Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

BACKGROUND: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine.¦OBJECTIVE: In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms.¦METHODS: Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically.¦RESULTS: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor.¦CONCLUSION: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

Plasma Level of Mmp-9 as Predictive Factor for Response and Progression Free Survival in Patients Treated with Bevacizumab (Bev) and Pegylated Liposomal Doxorubicin (Pld): Sakk 24/06

Background: There is currently no identified marker predicting benefit from Bev in patients with breast cancer (pts). We monitored prospectively 6 angiogenesis-related factors in the blood of advanced stage pts treated with a combination of Bev and PLD in a phase II trial of the Swiss Group for Clinical Cancer Research, SAKK.Methods: Pts received PLD (20 mg/m2) and Bev (10 mg/kg) every 2 weeks for a maximum of 12 administrations, followed by Bev monotherapy until progression or severe toxicity. Blood samples were collected at baseline, during treatment and at treatment discontinuation. Enzyme-linked immunosorbent assays (Quantikine, R&DSystems and Reliatech) were used to measure vascular endothelial growth factor (VEGF), placental growth factor (PlGF), matrix metalloproteinase 9 (MMP-9) and soluble VEGF receptors -1, -2 and -3. The natural log-transformed (ln) data for each factor was analyzed by analysis of variance (ANOVA) model to investigate differences between the mean values of the subgroups of interest (where a = 0.05), based on the best tumor response by RECIST.Results: 132 samples were collected in 41 pts. The mean of baseline ln MMP-9 levels was significantly lower in pts with tumor progression than those with tumor response (p=0.0202, log fold change=0.8786) or disease control (p=0.0035, log fold change=0.8427). Higher MMP-9 level was a significant predictor of superior progression free survival (PFS): p=0.0417, hazard ratio=0.574, 95% CI=0.336-0.979. In a multivariate cox proportional hazards model, containing performance status, disease free interval, number of tumor sites, visceral involvement and prior adjuvant chemotherapy, using stepwise regression baseline MMP-9 was still a statistically 117P Table 1. SOLTI-0701* AC01B07* NU07B1* SOR+CAP N=20 PL+CAP N=33 SOR+ GEM/CAP N=23 PL+ GEM/CAP N=27 SOR+PAC N=48 PL+PAC N=46 Baseline characteristics Age, median (range), y 49 (32-72) 53 (30-78 54 (32-69) 57 (31-82) 50 (27-80) 52 (23-74) AJCC stage, n (%) IIIB/IIIC 3 (15) 6 (18) 0 (0) 3 (11) 8 (17) 9 (20) IV 17 (85) 27 (82) 23 (100) 24 (89) 40 (83) 37 (80) Metastatic site, n (%) Non-visceral 3 (15) 6 (18) 7 (30) 6 (22) 9 (19) 17 (37) Visceral 17 (85) 27 (82) 16 (70) 21 (78) 39 (81) 29 (63) Prior metastatic chemo, n (%) 8 (40) 15 (45) 21 (91) 25 (93) - - Efficacy PFS, median, mo 4.3 2.5 3.1 2.6 5.6 5.5 HR (95% CI)_ 0.60 (0.31, 1.14) 0.57 (0.30, 1.09) 0.86 (0.50, 1.45) 1-sided P value_ 0.055 0.044 0.281 Overall survival, median, mo 17.5 16.1 Pending 14.7 18.2 HR (95% CI)_ 0.98 (0.50, 1.89) 1.11 (0.64, 1.94) 1-sided P value_ 0.476 0.352 Safety N=20 N=33 N=22 N=27 N=46 N=46 Tx-emergent Grade 3/4, n (%) 15 (75) 16 (48) 20 (91) 17 (63) 36 (78) 16 (35) Grade 3§ hand-foot skin reaction/ syndrome 8 (40) 5 (15) 8 (36) 0 (0) 14 (30) 2 (4) *Efficacy results based on intent-to-treat population and safety results based on safety population (pts who received study drug[s]); _Cox regression within each subgroup; _log-rank test within each subgroup; §maximum toxicity grade for hand-foot skin reaction/syndrome; AJCC, American Joint Committee on Cancer mittedabstractsª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from annonc.oxfordjournals.org at Bibliotheque Cantonale et Universitaire on June 6, 2011 significant factor (p=0.0266). The results of the other measured factors were presented elsewhere.Conclusions: Higher levels of MMP-9 could predict tumor response and superior PFSin pts treated with a combination of Bev and PLD. These exploratory results justify further investigations of MMP-9 in pts treated with Bev combinations in order to assess its role as a prognostic and predictive factor.Disclosure: K. Zaman: Participation in advisory board of Roche; partial sponsoring ofthe study by Roche (the main sponsor was the Swiss Federation against Cancer (Oncosuisse)). B. Thu¨rlimann: stock of Roche; Research grants from Roche. R. vonMoos: Participant of Advisory Board and Speaker honoraria

