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The human skin microbiota plays a key role in the maintenance of healthy skin, ensuring protection and biological barrier by competing with pathogens and by closely communicating with the immune system. The development of approaches which preserve or restore the skin microbiota represents a novel target for skincare applications. Prebiotics could be applied to balance almost any microbial community to achieve advantageous effects. However, information about their effectiveness as skin microbiota modulators is limited. The objective of the current study was to evaluate the effects of short chain fructo-oligosaccharides (scFOS) from sugar beet (DP 3–5), well-recognised prebiotics, on some representative bacterial strains of the skin microbiota. We measured the growth and competitive activity of these specific bacteria for the use of scFOS as energy source in minimal medium and in a reconstructed human epithelium (RHE) in vitro model. In minimal growth medium, scFOS promoted and sustained the growth of Staphylococcus epidermidis up to 24 h, considered a beneficial skin commensal bacterium, while inhibiting both Cutibacterium acnes and Staphylococcus aureus growth, regarded as opportunistic pathogens. S. epidermidis showed the highest colonization potential and 1% scFOS was effective in shifting the competition in favour of S. epidermidis with respect to C. acnes in the RHE model. This latter effect was observed following 24 h of exposure, suggesting a long-term effect of scFOS in a highly skin dynamic environment. Therefore, scFOS could be effectively implemented in skincare formulations for recovering skin microbiota homeostasis.

The complexity and costs associated with traditional randomized, controlled trials have increased exponentially over time, and now threaten to stifle the development of new drugs and devices. Nevertheless, the growing use of electronic health records, mobile applications, and wearable devices offers significant promise for transforming clinical trials, making them more pragmatic and efficient. However, many challenges must be overcome before these innovations can be implemented routinely in randomized, controlled trial operations. In October of 2018, a diverse stakeholder group convened in Washington, DC, to examine how electronic health record, mobile, and wearable technologies could be applied to clinical trials. The group specifically examined how these technologies might streamline the execution of clinical trial components, delineated innovative trial designs facilitated by technological developments, identified barriers to implementation, and determined the optimal frameworks needed for regulatory oversight. The group concluded that the application of novel technologies to clinical trials provided enormous potential, yet these changes needed to be iterative and facilitated by continuous learning and pilot studies.

Dietary fibres are important in the human diet with multiple health benefits. This study aimed to determine the gastrointestinal tolerance of short-chain fructo-oligosaccharides (scFOS), well-known prebiotic fibres, at doses up to 40 g/d. An observational, connected, dose-ranging trial was conducted in 116 healthy volunteers. During the first week, the participants were instructed to consume their usual diet. During the second week, half of the subjects consumed 15 g scFOS per day, and the other half consumed 20 g scFOS per day. For the third week, the scFOS dose was doubled for all subjects. Gastrointestinal symptom severity was reported daily, as well as stool consistency and frequency. The results show that scFOS are well tolerated up to 40 g/d; all reported symptoms remained very mild from a clinical perspective. Stool consistency stayed normal, between 3 and 5 on the Bristol stool scale, confirming that no diarrhoea appeared after scFOS intake. Stool frequency also remained within the normal range. In conclusion, scFOS intake is well tolerated up to 40 g/d in healthy subjects. Thanks to their short chains and unique composition, scFOS prebiotic fibres are much better tolerated than other types of inulin-type fructans with longer chains. The digestive tolerance of fibres should be considered when added to foods and beverages.

