Photon correlations in the collective emission of hybrid gold-(CdSe/CdS/CdZnS) nanocrystal supraparticles.

We investigate the photon statistics of the light emitted by single self-assembled hybrid gold-CdSe/CdS/CdZnS colloidal nanocrystal supraparticles through the detailed analysis of the intensity autocorrelation functiong(2)(τ). We first reveal that, despite the large number of nanocrystals involved in the supraparticle emission, antibunching can be observed. We then present a model based on non-coherent Förster energy transfer and Auger recombination that well captures photon antibunching. Finally, we demonstrate that some supraparticles exhibit a bunching effect at short time scales corresponding to coherent collective emission.

Fluorescence decay enhancement and FRET inhibition in self-assembled hybrid gold CdSe/CdS/CdZnS colloidal nanocrystal supraparticles.

We report on the synthesis of hybrid light emitting particles with a diameter ranging between 100 and 500 nm, consisting in a compact semiconductor CdSe/CdS/CdZnS nanocrystal aggregate encapsulated by a controlled nanometric size silica and gold layers. We first characterize the Purcell decay rate enhancement corresponding to the addition of the gold nanoshell as a function of the particle size and find a good agreement with the predictions of numerical simulations. Then, we show that the contribution corresponding to Förster resonance energy transfer is inhibited.

Unraveling the time cross correlations of an emitter switching between two states with the same fluorescence intensity.

The autocorrelation function of the fluorescence intensity of a nanoemitter is measured with the standard Hanbury-Brown and Twiss setup. Time-tagging of the photodetection events during all the experiment has opened new possibilities in terms of post-selection techniques that enable to go beyond the blinking and antibunching characterization. Here, we first present a new method developed to investigate in detail the antibunching of a fluorophore switching between two emitting states. Even if they exhibit the same fluorescence intensity, their respective amount of antibunching can be measured using the gap between their respective decay rates. The method is then applied to a nanoemitter consisting in a colloidal quantum dot coupled to a plasmonic resonator. The relative quantum efficiency of the charged and neutral biexcitons are determined.

Enhancing fluorescence in vivo imaging using inorganic nanoprobes.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities.

Electron cotunneling transport in gold nanocrystal arrays.

We describe current-voltage (I-V) characteristics of alkyl-ligated gold nanocrystals ~5 nm arrays in a long screening length limit. Arrays with different alkyl ligand lengths have been prepared to tune the electronic tunnel coupling between the nanocrystals. For long ligands, electronic diffusion occurs through sequential tunneling and follows activated laws, as a function of temperature σ∝e(-T(0)/T) and as a function of electric field I∝e(-E(0)/E). For better conducting arrays, i.e., with small ligands, the transport properties cross over to the cotunneling regime and follow Efros-Shklovskii laws as a function of temperature σ∝e(-(T(ES)/T)(1/2) and as a function of electric field I∝e(-(E)(ES)/E)(1/2). The data show that electronic transport in nanocrystal arrays can be tuned from the sequential tunneling to the cotunneling regime by increasing the tunnel barrier transparency.

(31)P Solid-State NMR study of the chemical setting process of a dual-paste injectable brushite cements.

The composition and evolution of a brushite-type calcium phosphate cement was investigated by Solid-State NMR and X-ray during the setting process. The cement is obtained by mixing beta-tricalcium phosphate [Ca(3)(PO(4))(2), beta-TCP] and monocalcium phosphate monohydrate [Ca(H(2)PO(4))(2).H(2)O, MCPM] in presence of water, with formation of dicalcium phosphate dihydrate or brushite [CaHPO(2).2H(2)O, DCPD]. Analysis of the initial beta-TCP paste has shown the presence of beta-calcium pyrophosphate [Ca(2)P(2)O(7), beta-CPy] and that of the initial MCPM a mixture of MCPM and dicalcium phosphate [CaHPO(4), DCP]. Follow-up of the chemical composition by (31)P Solid-State NMR enables to show that the chemical setting process appeared to reach an end after 20 min. The constant composition observed at the end of the process was similarly determined.

Microstructure evolution of hydrated cement pastes.

We propose an original method based on both proton nuclear magnetic relaxation dispersion and high-resolution NMR spectra to investigate the microstructure of synthesized Ca3SiO5-hydrated cement paste. This method allows a clear assessment of the local proton chemical sites as well as the determination of dynamical information of moving proton species in pores. We show also how the microstructure evolves during and after completion of hydration in a range of length scales between 2 and 500 nm. In particular, we show how the pore size distribution of the cement paste reaches progressively a power-law characteristic of a surface-fractal distribution with a dimension Df = 2.6, which takes into account the hierarchical order in the material. Last, we study how this pore size distribution is modified during setting by varying either the water-to-cement ratio or addition of ultrafine particles. This shows that our method could be relevant to relate the mechanical properties to the microstructure of the material. This proposed NMR method is general enough for the characterization of microstructure of any porous media with reactive surface involving water confinement.

