Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells.

Liquid deposit mimicking surface aerosolization in the airway is a promising strategy for targeting bronchopulmonary tumors with reduced doses of nanoparticle (NPs). In mimicking and studying such delivery approaches, the use of human in vitro 3D culture models can bridge the gap between 2D cell culture and small animal investigations. Here, we exposed airway epithelia to liquid-apical gadolinium-based AGuIX® NPs in order to determine their safety profile. We used a multiparametric methodology to investigate the NP's distribution over time in both healthy and tumor-bearing 3D models. AGuIX® NPs were able to target tumor cells in the absence of specific surface functionalization, without evidence of toxicity. Finally, we validated the therapeutic potential of this hybrid theranostic AGuIX® NPs upon radiation exposure in this model. In conclusion, 3D cell cultures can efficiently mimic the normal and tumor-bearing airway epitheliums, providing an ethical and accessible model for the investigation of nebulized NPs.

Quantitative Flow Cytometric Evaluation of Oxidative Stress and Mitochondrial Impairment in RAW 264.7 Macrophages after Exposure to Pristine, Acid Functionalized, or Annealed Carbon Nanotubes.

Conventional nanotoxicological assays are subjected to various interferences with nanoparticles and especially carbon nanotubes. A multiparametric flow cytometry (FCM) methodology was developed here as an alternative to quantify oxidative stress, mitochondrial impairment, and later cytotoxic and genotoxic events. The experiments were conducted on RAW264.7 macrophages, exposed for 90 min or 24 h-exposure with three types of multiwalled carbon nanotubes (MWCNTs): pristine (Nanocyl™ CNT), acid functionalized (CNTf), or annealed treatment (CNTa). An original combination of reactive oxygen species (ROS) probes allowed the simultaneous quantifications of broad-spectrum ROS, superoxide anion (O2•-), and hydroxyl radical (•OH). All MWCNTs types induced a slight increase of broad ROS levels regardless of earlier antioxidant catalase activity. CNTf strongly stimulated the O2•- production. The •OH production was downregulated for all MWCNTs due to their scavenging capacity. The latter was quantified in a cell-free system by electron paramagnetic resonance spectroscopy (EPR). Further FCM-based assessment revealed early biological damages with a mitochondrial membrane potential collapse, followed by late cytotoxicity with chromatin decondensation. The combined evaluation by FCM analysis and cell-free techniques led to a better understanding of the impacts of MWCNTs surface treatments on the oxidative stress and related biological response.

Effects of heparin and derivatives on podocytes: An in vitro functional and morphological evaluation.

Unfractionated heparin (UFH) and low molecular heparin derivatives (LMWH) display numerous biological properties in addition to their anticoagulant effects. However, due to the physicochemical heterogeneity of these drugs, a better understanding concerning their effects on human cells is clearly needed. Considering that heparins are mainly excreted by the kidney, we focused our attention on the effect of UFH and LMWH on human podocytes by functional and morphological/phenotypic in vitro analyses. We demonstrated that these products differentially modulate the permeability of podocyte monolayer to albumin. The functional perturbations observed were correlated to significant cellular morphological and cytoskeletal changes, as well as a decrease in the expression of proteins involved in podocyte adherence to the extracellular matrix or intercellular interactions. This point confirms that UFH and the different LMWHs exert specific effects on podocyte permeability and underlines the need of in vitro tests to evaluate new biological nonanticoagulant properties of LMWH.

Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP-/- Mice, Along With Faster Hematopoietic Recolonization.

The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin- (×3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi , ×2) and hematopoietic stem cells (HSC: Flt3- LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528-2537, 2017. © 2016 Wiley Periodicals, Inc.

Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.

Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes…). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification.

Absence of bone sialoprotein (BSP) alters profoundly hematopoiesis and upregulates osteopontin.

Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral and are thus in a key position to regulate the microenvironment of the bone tissue, including its hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (-/-) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in -/- bone marrow, and its composition is shifted toward more mature lymphocyte stages. BSP-null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in -/- mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the -/-. Thus, a BSP-null bone microenvironment affects the hematopoietic system both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP.

Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorption in vitro.

Bone sialoprotein (BSP) and osteopontin (OPN) belong to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members interact with bone cells and bone mineral. Previously, we showed that BSP knockout (BSP(-/-) ) mice have a higher bone mass than wild type (BSP(+/+) ) littermates, with very low bone-formation activity and reduced osteoclast surfaces and numbers. Here we report that approximately twofold fewer tartrate-resistant acid phosphatase (TRACP)-positive cells and approximately fourfold fewer osteoclasts form in BSP(-/-) compared with BSP(+/+) spleen cell cultures. BSP(-/-) preosteoclast cultures display impaired proliferation and enhanced apoptosis. Addition of RGD-containing proteins restores osteoclast number in BSP(-/-) cultures to BSP(+/+) levels. The expression of osteoclast-associated genes is markedly altered in BSP(-/-) osteoclasts, with reduced expression of cell adhesion and migration genes (αV integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K). The migration of preosteoclasts and mature osteoclasts is impaired in the absence of BSP, but resorption pit assays on dentine slices show no significant difference in pit numbers between BSP(+/+) and BSP(-/-) osteoclasts. However, resorption of mineral-coated slides by BSP(-/-) osteoclasts is markedly impaired but is fully restored by coating the mineral substrate with hrBSP and partly restored by hrOPN coating. In conclusion, lack of BSP affects both osteoclast formation and activity, which is in accordance with in vivo findings. Our results also suggest at least some functional redundancy between BSP and OPN that remains to be clarified.

Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells.

BACKGROUND: Mitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate. METHODOLOGY/PRINCIPAL FINDINGS: Mitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H2O2 level at the onset of differentiation. CONCLUSION/SIGNIFICANCE: A defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.

Cytostatic factor proteins are present in male meiotic cells and beta-nerve growth factor increases mos levels in rat late spermatocytes.

BACKGROUND: In co-cultures of pachytene spermatocytes with Sertoli cells, beta-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells. METHODOLOGY/PRINCIPAL FINDINGS: We show here, that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF, and their respective mRNAs, are present in male rat meiotic cells; this was assessed by using Western blotting, immunocytochemistry and reverse transcriptase PCR. We measured the relative cellular levels of Mos, Emi2, Cyclin E and Cdk2 in the meiotic cells by flow cytometry and found that the four proteins increased throughout the first meiotic prophase, reaching their highest levels in middle to late pachytene spermatocytes, then decreased following the meiotic divisions. In co-cultures of pachytene spermatocytes with Sertoli cells, beta-NGF increased the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes. CONCLUSION/SIGNIFICANCE: Our results suggest that CSF is not restricted to the oocyte. In addition, they reinforce the view that NGF, by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells.

Control of mitochondrial biogenesis, ROS level, and cytosolic Ca2+ concentration during the cell cycle and the onset of differentiation in L6E9 myoblasts.

Mitochondria can sense signals linked to changes in energy demand to affect nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. We have investigated the regulation of mitochondrial biogenesis and production of putative retrograde signaling agents [hydrogen peroxide (H(2)O(2)) and Ca(2+)] during the cell cycle and the onset of differentiation in L6E9 muscle cells. The biosynthesis of cardiolipin and mitochondrial proteins was mainly achieved in S phase, whereas the expression of mitochondrial biogenesis factors [peroxisome proliferator-activated receptor (PPAR)-alpha, PPAR-delta, and neuronal nitric oxide synthase 1] was regularly increased from G(1) to G(2)M phase. In agreement with the increase in mitochondrial membrane potential, mitochondria in S and G(2)M phases have a significantly higher H(2)O(2) level when compared with G(1) phase. By contrast, the onset of differentiation was characterized by a marked reduction in mitochondrial protein expression and mitochondrial H(2)O(2) level. The capacity of mitochondria to release Ca(2+) in response to a metabolic challenge was significantly decreased at the onset of differentiation. Finally, an increase in calmodulin expression in S and G(2)M phases and a transitory increase in phosphorylated nuclear factor of activated T cells (NFAT) c3 in S phase was observed. NFATc3 phosphorylation was markedly decreased at the onset of differentiation. Our data point to functional links between the control of mitochondrial biogenesis and the regulation of the level of retrograde signaling agents during the cell cycle and the onset of differentiation in L6E9 muscle cells.

