Increased metabolic rate and insulin sensitivity in male mice lacking the carcino-embryonic antigen-related cell adhesion molecule 2.

AIMS/HYPOTHESIS: The carcino-embryonic antigen-related cell adhesion molecule (CEACAM)2 is produced in many feeding control centres in the brain, but not in peripheral insulin-targeted tissues. Global Ceacam2 null mutation causes insulin resistance and obesity resulting from hyperphagia and hypometabolism in female Ceacam2 homozygous null mutant mice (Cc2 [also known as Ceacam2](-/-)) mice. Because male mice are not obese, the current study examined their metabolic phenotype. METHODS: The phenotype of male Cc2(-/-) mice was characterised by body fat composition, indirect calorimetry, hyperinsulinaemic-euglycaemic clamp analysis and direct recording of sympathetic nerve activity. RESULTS: Despite hyperphagia, total fat mass was reduced, owing to the hypermetabolic state in male Cc2(-/-) mice. In contrast to females, male mice also exhibited insulin sensitivity with elevated ß-oxidation in skeletal muscle, which is likely to offset the effects of increased food intake. Males and females had increased brown adipogenesis. However, only males had increased activation of sympathetic tone regulation of adipose tissue and increased spontaneous activity. The mechanisms underlying sexual dimorphism in energy balance with the loss of Ceacam2 remain unknown. CONCLUSIONS/INTERPRETATION: These studies identified a novel role for CEACAM2 in the regulation of metabolic rate and insulin sensitivity via effects on brown adipogenesis, sympathetic nervous outflow to brown adipose tissue, spontaneous activity and energy expenditure in skeletal muscle.

In situ assembly of linked geometrically coupled microdevices.

Complex systems require their distinct components to function in a dynamic, integrated, and cooperative fashion. To accomplish this in current microfluidic networks, individual valves are often switched and pumps separately powered by using macroscopic methods such as applied external pressure. Direct manipulation and control at the single-device level, however, limits scalability, restricts portability, and hinders the development of massively parallel architectures that would take best advantage of microscale systems. In this article, we demonstrate that local geometry combined with a simple global field can not only reversibly drive component assembly but also power distinct devices in a parallel, locally uncoupled, and integrated fashion. By employing this single approach, we assemble and demonstrate the operation of check valves, mixers, and pistons within specially designed microfluidic environments. In addition, we show that by linking these individual components together, more complex devices such as pumps can be both fabricated and powered in situ.

Platelets contribute to the initiation of colitis-associated cancer by promoting immunosuppression.

Essentials Inflammation plays a key role in the development of colorectal cancer. Understanding mechanisms of cancer initiation might reveal new anticancer preventive strategy. Hyperactive platelets promote tumor formation by fostering immune evasion of cancer. Platelet inhibition by clopidogrel prevents carcinogenesis by restoring antitumor immunity. SUMMARY: Background Clinical and experimental evidence support a role for inflammation in the development of colorectal cancer, although the mechanisms are not fully understood. Beyond thrombosis and hemostasis, platelets are key actors in inflammation; they have also been shown to be involved in cancer. However, whether platelets participate in the link between inflammation and cancer is unknown. Objective To investigate the contribution of platelets and platelet-derived proteins to inflammation-elicited colorectal tumor development. Methods We used a clinically relevant mouse model of colitis-associated cancer. Platelet secretion and platelet reactivity to thrombin were assessed at each stage of carcinogenesis. We conducted an unbiased proteomic analysis of releasates of platelets isolated at the pretumoral stage to identify soluble factors that might act on tumor development. Plasma levels of the identified proteins were measured during the course of carcinogenesis. We then treated the mice with clopidogrel to efficiently inhibit platelet release reaction. Results At the pretumoral stage, hyperactive platelets constituted a major source of circulating protumoral serum amyloid A (SAA) proteins. Clopidogrel prevented the early elevation of the plasma SAA protein level, decreased colitis severity, and delayed the formation of dysplastic lesions and adenocarcinoma. Platelet inhibition hindered the expansion and function of immunosuppressive myeloid cells, as well as their infiltration into tumors, but increased the number of tissue CD8+ T cells. Platelets and releasates of platelets from mice with cancer were both able to polarize myeloid cells towards an immunosuppressive phenotype. Conclusions Thus, platelets promote the initiation of colitis-associated cancer by enhancing myeloid cell-dependent immunosuppression. Antiplatelet agents may help to prevent inflammation-elicited carcinogenesis by restoring antitumor immunity.

Restoration of SHIP-1 activity in human leukemic cells modifies NF-kappaB activation pathway and cellular survival upon oxidative stress.