Micropore size analysis by NMR in hydrated cement.

The understanding of the microstructure of cement remains incomplete. Especially, the progressive setting of the material is still unclear. Micropore size distribution (microstructure) has been investigated by both standard proton nuclear magnetic relaxation (1H-NMR) and field-cycling relaxation in C3S hydrated paste. The non-exponential decay was interpreted as a distribution of discrete relaxation rates. The attribution of T1 is supported by both a spectral and a dispersion curve analyses. These experiments allow us to follow the structuration of the material during setting.

Micropore size analysis in hydrated cement paste by NMR.

We present a time evolution of 1H spin-lattice relaxation rates in the laboratory (1/T(1)) and in the rotating frame (1/T(1rho)) of a synthetic cement paste. The typical results found for both rates allows us to follow the main hydration stages of the cement paste and the refinement of its microporosity. In particular the texturation of the porosity and the structuration of the surface of the material is evidenced.

[Determination, by in situ hybridization on interphasic nucleus, a cytogenetic DNA index; application to breast cancer; comparison of this DNA index to DNA indexes determined by imaging and flow cytometry]. / Détermination, par hybridation in situ sur noyau interphasique, d'un index d'ADN cytogénétique; application au cancer du sein; comparaison de cet index d'ADN aux index d'ADN determinés par cytométrie en image et en flux.

In oncology, flow cytometry (FCM) and image cytometry (ICM) are commonly used to detect DNA aneuploid cell populations in solid tumors. Agreement between these two approaches is good. The use of both techniques in association minimizes the rate of FCM and ICM false negatives and gives better DNA pattern characterization, particularly for detection of any tumoral component in the FCM DNA diploid peak. Nevertheless, discrepancies exist between the FCM and the ICM DNA index values: the ICM DNA index is often greater than the FCM DNA index. The aim of the present study was to establish a cytogenetic DNA index by determining the chromosomal ploidy using a molecular cytogenetic approach and to compare it to the FCM and ICM DNA indexes. We present here the fluorescence in situ hybridization (FISH) technique we have adapted to the study of breast cancer in order to count the number of copies of the 22 + X human chromosomes in interphasic nuclei. This was achieved using a panel of 21 indirect FITC labeled probes which recognize specific chromosomic DNA sequences. Preliminary results obtained from DNA diploid and DNA aneuploid tumors are discussed.

Immunocytochemical assessment of sigma-1 receptor and human sterol isomerase in breast cancer and their relationship with a series of prognostic factors.

The purpose of this study was to immunocytochemically investigate two new markers, the sigma-1 receptor and the human sterol isomerase (hSI), in comparison with a series of clinicopathological and immunocytochemical prognostic factors in a trial including 95 patients with operable primary breast cancers. Our results showed no statistically significant relationship between these two markers and the age of the patients, their menopausal status, the tumour size and its histological grade, the nodal status and the expression of the Ki-67 proliferative marker. However, we evidenced a close correlation between the sigma-1 receptor expression and the hormonal receptor positivity (P = 0.008), essentially due to a link with the progesterone receptor status (P = 0.01). By contrast there was an inverse relationship between hSI expression and the oestrogen receptor and/or progesterone receptor positivity (P = 0.098). A significant relationship was shown between both the sigma-1 receptor, hSI expressions and Bcl2 expression, with P= 0.017 and 0.035 respectively. We also assessed whether the expression of the sigma-1 receptor or hSI might be linked with disease-free survival (DFS) and found that the presence of hSI and the absence of sigma-1 receptor expression were associated with a poorer disease-free survival (P= 0.007). Altogether these results suggest that in primary breast carcinomas in association with the evaluation of the steroid receptor status, the sigma-1 receptor and hSI may be interesting new markers useful to identify those patients who might be able to benefit from an adjuvant therapy.

Prostaglandin H synthase 1 and 2 immunoreactivities in the bronchial mucosa of asthmatics.