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis.

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

A peripheral dose calculation model for estimating second cancer risk after breast radiotherapy

AbstractBreast cancer is one of the most common cancers affecting one in eight women during their lives. Survival rates have increased steadily thanks to early diagnosis with mammography screening and more efficient treatment strategies. Post-operative radiation therapy is a standard of care in the management of breast cancer and has been shown to reduce efficiently both local recurrence rate and breast cancer mortality. Radiation therapy is however associated with some late effects for long-term survivors. Radiation-induced secondary cancer is a relatively rare but severe late effect of radiation therapy. Currently, radiotherapy plans are essentially optimized to maximize tumor control and minimize late deterministic effects (tissue reactions) that are mainly associated with high doses (» 1 Gy). With improved cure rates and new radiation therapy technologies, it is also important to evaluate and minimize secondary cancer risks for different treatment techniques. This is a particularly challenging task due to the large uncertainties in the dose-response relationship.In contrast with late deterministic effects, secondary cancers may be associated with much lower doses and therefore out-of-field doses (also called peripheral doses) that are typically inferior to 1 Gy need to be determined accurately. Out-of-field doses result from patient scatter and head scatter from the treatment unit. These doses are particularly challenging to compute and we characterized it by Monte Carlo (MC) calculation. A detailed MC model of the Siemens Primus linear accelerator has been thoroughly validated with measurements. We investigated the accuracy of such a model for retrospective dosimetry in epidemiological studies on secondary cancers. Considering that patients in such large studies could be treated on a variety of machines, we assessed the uncertainty in reconstructed peripheral dose due to the variability of peripheral dose among various linac geometries. For large open fields (> 10x10 cm2), the uncertainty would be less than 50%, but for small fields and wedged fields the uncertainty in reconstructed dose could rise up to a factor of 10. It was concluded that such a model could be used for conventional treatments using large open fields only.The MC model of the Siemens Primus linac was then used to compare out-of-field doses for different treatment techniques in a female whole-body CT-based phantom. Current techniques such as conformai wedged-based radiotherapy and hybrid IMRT were investigated and compared to older two-dimensional radiotherapy techniques. MC doses were also compared to those of a commercial Treatment Planning System (TPS). While the TPS is routinely used to determine the dose to the contralateral breast and the ipsilateral lung which are mostly out of the treatment fields, we have shown that these doses may be highly inaccurate depending on the treatment technique investigated. MC shows that hybrid IMRT is dosimetrically similar to three-dimensional wedge-based radiotherapy within the field, but offers substantially reduced doses to out-of-field healthy organs.Finally, many different approaches to risk estimations extracted from the literature were applied to the calculated MC dose distribution. Absolute risks varied substantially as did the ratio of risk between two treatment techniques, reflecting the large uncertainties involved with current risk models. Despite all these uncertainties, the hybrid IMRT investigated resulted in systematically lower cancer risks than any of the other treatment techniques. More epidemiological studies with accurate dosimetry are required in the future to construct robust risk models. In the meantime, any treatment strategy that reduces out-of-field doses to healthy organs should be investigated. Electron radiotherapy might offer interesting