Could 99mTc-glucarate be used to evaluate tumour necrosis? In vitro and in vivo studies in leukaemic tumour cell line U937.

PURPOSE: The aim of this study was to determine whether (99m)Tc-glucarate ((99m)Tc-GLA) is a powerful and discriminant tumour necrosis marker. MATERIALS AND METHODS: The induction of apoptosis and secondary necrosis (by a chemotherapeutic agent) and necrosis (by intense hyperthermia) was studied on an in vitro and in vivo leukaemic cell line model (U937). The percentage of apoptosis/necrosis in vitro was determined by flow cytometry after staining cells with annexin-V-fluorescein/propidium iodide. The uptake of (99m)Tc-GLA was studied after treatments that produce an optimal of necrosis cells or apoptotic cells. Three populations of interest: viable, apoptotic and necrotic cells were sorted by flow cytometry. The uptake and the intracellular distribution of (99m)Tc-GLA on each population have been studied. We also investigated the influence of necrosis on (99m)Tc-GLA uptake in a model of U937 xenografts in nude mice. RESULTS: The accumulation of (99m)Tc-GLA in untreated and apoptotic cells was lower than in necrotic cells. Cell sorting discriminated each cellular population and showed a 14% accumulation in necrotic cells and no more than a 3% in apoptotic cells. In apoptotic and viable cells, (99m)Tc-GLA is distributed between the cytosolic/membrane and the nucleus fractions. In necrotic cells, (99m)Tc-GLA is mainly found in the nucleus fraction. In vivo investigations showed a higher (99m)Tc-GLA uptake in necrotic tumour than in apoptotic and control ones. CONCLUSIONS: (99m)Tc-GLA may be a useful agent to specifically evaluate tumour necrosis and may be helpful for the follow-up of patients with cancer.

Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells.

Progression of germ cells through meiosis is regulated by phosphorylation events. We previously showed the key role of cyclin dependent kinases in meiotic divisions of rat spermatocytes co-cultured with Sertoli cells (SC). In the present study, we used the same culture system to address the role of mitogen-activated protein kinases (MAPKs) in meiotic progression. Phosphorylated ERK1/2 were detected in vivo and in freshly isolated SC and in pachytene spermatocytes (PS) as early as 3 h after seeding on SC. The yield of the two meiotic divisions and the percentage of highly MPM-2-labeled pachytene and secondary spermatocytes (SII) were decreased in co-cultures treated with U0126, an inhibitor of the ERK-activating kinases, MEK1/2. Pre-incubation of PS with U0126 resulted in a reduced number of in vitro formed round spermatids without modifying the number of SII or the MPM-2 labeling of PS or SII. Conversely, pre-treatment of SC with U0126 led to a decrease in the percentage of highly MPM-2-labeled PS associated with a decreased number of SII and round spermatids. These results show that meiotic progression of spermatocytes is dependent on SC-activated MAPKs. In addition, high MPM-2 labeling was not acquired by PS cultured alone in Sertoli cell conditioned media, indicating a specific need for cell-cell contact between germ cells and SC.

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis.