Prostaglandin H synthases or cyclooxygenases 1 (PGHS-1) and 2 (PGHS-2) catalyze the conversion of arachidonic acid to prostaglandin endoperoxides, leading to the formation of prostaglandin and thromboxane mediators of inflammation. The expression of these enzymes in the respiratory epithelium has not been determined, although they may be relevant to the pathophysiology of inflammatory disorders such as asthma and chronic bronchitis (CB). We studied PGHS-1 and PGHS-2 immunoreactivity in bronchial biopsies obtained from 22 patients with chronic stable asthma, seven patients with CB, and 12 normal subjects. Both types of PGHS were mainly expressed in the epithelium (basal and ciliated cells), and PGHS-1 and PGHS-2 were found in 21 of 41 and 34 of 41 biopsies, respectively. We did not find any differences in PGHS expression between the patient populations. There were no correlations between any of the clinical parameters studied or the pathologic patterns and the presence and characteristics of the PGHS immunoreactivities. Thus, both PGHS enzymes are expressed in normal human respiratory epithelium and are not quantitatively upregulated in the main bronchi in stable asthma and CB.

Expression of the interleukin 6 receptor in primary renal cell carcinoma.

AIMS: Interleukin 6 (IL-6) is expressed in the majority of renal cell carcinomas and has an important role in the proliferation of some renal cell carcinoma cell lines. This action is mediated by two membrane proteins, gp80 (the IL-6 receptor; IL-6R), which binds IL-6, and gp130, which transduces the signal. The soluble form of gp80 (sIL-6R) is able to activate gp130 when complexed to the IL-6 molecule. These considerations prompted an investigation of IL-6R expression in this malignancy. IL-6, C reactive protein (CRP), and sIL-6R were also measured in serum and correlated to clinical and pathological features. METHODS: Immunostaining was performed on cryostat sections from renal cell carcinoma tumours with M91, an anti-IL-6R monoclonal antibody, using the alkaline phosphatase antialkaline phosphatase technique. The proliferation index was measured using the KI-67 monoclonal antibody. CRP, IL-6, and sIL-6R were measured in serum before nephrectomy, using an immunoenzymatic or immunoradiometric assay. RESULTS: There were significant differences in survival in patients with tumours larger than 8 cm, metastasis at diagnosis, high nuclear grade tumours, detectable serum concentrations of IL-6 (correlated to CRP serum concentration), more than 4% proliferating cells, and the presence of the IL-6R in situ. Furthermore, the serum IL-6 concentration correlated with tumour size and stage. The mean serum sIL-6R concentration was not significantly different from that observed in 40 normal subjects. Tumour IL-6R expression was present in 10 samples. There was a significant association between the presence of the IL-6 receptor in tumours and tumour stage, nuclear grade, proliferation index, and serum IL-6. CONCLUSIONS: This study revealed the importance of IL-6/CRP and IL-6R expression in situ as potential new prognostic factors and opens the way to new therapeutic strategies in renal cell carcinoma.

Assessment of inflammation in noninfectious chronic maxillary sinusitis.

BACKGROUND AND AIM: Pathologic examination of the sinus mucosa and titration of inflammatory mediators in the sinus fluid were carried out to characterize inflammation in chronic sinusitis and determine whether patients with chronic allergic rhinitis (CAR) and sinusitis differ from patients with chronic nonallergic rhinitis (CNAR) and sinusitis. METHODS: Nine control subjects (patients requiring ear, nose, and throat surgery not related to sinusitis), 12 patients with CAR and sinusitis, and 13 patients with CNAR and sinusitis were investigated. Eosinophil cationic protein, tryptase, myeloperoxidase, histamine, and prostaglandin D2 were measured in the sinus lavage fluids, and cells were enumerated. The cellular infiltrate was studied by immunohistochemistry with monoclonal antibodies against eosinophil cationic protein (eosinophils), tryptase (mast cells), neutrophil elastase (neutrophils), CD3 (lymphocytes), CD68 (macrophages), and proliferating cell nuclear antigens. RESULTS: Neutrophils were not increased in sinusitis. In comparison with control subjects, patients with CAR and CNAR with sinusitis showed significant increases in eosinophils and macrophages in biopsy specimens and in eosinophil cationic protein in sinus lavage fluids. In comparison with patients with CNAR, patients with CAR had an increased number of intraepithelial mast cells and lymphocytes. CONCLUSIONS: These findings suggest that patients with CNAR and sinusitis can be distinguished from patients with CAR and sinusitis, which resembles nonallergic rhinitis with eosinophilia syndrome.

Cell proliferation in the bronchial mucosa of asthmatics and chronic bronchitics.