Background Challenges in clinical trial recruitment threaten the successful development of improved therapies. This is particularly true in Parkinson's disease (PD) studies of disease modification where the population of interest is difficult to find and study design is more complex. Objective This paper seeks to understand how STEADY PD III, a National Institute of Neurological Disorders and Stroke (NINDS) funded phase 3 trial evaluating the efficacy of isradipine as a disease modifying agent for PD, was able to recruit their full target population 6 months ahead of schedule. Methods STEADY PD III aimed to enroll 336 individuals with early stage idiopathic PD within 18 months using 57 sites across the United States and Canada. The study included a 10% NIH minority recruitment goal. Eligible participants agreed to be followed for up to 36 months, complete 12 in-person visits and 4 telephone visits. A Recruitment Committee of key stakeholders was critical in the development of a comprehensive recruitment strategy involving: multi-modal outreach, protocol modifications and comprehensive site selection and activation. Efforts to increase site-specific minority recruitment strategies were encouraged through additional funding. Results A total of 336 individuals, including 34 minorities, were enrolled within 12 months - 6 months ahead of the projected timeline. Quantitative analysis of recruitment activity questionnaires found that of the sites that completed them (n = 54), (20.4%) met goals, (24.1%) exceeded goals, and (55.6%) fell below projected goals. Referral sources completed at time of screening indicate top four study referral sources as: site personnel (53.8%); neurologists (24%); Fox Trial Finder (10.2%); and communications from The Michael J. Fox Foundation (3.9%). Conclusions STEADY PD III serves as an important example of methods that can be used to increase clinical trial recruitment. This research highlights a continued need to improve site infrastructure and dedicate more resources to increased participation of minorities in clinical research.

The resistance of glioblastomas to treatments is mainly the consequence of their invasive capacities. Therefore, in order to better treat these tumors, it is important to understand the molecular mechanisms which are responsible for this behavior. Previous work suggested that gap junction proteins, the connexins, facilitate the aggressive nature of glioma cells. Here, we show that one of them&mdash, connexin43 (Cx43)&mdash, is implicated in the formation and function of invadopodia responsible for invasion capacity of U251 human glioblastoma cells. Immunofluorescent approaches&mdash, combined with confocal analyses&mdash, revealed that Cx43 was detected in all the formation stages of invadopodia exhibiting proteolytic activity. Clearly, Cx43 appeared to be localized in invadopodia at low cell density and less associated with the establishment of gap junctions. Accordingly, lower extracellular matrix degradation correlated with less mature invadopodia and MMP2 activity when Cx43 expression was decreased by shRNA strategies. Moreover, the kinetics of invadopodia formation could be dependent on Cx43 dynamic interactions with partners including Src and cortactin. Interestingly, it also appeared that invadopodia formation and MMP2 activity are dependent on Cx43 hemichannel activity. In conclusion, these results reveal that Cx43 might be involved in the formation and function of the invadopodia of U251 glioblastoma cells.

Naringin (4',5,7-trihydroxyflavanone 7-rhamnoglucoside), a natural flavonoid, has pharmacological properties. In the present study, we investigated the anti-metastatic activity of naringin and its molecular mechanism(s) of action in human glioblastoma cells. Naringin exhibits inhibitory effects on the invasion and adhesion of U87 cells in a concentration-dependent manner by Matrigel Transwell and cell adhesion assays. Naringin also inhibited the migration of U87 cells in a concentration-dependent manner by wound-healing assay. Additional experiments showed that naringin treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase (MMP)-2 and MMP-9 using a gelatin zymography assay and western blot analyses. Furthermore, naringin was able to reduce the protein phosphorylation of extracellular signal-regulated kinase ERK, p38 mitogen-activated protein kinase and c-Jun N-terminal kinase by western blotting. Collectively, our data showed that naringin attenuated the MAPK signaling pathways including ERK, JNK and p38 and resulted in the downregulation of the expression and enzymatic activities of MMP-2, MMP-9, contributing to the inhibition of metastasis in U87 cells. These findings proved that naringin may offer further application as an antimetastatic agent.

Sertoli cells were discovered in the seminiferous tubules by Enrico Sertoli in 1865 (Morgagni 7:31-33, 1865). Intense phagocytosis is, in the context of spermatogenesis cycle, morphologically the most noticeable function of Sertoli cells. In this chapter the major principles of phagocytosis machinery and its specificities in the seminiferous tubules will be briefly reviewed, guidelines of analysis of main phagocytosis steps by confocal and transmission electron microscopy will be described, and a simplified method to assess phagocytosis rate in routine experiments will be given.