Glial cell-line-derived neurotropic factor (GDNF) and its receptors glial cell-line-derived neurotropic factor alpha (GFR1alpha) and rearranged during transformation (RET) have been localized in the rat testis during postnatal development. The three mRNAs, and GDNF and GFR1alpha proteins were detected in testis extracts from 1- to 90-day-old rats by reverse transcriptase PCR and Western blotting respectively. The three mRNAs were present in Sertoli cells from 20- and 55-day-old rats, pachytene spermatocytes (PS), and round spermatids (RS). The GDNF and GFR1alpha proteins were detected in PS, RS, and Sertoli cells. GDNF and GFR1alpha were also detected using flow cytometry in spermatogonia and preleptotene spermatocytes, and in secondary spermatocytes. The localization of GDNF and GFR1alpha in germ and Sertoli cells was confirmed by immunocytochemistry. The hypothesis that GDNF may control DNA synthesis of Sertoli cells and/or spermatogonia in the immature rat was addressed using cultures of seminiferous tubules from 7- to 8-day-old rats. Addition of GDNF for 48 h resulted in a twofold decrease in the percentage of spermatogonia able to duplicate DNA, whereas Sertoli cells were not affected. These results are consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia. Moreover, the wide temporal and spatial expression of GDNF and its receptors in the rat testis suggest that it might act at several stages of spermatogenesis.

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction.

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro.

BACKGROUND: TGF beta and its receptors are present in both germ cells and somatic cells of the male gonad. However, knock-out strategies for studying spermatogenesis regulation by TGF beta have been disappointing since TGF beta-or TGF beta receptor-null mice do not survive longer than a few weeks. METHODS: In the present study, we addressed the role of TGF beta-1 on the completion of meiosis by rat pachytene spermatocytes (PS) cocultured with Sertoli cells. Identification and counting of meiotic cells were performed by cytology and cytometry. RESULTS: Under our culture conditions, some PS differentiated into round spermatids (RS). When TGF beta-1 was added to the culture medium, neither the number of PS or of secondary spermatocytes nor the half-life of RS was modified by the factor. By contrast, the number of RS and the amount of TP1 mRNA were lower in TGF beta-1-treated cultures than in control cultures. Very few metaphase I cells were ever observed both in control and TGF beta-1-treated wells. Higher numbers of metaphase II were present and their number was enhanced by TGF beta-1 treatment. A TGF beta-like bioactivity was detected in control culture media, the concentration of which increased with the time of culture. CONCLUSION: These results indicate that TGF beta-1 did not change greatly, if any, the yield of the first meiotic division but likely enhanced a bottleneck at the level of metaphase II. Taken together, our results suggest strongly that TGF beta participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

Different DNA ploidy patterns for the differentiation of common subtypes of renal tumors.

OBJECTIVES: The common subtypes of renal tumors are conventional or clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma. Each subtype has its distinct histogenesis and clinical evolution. DNA ploidy is viewed as a marker of gross genomic aberrations. The aim of this study is to evaluate the DNA ploidy in the common subtypes of renal tumors to increase our understanding of renal tumor biology and to broaden clinical application of DNA ploidy. METHODS: 38 renal tumor samples (13 clear cell RCCs, 12 papillary RCCs, 7 chromophobe RCCs, and 6 oncocytomas) were studied. Five biopsies of different parts of each fresh tumor were subjected to a flow cytometric analysis of DNA ploidy. RESULTS: All tumors except one papillary RCC generated interpretable DNA histograms. Flow cytometric analysis of oncocytomas showed the diploid pattern (29/30 frequencies) while the chromophobe RCC never showed the diploid pattern (0/55 frequencies) (p<0.01). 3/7 chromopbobe RCCs possessed the hypodiploid stemline. The hypodiploid stemline appeared neither in conventional RCCs (0/63 frequencies) nor in papillary RCCs (0/50 frequencies). The diploid pattern was dominant in conventional and papillary RCCs. 10/13 (76.9%) of clear cell RCCs and 9/11 (81.8%) of papillary RCCs possessed a homogeneous DNA ploidy pattern while only 1/7 (14.3%) has a homogeneous DNA ploidy pattern. 6/7 chromophobe RCCs had multiple aneuploid stemlines. CONCLUSIONS: Flow cytometric analysis reveals that conventional and papillary RCCs are more homogeneous than chromophobe RCC. Each subtype of renal tumors possesses a specific DNA ploidy pattern. The analysis of DNA ploidy is useful for the differentiation of common subtypes of renal tumors in morphologically difficult cases.

