New insights into the tetraspanin Tspan5 using novel monoclonal antibodies.

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

A multicenter prospective observational study of the conformity of temozolomide prescriptions in France.

CONTEXT: Temozolomide (TMZ) is approved for the treatment of high-grade gliomas such as glioblastoma (GBM) multiforme and refractory anaplastic astrocytoma, but it is also used in indications not mentioned in the summary of product characteristics (SPC). The main objective of this study was to evaluate the conformity of TMZ prescriptions to the French SPC and prescription guidebook. METHODS: We conducted a prospective observational study of all consecutive patients treated with TMZ in 21 French hospitals between September 2006 and February 2007, accounting for 39% of total TMZ consumption in France. The conformity of TMZ prescriptions was evaluated in terms of the indication, dosage, treatment duration, and combination with other treatments, with respect to the SPC and prescription guidebook. RESULTS: We enrolled 831 patients (median age, 56 years) who received a total of 5982 TMZ treatment cycles. TMZ was mainly prescribed to patients with newly diagnosed GBM (384 patients), GBM in progression/relapse (28 patients), or anaplastic astrocytoma in progression/relapse (19 patients). Prescriptions conformed to the SPC in 51.9% of cases and to the prescription guidebook in 91.5% of cases. Global conformity with the SPC, in terms of the dosage, treatment duration, and combination with other treatments, was 62% for newly diagnosed GBM treated with radiotherapy plus TMZ, 72% for TMZ maintenance monotherapy, and 66% for GBM and anaplastic astrocytoma in progression/relapse. CONCLUSION/DISCUSSION: In France, routine TMZ prescriptions conform to the SPC and practice guidebook. This is one of the largest studies of drug use in neuro-oncology in terms of the number of patients and cycles analyzed.

Tetraspanins regulate ADAM10-mediated cleavage of TNF-alpha and epidermal growth factor.

Several cytokines and growth factors are released by proteolytic cleavage of a membrane-anchored precursor, through the action of ADAM (a disintegrin and metalloprotease) metalloproteases. The activity of these proteases is regulated through largely unknown mechanisms. In this study we show that Ab engagement of several tetraspanins (CD9, CD81, CD82) increases epidermal growth factor and/or TNF-alpha secretion through a mechanism dependent on ADAM10. The effect of anti-tetraspanin mAb on TNF-alpha release is rapid, not relayed by intercellular signaling, and depends on an intact MEK/Erk1/2 pathway. It is also associated with a concentration of ADAM10 in tetraspanin-containing patches. We also show that a large fraction of ADAM10 associates with several tetraspanins, indicating that ADAM10 is a component of the "tetraspanin web." These data show that tetraspanins regulate the activity of ADAM10 toward several substrates, and illustrate how membrane compartmentalization by tetraspanins can control the function of cell surface proteins such as ectoproteases.

Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

Multi-factorial modulation of colorectal carcinoma cells motility - partial coordination by the tetraspanin Co-029/tspan8.

Colorectal carcinoma cells Isreco1 display an ability to migrate controlled by a complex set of signals issued from the membrane. By comparing cells infected by mycoplasmas and mycoplasmas free cells, we have established that basal 2D migration is dependent on a double signal mediated by the collagen receptors integrins alpha1/2 and the Toll-Like receptor TLR2. The signal issued from mycoplasmas can be replaced by a TLR2 ligand and the functional effect is neutralized by silencing of MyD88. Following previous observation that downregulation of E-cadherin/p120 catenin increases cell motility, we now report that EGFR or CD44 inhibition have a similar effect on cell motility that is restricted to tetraspanin Co-029/tspan8 transduced IsrecoI cells (Is1-Co029). The modulation of cell migration linked to EGFR or CD44 can be neutralized by antagonizing Co-029 with the mAb Ts29.1 or by RNA interference. Altogether these data point to a crucial role of Co-029 in the modulation of colon cancer cell motility which could be related to the protumoral effect reported for this tetraspanin. Among surface molecules able to mediate Co-029 function, E-cadherin, EGFR and CD44 appear as likely candidates.

Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation.

The tetraspanins associate with various surface molecules and with each other to build a network of molecular interactions, the tetraspanin web. The interaction of tetraspanins with each other seems to be central for the assembly of the tetraspanin web. All tetraspanins studied, CD9, CD37, CD53, CD63, CD81, CD82 and CD151, were found to incorporate [3H]palmitate. By site-directed mutagenesis, CD9 was found to be palmitoylated at any of the four internal juxtamembrane regions. The palmitoylation of CD9 did not influence the partition in detergent-resistant membranes but contributed to the interaction with CD81 and CD53. In particular, the resistance of the CD9/CD81 interaction to EDTA, which disrupts other tetraspanin/tetraspanin interactions, was entirely dependent on palmitoylation.

[Clinical trials and outpatient dispensation: the pharmaceutical counseling place]. / Dispensation ambulatoire du médicament pour essai clinique: place du conseil pharmaceutique.

Drug dispensations in clinical trials essentially concern outpatients (90%), and several mistakes and bad uses have been noted. The aim of this work is to set up a pharmaceutical counselling for outpatients in clinical trials. The bibliographic research on this subject did not learn us about similar experiences, but several references helped us to select the support of information: personal meeting between patient and pharmacist to introduce a written leaflet. Pharmaceutical information mentioned in the leaflet has to be validated and has to respect the clinical trials regulations. In the same way, information does not interfere with the inclusion-exclusion criteria or with the physician's observations. Then, information has been deliberately limited to the practical aspects of medications. Anonymous questionnaires have been delivered to the thirty first patients concerned to value their satisfaction with our services. The need of a clear and practical pharmaceutical counselling in clinical trials has been clearly established. Such a pharmaceutical counselling in clinical trials is one component of the overall drug-use process and potentially increases drug compliance. This pharmaceutical counselling on drug management has been well accepted by patients and has been supported by physicians and clinical trial sponsors.

Effect of an anti-human Co-029/tspan8 mouse monoclonal antibody on tumor growth in a nude mouse model.

New therapeutic agents are needed in digestive tract tumors. Co-029/tspan8 is a tetraspanin frequently expressed on human colorectal tumors, In this work, we report the effects of the monoclonal antibody Ts29.2, targeting Co-029/tspan8, on colorectal tumor cells in vitro and after implantation in nude mice. HT29, Isreco1 and SW480 colorectal tumor cell lines were used for this study. HT29 has a strong endogenous expression of Co-029/tspan8, whereas Isreco1 cells don't express Co-029/tspan8 and SW480 has only a weak expression. Isreco1 and SW480 were transduced to express Co-029/tspan8 at the same level as HT29. In order to check the specificity of the effect of monoclonal antibody Ts29.2, low Co-029/tspan8 expressing SW480 cells were injected simultaneously with transduced cells in the back, on the left and right sides of the mice. With an early treatment, Ts29.2 mAb inhibited growth of tumors expressing Co-029/tspan8 up to 70%, whereas a delayed treatment was less efficient. No effect of the antibody on cell proliferation or apoptosis induction was detected in vitro. No increase of activated caspase 3 labeling was observed in vivo and areas occupied by vessels were not significantly different between treated mice and controls. This suggests that the action of Ts29.2 is linked neither to cellular toxicity nor to the inhibition of the previously reported angiogenic properties of Co-029/tspan8. An inhibition of cell proliferation in vivo is demonstrated by a reduction of the mitotic index in HT29 tumors of Ts29.2 treated mice. The discrepancy between in vitro and in vivo data on cell proliferation suggests that the binding of Ts29.2 to tumor cells may modify their response to signals issued from the microenvironment. Given the restricted pattern of tissue expression of the tetraspanin Co-029/tspan8, these preliminary results put forth for consideration the antibody targeting of this tetraspanin in further investigations for therapeutic applications.

Metabolic syndrome in French HIV-infected patients: prevalence and predictive factors after 3 years of antiretroviral therapy.