Background: Fox Trial Finder is an online registry for individuals with and without Parkinson disease (PD) interested in participating in PD research. However, distance or disability could prevent such individuals from participating in traditional, clinic-based research at major centers. Objective: Use videoconferencing to connect participants to specialists to: (1) demonstrate feasibility of virtual research visits within this population (2) collect phenotypic data of the participants, (3) validate self-reported diagnosis, and (4) gauge interest in virtual research visits. Methods: We solicited volunteers throughout the United States through Fox Trial Finder. Interested individuals with PD provided consent, were given web cameras if needed, completed baseline surveys, and downloaded videoconferencing software remotely. Participants had a test connection and assessment appointment which included the Montreal Cognitive Assessment (MoCA), then a virtual research visit with a neurologist who reviewed their history and assessed their PD using a modified Movement Disorders Society Unified Parkinson’s Disease Rating Scale. Neurologists assessed PD diagnosis and symptomatology. Physicians and participants were surveyed about their experience. Results: Of 204 individuals who consented, 166 (81% ) individuals from 39 states completed all visits. The mean age was 62 and mean disease duration was 8.0 years. Mean MoCA score was 26.5, and mean modified MDS-UPDRS motor score was 22.8 (out of a possible 124). Neurologists judged PD as the most likely diagnosis in 97% of cases. Overall satisfaction with the visits was 79% (satisfied or very satisfied) among neurologists and 93% among participants. Conclusions: Through virtual research visits, neurologists engaged, characterized, and validated self-reported diagnosis in individuals with PD over a broad geography. This model may facilitate future research participation.

BACKGROUND As in other therapeutic areas, clinical studies in Parkinson's disease (PD) face significant recruitment challenges. However, qualitative surveys suggest that individuals with PD are willing to participate in clinical research. The Michael J. Fox Foundation therefore established Fox Trial Finder in 2011 to facilitate connection between PD research teams and volunteers. OBJECTIVE Characterize the research volunteers (with and without PD) registered on Fox Trial Finder as of June 2014, and the published, recruiting studies to identify trends and highlight gaps between research requirements and available volunteers. METHODS Profiles of volunteers with and without PD were analyzed to explore trends in geography, demographics, family history and, for those volunteers with PD, disease progression and treatment history. Clinical study profiles were analyzed to determine study type, phase, sponsor, focus, location and eligibility criteria. The analysis focused on volunteers and studies based in the United States. RESULTS The database contained 26,261 US-based volunteers, including 19,243 volunteers (73%) with PD and 7,018 (27%) controls without PD. The average time since diagnosis for PD volunteers was 5.7 years and the average age at diagnosis was 58 years. Control volunteers were more likely than volunteers with PD to be female (67% vs. 35%) and to have a family history of PD (49% vs. 12%). CONCLUSIONS Fox Trial Finder's registration history to date demonstrates the high level of willingness among individuals affected by PD to participate in clinical research and provide a significant amount of personal health information to facilitate that participation.

Timely and efficient clinical trial recruitment is a chronic problem across diseases. The Michael J Fox Foundation for Parkinson‘s Research (MJFF) is a leading funder and sponsor of Parkinson‘s disease research. Solving recruitment challenges in Parkinson‘s disease is a priority for the MJFF and led to the development of an online tool – Fox Trial Finder – that connects participants to trials. As the sponsor of the Parkinson‘s Progression Markers Initiative, an observational biomarkers study, the MJFF developed first-hand expertise in recruitment planning and tactical implementation. Solving poor enrollment relies on all stakeholders involved – sponsors, funders, site personnel and patients – to work together to raise awareness of the problem and implement strategies to increase the number of people who participate in research.