Nuclear factor-kappa B (NF-kappaB) is an important prosurvival transcription factor activated in response to a large array of external stimuli, including reactive oxygen species (ROS). Previous works have shown that NF-kappaB activation by ROS involved tyrosine phosphorylation of the inhibitor IkappaBalpha through an IkappaB kinase (IKK)-independent mechanism. In the present work, we investigated with more details NF-kappaB redox regulation in human leukemic cells. By using different cell lines (CEM, Jurkat and the subclone Jurkat JR), we clearly showed that NF-kappaB activation by hydrogen peroxide (H2O2) is cell-type dependent: it activates NF-kappaB through tyrosine phosphorylation of IkappaBalpha in Jurkat cells, whereas it induces an IKK-mediated IkappaBalpha phosphorylation on S32 and 36 in CEM and Jurkat JR cells. We showed that this H2O2-induced IKK activation in CEM and Jurkat JR cells is mediated by SH2-containing inositol 5'-phosphatase 1 (SHIP-1), a lipid phosphatase that is absent in Jurkat cells. Indeed, the complementation of SHIP-1 in Jurkat cells made them shift to an IKK-dependent mechanism upon oxidative stress stimulation. We also showed that Jurkat cells expressing SHIP-1 are more resistant to H2O2-induced apoptosis than the parental cells, suggesting that SHIP-1 has an important role in leukemic cell responses to ROS in terms of signal transduction pathways and apoptosis resistance, which can be of interest in improving ROS-mediated chemotherapies.

[New approaches to the treatment of the HIV-infected patient]. / Nouvelles approches dans la prise en charge de l'infection à VIH.

HIV infection remains a major problem of public health in Belgium as well as globally. The number of new diagnosies of HIV infection in Belgium remains between two and three daily. Given the dramatic effect of antiretroviral therapy on the mortality due to HIV infection, the number of patients is constantly increasing. The different problems related to HIV care are also changing. Aging of the patients and chronic exposure to antiretroviral medications have induced new complications. We will present in this brief article several new experimental and clinical approaches in which our centre has participated during the last two years.

Targeting of C-type lectin-like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides.

Essentials CpG oligodeoxynucleotide (ODN) immuotherapeutics cause undesired platelet activating effects. It is crucial to understand the mechanisms of these effects to identify protective strategies. CpG ODN-induced platelet activation depends on C-type lectin-like receptor 2 (CLEC-2) and P2Y12. Targeting CLEC-2 or P2Y12 fully prevents CpG ODN-induced platelet activation and thrombosis. SUMMARY: Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoprotein VI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods In vitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor 2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. In vivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.

[Characterization of a Cl. Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture]. / Caractérisation d'une souche de Cl. Perfringens type D, isolée de terrain et optimisation de la biosynthèse en biofermenteur de la toxine epsilon.

A field strain of cl. perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case. The isolate characterized as Cl. perfringens, type D was based on its cultural and biochemical characters and its factors of virulence. The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat ... were optimized. Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times. The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type. The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin. It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart. The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.

SHIP-1 inhibits CD95/APO-1/Fas-induced apoptosis in primary T lymphocytes and T leukemic cells by promoting CD95 glycosylation independently of its phosphatase activity.

SHIP-1 (SH2 (Src homology 2)-containing inositol 5'-phosphatase-1) functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by phosphoinositide-3 (PI 3)-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B-cell lymphoma. On the other hand, SHIP-1-deficient mice have a reduced T-cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1(-/-) mice and T leukemic cell lines, we report that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum (ER), in which it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired death-inducing signaling complex (DISC) formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1-negative leukemic T cells.

High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins.

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors.

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.

Decreased protein levels of the c-Cbl protooncogene in murine AIDS.

Murine acquired immunodeficiency syndrome (MAIDS) can be viewed as a lymphoproliferative disease which involves B cells as well as T cells from spleen and lymph nodes while thymus and Peyer's patches do not participate in the process. The 120-kDa protooncogene product c-Cbl was initially cloned from the murine Cas NS-1 B cell lymphoma. It is a main target of immunoreceptor (TCR and BCR)-mediated protein tyrosine kinase activity. Moreover, recent data suggest that c-Cbl might play a crucial role in the regulation of cell proliferation through regulation of GTP-binding proteins. Therefore, the involvement of c-Cbl was evaluated in the lymphoproliferative disease induced by the MAIDS virus. The expression of the c-Cbl protein was dramatically reduced in the lymph node of infected mice while it remained normal in the thymus. In contrast, the expression of actin, TCR-zeta chain, ZAP-70, and p59(fyn) remained similar in controls and infected mice. Identical results were obtained with sorted B cells and T cells. Surprisingly, a B cell lymphoma line derived from late stage MAIDS mice displayed a normal level of c-Cbl.