Treatment of HIV infection with highly active antiretroviral therapy can induce metabolic complications and increase the risk of developing the metabolic syndrome (MS). The purpose of this study was to report the prevalence and the risk factors for MS in HIV-infected patients who started highly active antiretroviral therapy (HAART) after 2000. SYMET is a prospective, multicentric, cohort study evaluating the prevalence of MS in 269 patients who had received continuous HAART for 1 to 4 years up to September 2007. MS was defined according to the American Heart Association (AHA) and the National Heart, Lung, and Blood Institute (NHLBI) 2005 criteria. Cross-sectional assessment included clinical examination and fasting evaluation of metabolic, inflammatory, and oxidative parameters. Data were analyzed with Chi-square, Student, or Wilcoxon tests. Univariate and multivariate logistic regressions were performed to identify predictive factors for MS. The prevalence of MS was 18.2% after a median duration of HAART of 29.8 months. In multivariate analysis, predictive factors of MS were high non-HDL-cholesterol (OR=1.87; p<0.0001), high-sensitivity C-reactive protein levels (hsCRP) (OR=1.56; p=0.01), coinfection with hepatitis C virus (HCV) (OR=5.67; p=0.02), as well as age (OR=1.04; p=0.02) and duration of exposure to protease inhibitors (PI) (OR=1.03; p=0.02) or to abacavir (ABC) (OR=1.03; p=0.02). In this cohort of patients exposed to less than 4 years of HAART, MS prevalence was 18.2%. Older age, high hsCRP, HCV coinfection, and elevated non-HDL-cholesterol were risk factors for the MS. There was also a moderate significant association of increased risk of MS with cumulative PI and ABC exposure.

Profiling of the tetraspanin web of human colon cancer cells.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.

Cholesterol contributes to the organization of tetraspanin-enriched microdomains and to CD81-dependent infection by malaria sporozoites.

Tetraspanins constitute a family of widely expressed integral membrane proteins that associate extensively with one another and with other membrane proteins to form specific membrane microdomains distinct from conventional lipid rafts. So far, because of the lack of appropriate tools, the functionality of these microdomains has remained largely unknown. Here, using a new monoclonal antibody that only binds to the tetraspanin CD81 associated with other tetraspanins, we show that membrane cholesterol contributes to the organization of tetraspanin microdomains on the surface of live cells. Furthermore, our data demonstrate involvement of host membrane cholesterol during infection by Plasmodium yoelii and Plasmodium falciparum sporozoites, which both depend on host CD81 expression for invasion, but not during CD81-independent infection by Plasmodium berghei sporozoites. Our results unravel a functional link between CD81 and cholesterol during infection by malaria parasites, and illustrate that tetraspanin microdomains constitute a novel type of membrane microdomains that could be used by pathogens for infection.

Multiple levels of interactions within the tetraspanin web.

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.

A physical and functional link between cholesterol and tetraspanins.

By interacting with each others, the tetraspanins are thought to assemble a network of molecular interactions, the tetraspanin web. These tetraspanin/tetraspanin interactions involve in part the palmitoylation of the proteins. We show that tetraspanins interact with cholesterol as indicated by the precipitation of tetraspanin/tetraspanin complexes by digitonin, a cholesterol-precipitating reagent, and the labeling of the tetraspanins CD9, CD81 and CD82 with a photoactivatable cholesterol in vivo. Cholesterol may participate to the interaction of tetraspanins with each other since digitonin-precipitation of tetraspanins was correlated with their mutual interaction, and because these interactions were disrupted following cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment, or cholesterol sequestration by saponin. A mutant CD9 molecule lacking all palmitoylation sites was not precipitated by digitonin under conditions in which wild-type CD9 was precipitated, indicating a role of palmitoylation for the interaction with cholesterol. Finally, upon ligation of tetraspanins on the surface of a lymphoid B cell line, the tyrosine phosphorylation of several proteins, including the vav nucleotide exchange factor, was inhibited when cells were pretreated with MbetaCD, and increased when they were treated with MbetaCD/cholesterol complexes. Thus, there is a physical and functional link between tetraspanins and cholesterol.

EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.