IF : 2,03; International audience; Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor in adults. This cancer has an infiltrative nature and the median survival of patients is about one year. Vasoactive intestinal peptide (VIP) belongs to a structurally related family of polypeptides and is a major regulatory factor in the central and peripheral nervous systems. VIP regulates proliferation of astrocytes and of numerous cancer cell lines and modulates migration in prostatic and colonic cancer cell lines. Little is known about the involvement of VIP and its receptors (VIP-receptor system) in proliferation or migration of GBM cells. The effects of VIP, PACAP and of synthetic VIP antagonists were tested in two human GBM cell lines, M059K and M059J, established from two different parts of a single tumor. In these cells, the data revealed that the VIP-receptor system did not affect proliferation but controlled cell migration. Indeed, in M059K cells which express components of the VIP receptor system, the VIP receptor antagonists and a PACAP antibody enhanced migration. The VIP receptor antagonists increased generation of typical migration-associated processes: filopodia and lamellipodia, and activation of Rac1 and Cdc42 GTPases. Reciprocally, in M059J cells which poorly express the VIP-receptor system, treatments with the agonists VIP and PACAP resulted in decreased cell migration. Furthermore, the peptides appeared to act through a subclass of binding sites displaying an uncommon very high affinity for these ligands. Taken together, these observations suggest that components of the VIP-receptor system negatively regulate cell migration, thus showing potential anti-oncogenic properties.

Cet article tente d’explorer une des nombreuses avenues qui s’ouvrent aux applications pédagogiques de l’ordinateur, soit celle du vidéotex. Comment tenir compte des acquis de la pédagogie des médias devenus « traditionnels » tout en respectant la spécificité de ce nouveau média? Le texte met l’accent sur l’influence des modes de présentation et des systèmes symboliques des médias sur l’apprentissage. Il fait ressortir aussi l’importance de connaître les pratiques culturelles du destinataire avant de concevoir les contenus qui lui sont destinés. Enfin, le texte rend compte d’un projet entrepris à partir de ces présupposés., This article attempts to explore one of the many avenues that have opened for computer assisted instruction, that of the videotex. How can one take into account the development of teaching medias that have become more "traditional" while at the same time considering the specificity of this new media? This paper emphasizes the influence of modes of presentation and of symbol systems of media on learning. As well, the author points out the importance of understanding the cultural practices of the user before conceptualizing the content. A report on a project based on these principles follows., Este artículo trata de explorar una de las numerosas avenidas que se ofrecen a las aplicaciones pedagógicas del computador, o sea la del videotex. ¿ Cómo considerar las acquisiciones de la pedagogía de los medios de communicación convertidas en "tradicionales", y respetar a la vez, la especificidad de este nuevo medio? El texto acentuá la influencia de las formas de presentación y de los sistemas simbólicos de los medios sobre el aprendizaje. Hace sobresalir, también, la importancia de conocer las prácticas culturales del destinatario antes de concebir los contenidos que le son enviados. Enfin, el texto refiere a un projecto realizado en estas bases., Dieser Artikel sucht einen der zahlreichen Wege zu erforschen, die sich der pädagogischen Anwendung des Computers eröffnen, nämlich den des "Videotex". Wie kann man die Errungenschaften der Pädagogik der inzwischen zur "Tradition" gewordenen Medien benutzen und zugleich die Besonderheit dieses neuen Kommunikationsmittels berücksichtigen? Der Text unterstreicht den Einfluss, den die Darstellungsweisen und die Symbolsysteme der Medien auf das Lernen haben. Er betont auch, wie wichtig es ist, die kulturellen Gebraüche des Empfängers zu kennen, ehe man die ihm bestimmten Inhalte konzipiert. Schliesslich wird über ein in diesem Sinn durchgeführtes Projekt berichtet.