Differential production of immunoglobulin classes and subclasses by mucosal-type human B-lymphocytes exposed in vitro to CpG oligodeoxynucleotides.

As B-lymphocytes play an important role in innate and adaptive immunity, we aimed to examine the effects of CpG oligodeoxynucleotides (ODNs) on purified tonsil-originating CD19+ B-cells, representing mucosal B-cells. We screened various K-type ODNs, reactive with human B-cells, and tested for the production of immunoglobulins in vitro. Using one CpG-ODN, DSP30, we observed that it could upregulate not only Toll-like receptor 9 (TLR9) mRNA expression in activated B-cells, but also the early expression of CD69 followed by the sequential expression of CD80, CD86 and the nuclear factor (NF)-kappaB pathway. Furthermore, mRNA expression of certain B-cell-derived cytokines was influenced by exposure to DSP30, with a strong upregulation of interleukin 6 (IL-6) and downregulation of IL1-beta. Stimulation of B-cells, co-stimulated with IL-2, IL-10 and soluble CD40 ligand (sCD40L) with different CpG-ODNs, had differing effects on the terminal differentiation in vitro of B-cells into immunoglobulin-secreting cells. TLR9 is involved in innate immunity and the recognition of bound CpG DNA from invading bacterial pathogens. As tonsillar B-cells are mucosal-type B-lymphocytes, this study suggests that CpG-ODNs show promise as mucosal adjuvants in modulating the local production of immunoglobulins of certain classes and subclasses, a crucial issue in vaccine perspectives.

Mitochondrial-dependent regulation of myoblast proliferation.

The aim of the present study was to determine whether mitochondrial activity could regulate myoblast proliferation. We demonstrate that an increase in mitochondrial activity of L6E9 myoblasts can be easily obtained by simply raising extracellular pyruvate concentration in the culture dish. Under this condition, L6E9 myoblasts underwent a rapid growth arrest in G1 + S phases concomitant to a marked cellular hypertrophy. No sign of myoblast fusion was evident. This was accompanied by the down-regulation of proliferating cell nuclear antigen expression and an increase in p21 expression. Mitochondrial biogenesis was also stimulated, as indicated by a twofold increase in mitochondrial content. These cells exhibited a large increase in the production of reactive oxygen species that could contribute to the observed phenotypic alterations. However, exposure of pyruvate-treated cells to antioxidants did not reverse growth arrest. Similarly, exposure of control cells to oxidants did not induce growth arrest. Our observations suggest that mitochondrial activity appears to play a central role in regulating myoblast proliferation. They also argue strongly in favor of a retrograde communication establishing a mitochondrial control of nuclear gene expression that could be modulated by mitochondrial activity.

HIV-1 infection of Langerhans cells in a reconstructed vaginal mucosa.

We have developed an in vitro reconstructed vaginal mucosa integrating Langerhans cells (LCs), obtained by differentiation of umbilical cord blood CD34(+) hematopoietic progenitor cells, and, in this model, we have investigated the infection of LCs by human immunodeficiency virus type 1 (HIV-1). Proviral DNA of X4 (LAI and NL4-3) and R5 (Ba-L) HIV-1 strains were detected in LCs integrated in the reconstituted mucosa. Infection of LCs could be specifically inhibited by the chemokines stromal cell-derived factor 1 (SDF-1) and RANTES (regulated on activation, normally T cell-expressed and -secreted), confirming the presence of functional coreceptors on LCs generated in vitro. A complete inhibition of LCs, by use of azidothymidine, a reverse-transcriptase inhibitor, was also observed. Moreover, HIV-1-infected LCs of the reconstructed mucosa were able to transmit R5 or X4 HIV-1 strains to activated peripheral blood mononuclear cells. Such a model could be a useful tool to study the mechanisms involved in transmission of HIV in the female genital tract.