In chronic inflammatory diseases, cells are recruited but may also derive from local proliferation. In normal bronchial epithelium, under 5% of cells are in cycle but in asthma and chronic bronchitis, proliferation may occur. Cycling cells can be identified by immunohistochemistry using PC10 monoclonal antibody (Proliferating Cell Nuclear Antigen, PCNA). We enumerated PCNA-positive cells (labeling index = LI) in bronchial biopsies of 11 healthy non-smokers (HNS), seven healthy smokers (HS), 30 non-smoking asthmatics (NSA), six smoking asthmatics (SA) and 18 chronic bronchitics (CB). Twenty non-small cell lung cancer patients were used as positive control subjects. Ciliated and secretory cells were PCNA-negative. Basal cells were PCNA-positive in one of the 11 HNS (LI = 0.18 +/- 0.60), none of the seven HS, two of the 30 NSA (LI = 0.05 +/- 0.20), two of the six SA (LI = 2.4 +/- 4.3) and 11 of the 18 CB (LI = 12 +/- 20). In smokers, PCNA positivity correlated with tobacco consumption (Rho = 0.62, p < 0.0008) and in patients with chronic bronchitis, with the degree of metaplasia (tau = 0.815, p < 0.0001). The submucosa of most subjects showed no PCNA immunoreactivity. These findings suggest that the bronchial mucosa of nonsmokers is not hyperproliferative, even in asthmatics. Tobacco smoking increases PCNA immunoreactivity, possibly leading to the metaplasia of chronic bronchitis.

Quantification of ERBB2 protein expression in breast cancer: three levels of expression defined by their clinico-pathological correlations.

Activation of the ERBB2 oncogene seems to be an early event in breast cancer progression and prevalent in in situ carcinomas. However, its prognosis value, albeit recognized for node-positive patients, remains controversial for those without apparent nodal involvement. One possible reason for this problem is likely to be the difficulty of defining threshold levels for ERBB2 protein overexpression. ERBB2 protein expression was therefore analyzed in primary invasive breast tumors. Quantification of the gene product by a commercial ELISA test was compared to results obtained by immunohistochemistry and western blotting, as well as to gene amplification status determined by Southern blotting. Correlations between results obtained by the different techniques were highly significant (P value < 10(-6)). Nevertheless, ELISA permitted us to determine three levels of protein expression corresponding to distinct tumor subsets. 1) Tumors with p185/ERBB2 expression levels exceeding 10 U/microgram exhibited in most cases amplification of the gene (83% of cases), DNA aneuploidy (81%) and absence of estrogen receptor (ER) (44%). Such high levels of protein expression were exclusively observed in invasive ductal carcinomas and were prevalent in those showing a significant in situ component. 2) "Intermediate" levels of expression (3-10 U/micrograms) were rarely observed in tumors exhibiting gene amplification (9%), but were preferentially found in cancers of more favorable prognosis (only 49% were aneuploid and 9% estrogen receptor negative). 3) Levels of p185/ERBB2 below 3 U/micrograms were detected in benign mastopathies and, thus, carcinomas presenting such levels were scored ERBB2 negative. Interestingly, invasive lobular carcinomas were rarely ERBB2 positive, and if so, only at intermediate levels. Moreover, our data show a complex interrelationship between p185/ERBB2 expression and ER levels. Indeed, tumors with more than 10 U/micrograms of p185 were prevalently ER, whereas those with p185 ranging from 3 to 10 U presented elevated levels of ER.

Cytofluorometric detection of human endothelial cells in whole blood using S-Endo 1 monoclonal antibody.

It has long been debated whether endothelial cells are present at very low frequency in peripheral blood. Elevated concentrations of such circulating cells may represent a good marker of vascular injury. We have therefore designed an immunocytometric assay for the detection of rare endothelial cells in whole blood. This assay is based on a new monoclonal antibody (MAb) S-Endo 1, made against human umbilical vein endothelial cells (HUVEC) and specific for endothelial cells of various origins without detectable reactivity with blood cells. First, the sensitivity of the assay was established by using normal blood samples with admixed HUVEC as an in vitro model. A good correlation was obtained between added and counted endothelial cells; the recovery was greater than 90% and the minimum detectable concentration of HUVEC was about 0.2 cells/microliters whole blood. Using this rapid counting technique, no detectable levels of endothelial cells were found in the blood of normal individuals (CE less than or equal to 0.1 cells/microliters) while elevated concentrations (up to 8 cells/microliters) were detected in a human model of vascular injury corresponding to a traumatic venepuncture. Thus, this new whole blood immunocytometric assay using S-Endo 1 MAb may be useful in determining the levels of circulating endothelial cells in vascular disorders.