Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.

OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

Reovirus-induced apoptosis is mediated by TRAIL.

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

Engineering tolerance into transplanted beta cell lines.

In this paper we explore the possibility of improving, by genetic engineering, the resistance of insulin-secreting cells to the metabolic and inflammatory stresses that are anticipated to limit their function and survival when encapsulated and transplanted in a type 1 diabetic environment. We show that transfer of the Bcl-2 antiapoptotic gene, and of genes specifically interfering with cytokine intracellular signaling pathways, greatly improves resistance of the cells to metabolic limitations and inflammatory stresses.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

The anti-apoptotic role of PPARbeta contributes to efficient skin wound healing.

PPARalpha and PPARbeta are expressed in the mouse epidermis during fetal development, but their expression progressively disappears after birth. However, the expression of PPARbeta is reactivated in adult mice upon proliferative stimuli, such as cutaneous injury. We show here that PPARbeta protects keratinocytes from growth factor deprivation, anoikis and TNF-alpha-induced apoptosis, by modulating both early and late apoptotic events via the Akt1 signaling pathway and DNA fragmentation, respectively. The control mechanisms involve direct transcriptional upregulation of ILK, PDK1, and ICAD-L. In accordance with the anti-apoptotic role of PPARbeta observed in vitro, the balance between proliferation and apoptosis is altered in the epidermis of wounded PPARbeta mutant mice, with increased keratinocyte proliferation and apoptosis. In addition, primary keratinocytes deleted for PPARbeta show defects in both cell-matrix and cell-cell contacts, and impaired cell migration. Together, these results suggest that the delayed wound closure observed in PPARbeta mutant mice involves the alteration of several key processes. Finally, comparison of PPARbeta and Akt1 knock-out mice reveals many similarities, and suggests that the ability of PPARbeta to modulate the Akt1 pathway has significant impact during skin wound healing.

An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

Differences in low-grade chronic inflammation and insulin resistance in women with previous gestational diabetes mellitus and women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) and gestational diabetes mellitus (GDM) are both characterized by an increase in insulin resistance. Our goal in the present study was to measure insulin resistance (as estimated by homeostasis model assessment, sex hormone-binding globulin (SHBG) and adiponectin concentrations) and parameters of low-grade inflammation in non-diabetic, non-hyperandrogenic ovulatory women with previous GDM (pGDM) and in non-diabetic women with classic PCOS, characterized by hyperandrogenism and oligo/anovulation. PATIENTS AND DESIGN: We evaluated 20 women with PCOS, 18 women with pGDM and 19 controls, all matched according to body mass index (BMI). Fasting blood samples were drawn in all women 3-6 days after spontaneous or dydrogesterone-induced withdrawal bleeding. Body fat distribution was assessed using dual-energy X-ray absorptiometry in all women. RESULTS: After adjusting for age and percent body fat, measures of insulin resistance such as SHBG and adiponectin concentrations were decreased and central obesity was increased in women with PCOS and pGDM compared with controls (all p < 0.05). Parameters of low-grade inflammation such as serum tumor necrosis factor-alpha and highly sensitive C-reactive protein concentrations, white blood cell and neutrophil count were increased only in women with PCOS compared with BMI-matched controls (all p < 0.05). CONCLUSIONS: Certain markers of insulin resistance are increased in both women with PCOS and women with pGDM, while low-grade inflammation is increased only in PCOS. PCOS and GDM might represent specific phenotypes of one disease entity with an increased risk of cardiovascular disease, whereby women with PCOS demonstrate an augmented cardiovascular risk profile.

Rôle du pathologiste dans la prise en charge des tumeurs stromales gastro-intestinales (GIST) [The role of the pathologist in the management of gastrointestinal stromal tumors (GIST)]

Considerable progress was realized these last years in the understanding of the molecular mechanisms and the treatment of the GIST. Their diagnosis remains based on the morphology and immunohistochemistry. The evaluation of GIST prognosis was till know difficult to establish but a new histopronostic classification currently used allows a better therapeutic approach. The search for KIT and PDGFRA mutations is recommended to adapt a targeted therapy by KIT inhibitors. The pathologist plays a crucial role in the management of the GIST because it is on him that is based the diagnosis, the evaluation of the prognosis and the treatment (surgery and kit inhibitors).

The efficacy and safety of a third anti-TNF monoclonal antibody in Crohn's disease after failure of two other anti-TNF antibodies.

BACKGROUND: Adalimumab (ADA) and certolizumab pegol (CZP) have demonstrated efficacy in Crohn's disease (CD) patients previously treated with infliximab (IFX). AIM: To assess the efficacy and tolerability of a third anti-TNF in CD after failure of and/or intolerance to two different anti-TNF antibodies. METHODS: Crohn's disease patients who received ADA or CZP after loss of response and/or intolerance to two anti-TNF agent were included in this retrospective study. Data were collected using a standardized questionnaire. Clinical response, duration, safety and reasons for discontinuation were assessed. RESULTS: Sixty-seven patients treated with CZP (n = 40) or ADA (n = 27) were included. A clinical response was observed in 41 (61%) at week 6 and 34 patients (51%) at week 20. The probability of remaining under treatment at 3 months, 6 months and 9 months was 68%, 60% and 45%, respectively. At the end of follow-up, the third anti-TNF had been stopped in 36 patients for intolerance (n = 13), or failure (n = 23). Two deaths were observed. CONCLUSIONS: The treatment with a third anti-TNF (CZP or ADA) agent of CD patients, who have experienced loss of response and/or intolerance to two anti-TNF antibodies, has favourable short-term and long-term efficacy. It is an option to be considered in patients with no other therapeutic options.

Role of MyD88 and Toll-like receptors 2 and 4 in the sensing of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88(-/-), TLR4(-/-), and TLR2(-/-) mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

TWEAK can induce cell death via endogenous TNF and TNF receptor 1.

TWEAK is a recently cloned novel member of the TNF ligand family. Here we show that soluble TWEAK is sufficient to induce apoptosis in Kym-1 cells within 18 h. TWEAK-induced apoptosis is indirect and is mediated by the interaction of endogenous TNF and TNF receptor (TNFR)1, as each TNFR1-Fc, neutralizing TNF-specific antibodies and TNFR1-specific Fab fragments efficiently antagonize cell death induction. In addition to this indirect mode of action, co-stimulation of Kym-1 cells with TWEAK enhances TNFR1-mediated cell death induction. In contrast to TNF, TWEAK does only modestly activate NF-kappaB or c-jun N-terminal kinase (JNK) in Kym-1 cells. Although TWEAK binding to Kym-1 cells is easily detectable by flow cytometric analysis, we found neither evidence for expression of the recently identified TWEAK receptor Apo3/TRAMP/wsl/DR3/LARD, nor indications for direct interactions of TWEAK with TNFR. Together, these characteristics of TWEAK-induced signaling in Kym-1 cells argue for the existence of an additional, still undefined non-death domain-containing TWEAK receptor in Kym-1 cells.