possibilities with this regard.RésuméLe cancer du sein affecte une femme sur huit au cours de sa vie. Grâce au dépistage précoce et à des thérapies de plus en plus efficaces, le taux de guérison a augmenté au cours du temps. La radiothérapie postopératoire joue un rôle important dans le traitement du cancer du sein en réduisant le taux de récidive et la mortalité. Malheureusement, la radiothérapie peut aussi induire des toxicités tardives chez les patients guéris. En particulier, les cancers secondaires radio-induits sont une complication rare mais sévère de la radiothérapie. En routine clinique, les plans de radiothérapie sont essentiellement optimisées pour un contrôle local le plus élevé possible tout en minimisant les réactions tissulaires tardives qui sont essentiellement associées avec des hautes doses (» 1 Gy). Toutefois, avec l'introduction de différentes nouvelles techniques et avec l'augmentation des taux de survie, il devient impératif d'évaluer et de minimiser les risques de cancer secondaire pour différentes techniques de traitement. Une telle évaluation du risque est une tâche ardue étant donné les nombreuses incertitudes liées à la relation dose-risque.Contrairement aux effets tissulaires, les cancers secondaires peuvent aussi être induits par des basses doses dans des organes qui se trouvent hors des champs d'irradiation. Ces organes reçoivent des doses périphériques typiquement inférieures à 1 Gy qui résultent du diffusé du patient et du diffusé de l'accélérateur. Ces doses sont difficiles à calculer précisément, mais les algorithmes Monte Carlo (MC) permettent de les estimer avec une bonne précision. Un modèle MC détaillé de l'accélérateur Primus de Siemens a été élaboré et validé avec des mesures. La précision de ce modèle a également été déterminée pour la reconstruction de dose en épidémiologie. Si on considère que les patients inclus dans de larges cohortes sont traités sur une variété de machines, l'incertitude dans la reconstruction de dose périphérique a été étudiée en fonction de la variabilité de la dose périphérique pour différents types d'accélérateurs. Pour de grands champs (> 10x10 cm ), l'incertitude est inférieure à 50%, mais pour de petits champs et des champs filtrés, l'incertitude de la dose peut monter jusqu'à un facteur 10. En conclusion, un tel modèle ne peut être utilisé que pour les traitements conventionnels utilisant des grands champs.Le modèle MC de l'accélérateur Primus a été utilisé ensuite pour déterminer la dose périphérique pour différentes techniques dans un fantôme corps entier basé sur des coupes CT d'une patiente. Les techniques actuelles utilisant des champs filtrés ou encore l'IMRT hybride ont été étudiées et comparées par rapport aux techniques plus anciennes. Les doses calculées par MC ont été comparées à celles obtenues d'un logiciel de planification commercial (TPS). Alors que le TPS est utilisé en routine pour déterminer la dose au sein contralatéral et au poumon ipsilatéral qui sont principalement hors des faisceaux, nous avons montré que ces doses peuvent être plus ou moins précises selon la technTque étudiée. Les calculs MC montrent que la technique IMRT est dosimétriquement équivalente à celle basée sur des champs filtrés à l'intérieur des champs de traitement, mais offre une réduction importante de la dose aux organes périphériques.Finalement différents modèles de risque ont été étudiés sur la base des distributions de dose calculées par MC. Les risques absolus et le rapport des risques entre deux techniques de traitement varient grandement, ce qui reflète les grandes incertitudes liées aux différents modèles de risque. Malgré ces incertitudes, on a pu montrer que la technique IMRT offrait une réduction du risque systématique par rapport aux autres techniques. En attendant des données épidémiologiques supplémentaires sur la relation dose-risque, toute technique offrant une réduction des doses périphériques aux organes sains mérite d'être étudiée. La radiothérapie avec des électrons offre à ce titre des possibilités intéressantes.

