studies that fulfilled the three following criteria were included in a meta-analysis: (1) FDG PET used as a diagnostic tool for GCA and PMR; (2) American College of Rheumatology and Healey criteria used as the reference standard for the diagnosis of GCA and PMR, respectively; and (3) the use of a control group. RESULTS: We found 14 complete articles. A smooth linear or long segmental pattern of FDG uptake in the aorta and its main branches seems to be a characteristic pattern of GCA. Vessel uptake that was superior to liver uptake was considered an efficient marker for vasculitis. The meta-analysis of six selected studies (101 vasculitis and 182 controls) provided the following results: sensitivity 0.80 [95% confidence interval (CI) 0.63-0.91], specificity 0.89 (95% CI 0.78-0.94), positive predictive value 0.85 (95% CI 0.62-0.95), negative predictive value 0.88 (95% CI 0.72-0.95), positive likelihood ratio 6.73 (95% CI 3.55-12.77), negative likelihood ratio 0.25 (95% CI 0.13-0.46) and accuracy 0.84 (95% CI 0.76-0.90). CONCLUSION: We found overall valuable diagnostic performances for FDG PET against reference criteria. Standardized FDG uptake criteria are needed to optimize these diagnostic performances.

Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity.

Malignant cells are frequently recognized and destroyed by T cells, hence the development of T cell vaccines against established tumors. The challenge is to induce protective type 1 immune responses, with efficient Th1 and CTL activation, and long-term immunological memory. These goals are similar as in many infectious diseases, where successful immune protection is ideally induced with live vaccines. However, large-scale development of live vaccines is prevented by their very limited availability and vector immunogenicity. Synthetic vaccines have multiple advantages. Each of their components (antigens, adjuvants, delivery systems) contributes specifically to induction and maintenance of T cell responses. Here we summarize current experience with vaccines based on proteins and peptide antigens, and discuss approaches for the molecular characterization of clonotypic T cell responses. With carefully designed step-by-step modifications of innovative vaccine formulations, T cell vaccination can be optimized towards the goal of inducing therapeutic immune responses in humans.

Molecular imaging of matrix metalloproteinases in atherosclerotic plaques.

Ischaemic stroke and myocardial infarction often result from the sudden rupture of an atherosclerotic plaque. The subsequent arterial thrombosis occluding the vessel lumen has been widely indicated as the crucial acute event causing peripheral tissue ischaemia. A complex cross-talk between systemic and intraplaque inflammatory mediators has been shown to regulate maturation, remodeling and final rupture of an atherosclerotic plaque. Matrix metalloproteinases (MMPs) are proteolytic enzymes (released by several cell subsets within atherosclerotic plaques), which favour atherogenesis and increase plaque vulnerability. Thus, the assessment of intraplaque levels and activity of MMP might be of pivotal relevance in the evaluation of the risk of rupture. New imaging approaches, focused on the visualisation of inflammation in the vessel wall and plaque, may emerge as tools for individualised risk assessment and prevention of events. In this review, we summarize experimental findings of the currently available invasive and noninvasive imaging techniques, used to detect the presence and activity of MMPs in atherosclerotic plaques.

Increase of [(18)F]FLT Tumor Uptake In Vivo Mediated by FdUrd: Toward Improving Cell Proliferation Positron Emission Tomography.

PURPOSE: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D: -glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. METHODS: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. RESULTS: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. CONCLUSION: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Sorting live stem cells based on sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

Interstitial dendritic cell guidance by haptotactic chemokine gradients.

Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Carotid sparing in definitive irradiation of T1N0 squamous cell larnyx cancer using helical tomotherapy

Objective: To implement a carotid sparing protocol using helical Tomotherapy(HT) in T1N0 squamous-cell laryngeal carcinoma.Materials/Methods: Between July and August 2010, 7 men with stage T1N0 laryngeal carcinoma were included in this study. Age ranged from 47-74 years. Staging included endoscopic examination, CT-scan and MRI when indicated.Planned irradiation dose was 70 Gy in 35 fractions over 7 weeks. A simple treatment planning algorithm for carotidsparing was used: maximum point dose to the carotids 35 Gy, to the spinal cord 30 Gy, and 100% PTV volume to becovered with 95% of the prescribed dose. Carotid volume of interest extended to 1 cm above and below of the PTV.Doses to the carotid arteries, critical organs, and planned target volume (PTV) with our standard laryngealirradiation protocol was compared. Daily megavoltage scans were obtained before each fraction. When necessary, thePlanned Adaptive? software (TomoTherapy Inc., Madison, WI) was used to evaluate the need for a re-planning,which has never been indicated. Dose data were extracted using the VelocityAI software (Atlanta, GA), and datanormalization and dosevolume histogram (DVH) interpolation were realized using the Igor Pro software (Portland,OR).Results: A significant (p < 0.05) carotid dose sparing compared to our standard protocol with an average maximum point dose of 38.3 Gy (standard devaition [SD] 4.05 Gy), average mean dose of 18.59 Gy (SD 0.83 Gy) was achieved.In all patients, 95% of the carotid volume received less than 28.4 Gy (SD 0.98 Gy). The average maximum point doseto the spinal cord was 25.8 Gy (SD 3.24 Gy). PTV was fully covered with more than 95% of the prescribed dose forall patients with an average maximum point dose of 74.1 Gy and the absolute maximum dose in a single patient of75.2 Gy. To date, the clinical outcomes have been excellent. Three patients (42%) developed stage 1 mucositis that was conservatively managed, and all the patients presented a mild to moderate dysphonia. All adverse effectsresolved spontaneously in the month following the end of treatment. Early local control rate is 100% considering a 4-5months post treatment follow-up.Conclusions: HT allows a clinically significant decrease of carotid irradiation dose compared tostandard irradiation protocols with an acceptable spinal cord dose tradeoff. Moreover, this technique allows the PTV to be homogenously covered with a curative irradiation dose. Daily control imaging brings added security marginsespecially when working with high dose gradients. Further investigations and follow-up are underway to better evaluatethe late clinical outcomes especially the local control rate, late laryngeal and vascular toxicity, and expected potentialimpact on cerebrovascular events.

Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy.

We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

Correlation between subjective evaluation of symptoms and objective findings in early recurrent head and neck squamous cell carcinoma.

IMPORTANCE: This study addresses the value of patients' reported symptoms as markers of tumor recurrence after definitive therapy for head and neck squamous cell carcinoma. OBJECTIVE: To evaluate the correlation between patients' symptoms and objective findings in the diagnosis of local and/or regional recurrences of head and neck squamous cell carcinomas in the first 2 years of follow-up. DESIGN: Retrospective single-institution study of a prospectively collected database. SETTING: Regional hospital. PARTICIPANTS: We reviewed the clinical records of patients treated for oral cavity, oropharyngeal, laryngeal, and hypopharyngeal carcinomas between January 1, 2008, and December 31, 2009, with a minimum follow-up of 2 years. MAIN OUTCOMES AND MEASURES: Correlation between symptoms and oncologic status (recurrence vs remission) in the posttreatment period. RESULTS: Of the 101 patients included, 30 had recurrences. Pain, odynophagia, and dysphonia were independently correlated with recurrence (odds ratios, 16.07, 11.20, and 5.90, respectively; P < .001). New-onset symptoms had the best correlation with recurrences. Correlation was better between 6 to 12 and 18 to 21 months after therapy and in patients initially treated unimodally (P < .05). Primary stage and tumor site had no effect. CONCLUSIONS AND RELEVANCE: The correlation between symptoms and oncologic status is low during substantial periods within the first 2 years of follow-up. New-onset symptoms, especially pain, odynophagia, or dysphonia, better correlate with tumor recurrence, especially in patients treated unimodally.

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca(2+)-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca(2+) from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.