Evaluation of two (125)I-radiolabeled acridine derivatives for Auger-electron radionuclide therapy of melanoma.

We previously selected two melanin-targeting radioligands [(125)I]ICF01035 and [(125)I]ICF01040 for melanoma-targeted (125)I radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [(125)I]ICF01035 induced a similar survival fraction (A50) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [(125)I]ICF01040 induced a higher A50 in B16 cell lines compared to [(125)I]ICF01035 ones. [(125)I]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [(125)I]ICF01035 and [(125)I]ICF01040 are not exclusively reliant on DNA alterations compatible with γ rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [(125)I]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy.

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy.

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 µm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

Direct contact of platelets and their released products exert different effects on human dendritic cell maturation.

BACKGROUND: Dendritic cells (DCs) are antigen presenting cells capable of inducing innate and adaptive immune responses. According to the stimulus and their maturation state, DCs induce immunogenic or tolerogenic responses. Platelets (PLTs), which are involved in haemostasis and inflammation, can also interact with DCs. In this study, we examined the effect of PLTs on DC maturation in vitro. Human monocyte-derived DCs were co-cultured for 2 days with homologous PLTs either in the same well or in 0.4 mum-pore size filter-separated compartments. RESULTS: Confocal microscopy showed the attachment of PLTs to DC membranes. The DC receptor involved in this interactions was found to be CD162. In addition, we observed that DCs co-cultured with PLTs in filter-separated compartments acquired a mature phenotype (high CD80, CD86, and intermediate CD83 expression; IL-12(p70) production; efficient stimulation of autologous CD4+ T cell proliferation), while DCs co-cultured with PLTs in the same compartment did not undergo phenotypic maturation, did not secrete IL-12(p70) or IL-1beta, but instead induced moderate Th2-polarized T cell proliferation. CONCLUSION: These data indicate that (i) PLTs secrete a soluble DC-activating factor that was demonstrated not to be soluble CD40-Ligand (CD154; as could have been expected from in vivo and previous in vitro work) but to be nucleotide, and (ii) that cell-to-cell contact did not induce DC maturation, possibly because nucleotide release by PLTs was prevented by direct contact with DCs. This work demonstrates that PLTs are active elements of the immune system that might play a role in balancing the ability of DCs to polarize T cell responses, therefore making them critical factors in transfusion processes.

Visualization of X4- and R5-Tropic HIV-1 Viruses Expressing Fluorescent Proteins in Human Endometrial Cells: Application to Tropism Study.

Worldwide most HIV infections occur through heterosexual transmission, involving complex interactions of cell-free and cell-associated particles with cells of the female genital tract mucosa. The ability of HIV-1 to "infect" epithelial cells remains poorly understood. To address this question, replicative-competent chimeric constructs expressing fluorescent proteins and harboring the envelope of X4- or R5-tropic HIV-1 strains were used to "infect" endometrial HEC1-A cells. The virus-cell interactions were visualized using confocal microscopy (CM) at various times post infection. Combined with quantification of viral RNA and total HIV DNA in infected cells, the CM pictures suggest that epithelial cells do not support a complete viral replication cycle: X4-tropic viruses are imported into the nucleus in a non-productive way, whereas R5-tropic viruses transit through the cytoplasm without replication and are preferentially transmitted to susceptible activated peripheral blood mononuclear cells. Within the limit of experiments conducted in vitro on a continued cell line, these results indicate that the epithelial mucosa may participate to the selection of HIV-1 strains at the mucosal level.

Different glycosylation profile of serum IgA1 in IgA nephropathy according to the glomerular basement membrane thickness: normal versus thin.