To assess the evolution of triglyceride (TG) levels in HIV-infected patients receiving stable potent antiretroviral therapy treated with N-3 polyunsaturated fatty acids (PUFAs), a prospective double-blind randomized design for a reliable assessment of TG evolution was performed. One hundred twenty-two patients with TG levels2 g/L andor =10 g/L after a 4-week diet (baseline TG: 4.5 +/- 1.9 g/L) were randomized for 8 weeks to N-3 PUFAs (2 capsules containing 1 g of fish oil 3 times daily, n = 60), or placebo (1 g of paraffin oil capsules, n = 62). An 8-week open-label phase of N-3 PUFAs followed. Evaluation criteria were TG percent change at week 8, percentage of responders (normalization oror =20% TG decrease), and safety issues. Ten patients with baseline TG levels10 g/L were not randomized and received N-3 PUFAs as open treatment. The difference (PUFA - placebo) in TG percent change at week 8 was -24.6% (range: -40.9% to -8.4%; P = 0.0033), the median was -25.5% in the PUFA group versus 1% in the placebo group, and mean TG levels at week 8 were 3.4 +/- 1.8 g/L and 4.8 +/- 3.1 g/L, respectively. TG levels were normalized in 22.4% (PUFA) versus 6.5% (placebo) of patients (P = 0.013) with aor =20% reduction in 58.6% (PUFA) versus 33.9% (placebo) of patients (P = 0.007). Under the open-label phase of N-3 PUFAs, the decrease in TG levels was sustained at week 16 for patients in the PUFA group (mean TG: 3.4 +/- 1.7 g/L), whereas a 21.2% decrease in TG levels occurred for patients in the placebo group (mean TG: 3.3 +/- 1.4 g/L). No significant differences were observed between groups in the occurrence of adverse events. The median TG change at week 8 was -43.6% (range: Q1-Q3; 95% CI: -66.5% to -4.6%) for patients with baseline TG levels10 g/L. The difference in mean total cholesterol between groups (PUFA - placebo) at week 8 was -8.5% (P = 0.0117). This study demonstrated the efficacy of PUFAs to lower elevated TG levels in treated HIV-infected hypertriglyceridemic patients. N-3 PUFAs have a good safety profile.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides acting through VPAC1, VPAC2 and PAC1 receptors (referred here as the VIP-receptor system). In the central nervous system, VIP and PACAP are involved in neurogenesis, cell differentiation and migration, suggesting that they could be implicated in the development of glioblastoma (GBM). The infiltrative nature of GBM remains a major problem for the therapy of these tumors. We previously demonstrated that the VIP-receptor system regulated cell migration of the human cell lines M059J and M059K, derived from a single human GBM. Here, we evaluated the involvement of the VIP-receptor system in GBM cell invasion. In Matrigel invasion assays, M059K cells that express more the VIP-receptor system than M059J cells were less invasive. Invasion assays performed in the presence of agonists, antagonists or anti-PACAP antibodies as well as experiments with transfected M059J cells overexpressing the VPAC1 receptor indicated that the more the VIP-receptor system was expressed and activated, the less the cells were able to invade. Western immunoblotting experiments revealed that the VIP-receptor system inactivated the signaling protein AKT. Invasion assays carried out in the presence of an AKT inhibitor demonstrated the involvement of this signaling kinase in the regulation of cell invasion by the VIP-receptor system in M059K cells. The inhibition by VIP of invasion and AKT was also observed in U87 cells. In conclusion, VIP and PACAP act as anti-invasive factors in different GBM cell lines, a function mediated by VPAC1 inhibition of AKT signaling in M059K cells.

International audience; Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (α, γ and δ) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARα-null mice, we have found that PPARα in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARδ/α agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARα-null mice. These findings demonstrate that PPARδ can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARδ target gene in the small intestine. We propose that PPARδ contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