CD4+CD25-mTGFbeta+ T cells induced by nasal application of ovalbumin transfer tolerance in a therapeutic model of asthma.

Background: Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. Objectives: To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. Methods: After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in the induction of tolerance was analysed. Results: Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T(h)1 and T(h)2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4(+)CD25(-) T cells, which strongly expressed membrane-bound transforming growth factor beta (mTGF beta), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4(+)CD25(+)(Foxp3(+)) T cells during the process of tolerization was indispensable to CD4(+)CD25(-) T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4(+)CD25(-)mTGF beta(+) T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Conclusion: Nasal application of OVA in established asthma led to the induction of CD4(+)CD25(-)mTGF beta(+) T cells with regulatory properties, able to confer protection upon allergen re-exposure.

Oxidative stress and inflammation response after nanoparticle exposure : differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-a and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-a concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure. [Authors]

Effects of a dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240, on endocrine and renal functions in healthy volunteers.

OBJECTIVE: To investigate the endocrine and renal effects of the dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240. DESIGN: A randomized, placebo-controlled, crossover study was performed in 12 healthy volunteers. METHODS: MDL 100,240 was administered intravenously over 20 min at single doses of 6.25 and 25 mg in subjects with a sodium intake of 280 (n = 6) or 80 (n = 6) mmol/day. Measurements were taken of supine and standing blood pressure, plasma angiotensin converting enzyme activity, angiotensin II, atrial natriuretic peptide, urinary atrial natriuretic peptide and cyclic GMP excretion, effective renal plasma flow and the glomerular filtration rate as p-aminohippurate and inulin clearances, electrolytes and segmental tubular function by endogenous lithium clearance. RESULTS: Supine systolic blood pressure was consistently decreased by MDL 100,240, particularly after the high dose and during the low-salt intake. Diastolic blood pressure and heart rate did not change. Plasma angiotensin converting enzyme activity decreased rapidly and dose-dependently. In both the high- and the low-salt treatment groups, plasma angiotensin II levels fell and renin activity rose accordingly, while plasma atrial natriuretic peptide levels remained unchanged. In contrast, urinary atrial natriuretic peptide excretion increased dose-dependently under both diets, as did urinary cyclic GMP excretion. Effective renal plasma flow and the glomerular filtration rate did not change. The urinary flow rate increased markedly during the first 2 h following administration of either dose of MDL 100,240 (P < 0.001) and, similarly, sodium excretion tended to increase from 0 to 4 h after the dose (P = 0.07). Potassium excretion remained stable. Proximal and distal fractional sodium reabsorption were not significantly altered by the treatment. Uric acid excretion was increased. The safety and clinical tolerance of MDL 100,240 were good. CONCLUSIONS: The increased fall in blood pressure in normal volunteers together with the preservation of renal hemodynamics and the increased urinary volume, atrial natriuretic peptide and cyclic GMP excretion distinguish MDL 100,240 as a double-enzyme inhibitor from inhibitors of the angiotensin converting enzyme alone. The differences appear to be due, at least in part, to increased renal exposure to atrial natriuretic peptide following neutral endopeptidase blockade.

Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.Gene Therapy advance online publication, 27 June 2013; doi:10.1038/gt.2013.36.

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

Role of fibroblast growth factor (FGF) signaling in the neuroendocrine control of human reproduction.

Fibroblast growth factor (FGF) signaling is critical for a broad range of developmental processes. In 2003, Fibroblast growth factor receptor 1 (FGFR1) was discovered as a novel locus causing both forms of isolate GnRH Deficiency, Kallmann syndrome [KS with anosmia] and normosmic idiopathic hypogonadotropic hypogonadism [nIHH] eventually accounting for approximately 10% of gonadotropin-releasing hormone (GnRH) deficiency cases. Such cases are characterized by a broad spectrum of reproductive phenotypes from severe congenital forms of GnRH deficiency to reversal of HH. Additionally, the variable expressivity of both reproductive and non-reproductive phenotypes among patients and family members harboring the identical FGFR1 mutations has pointed to a more complex, oligogenic model for GnRH deficiency. Further, reversal of HH in patients carrying FGFR1 mutations suggests potential gene-environment interactions in human GnRH deficiency disorders.