BACKGROUND: Abnormal glycosylation of immunoglobulin A1 (IgA1) has been implicated in the pathophysiological characteristics of IgA nephropathy, leading to failure of normal clearance mechanisms and mesangial deposition of serum IgA1. Furthermore, systematic measurement of glomerular basement membrane (GBM) thickness by electron microscopy evidenced two different subgroups: IgA nephropathy with normal GBM (N-GBM) and thin GBM (T-GBM). This finding prompted us to study comparatively the profile of N- and O-glycosylation of IgA1 in the two subgroups. METHODS: Using lectin-binding properties, sialylation and galactosylation of serum IgA1, isolated on jacalin-conjugated agarose, were investigated in male and female patients with IgA nephropathy with T-GBM (n = 22) and N-GBM (n = 22) compared with matched (age and sex) healthy controls (n = 22). Sambacus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA) were designed to examine the detection of Neu5Acalpha2,6- and Neu5Acalpha2,3-linked galactose, respectively. Helix aspersa agglutinin (HAA) was used to examine the expression of terminal N-acetylgalactosamine of the O-linked glycans in the hinge region of IgA1. RESULTS: The following galactosylation abnormalities were confirmed in the common subgroup with N-GBM: a trend to an alpha2,6 oversialylation (SNA binding) of native IgA1 associated with a defect in its terminal galactose (HAA binding); these two findings were predominant in male patients (P < 0.05 and 0.01 for SNA and HAA, respectively). No change in MAA was observed. Conversely, no significant anomaly was found in the T-GBM variant, which could indicate the absence or low magnitude of galactosylation defects (not significant) or another yet unidentified defect. CONCLUSION: The present study evidenced differences in glycosylation profiles of serum IgA1 according to GBM thickness (N-GBM versus T-GBM) in patients with IgA nephropathy. These data raised the possibility of different mechanisms for IgA1 glomerular deposition.

Role of Siglec-7 in apoptosis in human platelets.

BACKGROUND: Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis. CONCLUSIONS/SIGNIFICANCE: The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention.

Selective transmigration of monocyte-associated HIV-1 across a human cervical monolayer and its modulation by seminal plasma.

OBJECTIVE: To analyse the transmigration of immune cells infected by HIV-1 across the epithelial monolayer using the endometrial human endometrial carcinoma (HEC)-1A cell line and to study the influence of seminal plasma in this process. DESIGN: After sexual intercourse involving a male partner infected by HIV-1, a selection process has been shown to lead to a predominant transmission of the R5 phenotype despite the presence of X4 and R5 strains in semen. Transmigration of HIV-infected monocytes present in semen may represent a pertinent mechanism that could explain this tropism selection. METHODS: Epithelial monolayer crossing was studied by using HEC-1A epithelial cells cultured on permeable support and monocyte-enriched or lymphocyte-enriched populations of cells infected or not by HIV R5 or X4 strains. Transmigrating cells were quantified and analysed for their ability to transmit HIV infection to immune target cells. The effect of HIV-negative seminal plasma on cell transmigration was analysed. RESULTS: A preferential passage of the R5 strain associated with monocyte-enriched populations was observed together with the ability of this strain to transmit infection. Seminal plasma was found able to decrease the epithelial crossing of immune cells by enhancing transepithelial resistance and by increasing the adherence of immune cells to the monolayer. CONCLUSION: The preferential transmigration of HIV R5 strains associated with monocytes across the endocervical monolayer may explain the predominant transmission of the R5 strains after sexual intercourse. By its capacity to modulate the tightness of the epithelial structure, seminal plasma reinforces this selection process.

Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae).

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.

Construction and tropism characterisation of recombinant viruses exhibiting HIV-1 env gene from seminal strains.

Genetic differences between blood and mucosal-derived HIV-1 strains have been widely reported. As amplification of HIV-1 strains from mucosal samples including semen or saliva by co-culture has low sensitivity, we developed the construction of chimeric viruses expressing wild-type seminal HIV-1 envelope protein. Chimeric viruses were produced by co-transfection of a V1-V3 deleted pNL 43 vector and PCR fragments spanning the deleted region, amplified from HIV-1 RNA positive seminal plasma samples. After an initial testing of co-receptor usage by a tropism recombinant test, replication capacity and amplification of these recombinant viruses were assessed using PBMC. Four chimeric replicative strains, all using CXCR4 as coreceptor, were produced. The interaction between cell-free viral particles and reporter cell lines was assessed by confocal microscopy. These replicative chimeras exhibiting HIV-1 env from seminal strains represent useful tools for the in vitro study of the heterosexual transmission of HIV-1 and testing of microbicide activity.