Importance We observed a significant correlation between cerebrospinal fluid (CSF) levels of tau proteins and α-synuclein, but not β-amyloid 1-42 (Aβ1-42), and lower concentration of CSF biomarkers, as compared with healthy controls, in a cohort of entirely untreated patients with Parkinson disease (PD) at the earliest stage of the disease studied so far. Objective To evaluate the baseline characteristics and relationship to clinical features of CSF biomarkers (Aβ1-42, total tau [T-tau], tau phosphorylated at threonine 181 [P-tau 181 ], and α-synuclein) in drug-naive patients with early PD and demographically matched healthy controls enrolled in the Parkinson’s Progression Markers Initiative (PPMI) study. Design, Setting, and Participants Cross-sectional study of the initial 102 research volunteers (63 patients with PD and 39 healthy controls) of the PPMI cohort. Main Outcomes and Measures The CSF biomarkers were measured by INNO-BIA AlzBio3 immunoassay (Aβ1-42, T-tau, and P-tau 181 ; Innogenetics Inc) or by enzyme-linked immunosorbent assay (α-synuclein). Clinical features including diagnosis, demographic characteristics, motor, neuropsychiatric, and cognitive assessments, and DaTscan were systematically assessed according to the PPMI study protocol. Results Slightly, but significantly, lower levels of Aβ1-42, T-tau, P-tau 181 , α-synuclein, and T-tau/Aβ1-42 were seen in subjects with PD compared with healthy controls but with a marked overlap between groups. Using multivariate regression analysis, we found that lower Aβ1-42 and P-tau 181 levels were associated with PD diagnosis and that decreased CSF T-tau and α-synuclein were associated with increased motor severity. Notably, when we classified patients with PD by their motor phenotypes, lower CSF Aβ1-42 and P-tau 181 concentrations were associated with the postural instability–gait disturbance–dominant phenotype but not with the tremor-dominant or intermediate phenotype. Finally, we found a significant correlation of the levels of α-synuclein with the levels of T-tau and P-tau 181 . Conclusions and Relevance In this first report of CSF biomarkers in PPMI study subjects, we found that measures of CSF Aβ1-42, T-tau, P-tau 181 , and α-synuclein have prognostic and diagnostic potential in early-stage PD. Further investigations using the entire PPMI cohort will test the predictive performance of CSF biomarkers for PD progression.

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.

(IF : 2,492); International audience; Vasoactive intestinal peptide (VIP) is a neuropeptide known to regulate proliferation and differentiation in normal and tumoral cells. We previously reported that VIP induced neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium. This neuritogenesis was associated with a regulated expression of neuronal cytoskeleton markers. To further characterize the neuroblastic cell differentiation induced by VIP in human SH-SY5Y cells, we investigated expression of synaptosomal-associated protein of 25 kDa (SNAP-25), a protein implicated in exocytosis associated with different processes, including neurite outgrowth. Western immunoblotting and real-time RT-PCR analyses revealed that VIP increased expression of the SNAP-25 protein and the level of both SNAP-25a and SNAP-25b mRNA isoforms. Immunofluorescence experiments indicated that SNAP-25 was mainly located in neurites and at the plasma membrane in SH-SY5Y cells treated with VIP. RNA interference experiments demonstrated that SNAP-25 was involved in VIP-induced neuritogenesis. In conclusion, SNAP-25 is up-regulated and implicated in neuritogenesis in human neuroblastoma SH-SY5Y cells treated with the neuropeptide VIP.

(IF: 2,368); International audience; The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Human colonic adenocarcinoma cell lines have conserved several features of the native tissue. Among these is the expression of cell surface receptors for hormones and neurotransmitters that may be involved in the regulation of proliferation and differentiation processes in these cancer cells. Here, we confirm that high-affinity binding sites for the Vasoactive Intestinal Polypeptide (VIP) and for the VIP analogue Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP), were expressed in 4 human colonic adenocarcinoma cell lines, HT29, SW403, DLD-1 and Caco-2, that spontaneously displayed variable phenotypic properties in culture. We demonstrated that after long-term treatments, VIP and PACAP significantly reduced cell proliferation in the 4 cell lines and modulated intracellular cAMP and cGMP levels. Furthermore, conspicuous differences were observed from one cell type to another concerning expression of the receptor subsets or the effects of the neuropeptides on cell growth and on cyclic nucleotides production.

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-α v β 3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus–lipofectAMINE–DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and α v β 3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

In addition to its commonly recognized status as a neuromodulator of virtually all vital functions, including neurobiological., the neuropeptide VIP plays a role in the control of cell growth and differentiation and of neuronal survival. Through, these actions, VIP, whose impact appears early in ontogeny, may possess developmental functions. VIP can be stimulatory or inhibitory on cell growth in function of the model considered. The growth regulatory actions of VIP, which are often independent of cAMP, are most likely significant when mitogenic or trophic factors, eventually released by nontarget cells, are simultaneously present in the extracellular medium. The intracellular mechanisms that mediate these actions of VIP may involve different transduction cascades triggered by subsets of VIP binding sites that may coexist in the same tissue.