The mycobacterial cord factor adjuvant analogue trehalose-6,6'-dibehenate (TDB) activates the Nlrp3 inflammasome.

The success of a vaccine consists in the induction of an innate immune response and subsequent activation of the adaptive immune system. Because antigens are usually not immunogenic, the addition of adjuvants that activate innate immunity is required. The mycobacterial cord factor trehalose-6,6'-dimycolate (TDM) and its synthetic adjuvant analogue trehalose-6,6'-dibehenate (TDB) rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger innate immunity. In this study, we show that stimulation of bone marrow-derived dendritic cells (BMDCs) with TDB induces Nlrp3 inflammasome-dependent IL-1β secretion. While Card9 is required for NF-κB activation by TDB, it is dispensable for TDB-induced activation of the Nlrp3 inflammasome. Additionally, efflux of intracellular potassium, lysosomal rupture, and oxygen radical (ROS) production are crucial for caspase-1 processing and IL-1β secretion by TDB. In an in vivo inflammation model, we demonstrate that the recruitment of neutrophils by TDB is significantly reduced in the Nlrp3-deficient mice compared to the wild-type mice, while the production of chemokines in vitro is not influenced by the absence of Nlrp3. These results identify the Nlrp3 inflammasome as an essential mediator for the induction of an innate immune response triggered by TDB.

Efavirenz in an obese HIV-infected patient - a report and an in vitro-in vivo extrapolation model indicate risk of underdosing.

Here, we report suboptimal efavirenz exposure in an obese patient treated with the standard 600 mg dose. Tripling the dose allowed attainment of therapeutic efavirenz concentrations. We developed an in vitro-in vivo extrapolation model to quantify dose requirements in obese individuals. Obesity represents a risk factor for antiretroviral therapy underdosing.

Implication of DNA methylation and PAX5 factor in the transcriptional regulation of hTERT, the human telomerase reverse transcriptase gene

RESUME La télomérase est une enzyme dite "d'immortalité" qui permet aux cellules de maintenir la longueur de leurs télomères, ce qui confère une capacité de réplication illimitée aux cellules reproductrices et cancéreuses. A l'inverse, les cellules somatiques normales, qui n'expriment pas la télomérase, ont une capacité de réplication limitée. La sous-unité catalytique de la télomérase, hTERT, est définie comme le facteur limitant l'activité télomérasique. Entre activateurs et répresseurs, le rôle de la méthylation de l'ADN et de l'acétylation des histones, de nombreux modèles ont été suggérés. La découverte de l'implication de CTCF dans la régulation transcriptionnelle de hTERT explique en partie le mécanisme de répression de la télomérase dans la plupart des cellules somatiques et sa réactivation dans les cellules tumorales. Dans les cellules télomérase-positives, l'activité inhibitrice de CTCF est bloquée par un mécanisme dépendent ou non de la méthylation. Dans la plupart des carcinomes, une hyperméthylation de la région 5' de hTERT bloque l'effet inhibiteur de CTCF, alors qu'une petite région hypométhylée permet un faible niveau de transcription du gène. Nous avons démontré que la protéine MBD2 se lie spécifiquement sur la région 5' méthylée de hTERT dans différentes lignées cellulaires et qu'elle est impliquée dans la répression partielle de la transcription de hTERT dans les cellules tumorales méthylées. Par contre, nous avons montré que dans les lymphocytes B normaux et néoplasiques, la régulation de hTERT est indépendante de la méthylation. Dans ces cellules, le facteur PAX5 se lie sur la région 5' de hTERT en aval du site d'initiation de la traduction (ATG). L'expression exogène de PAX5 dans les cellules télomérase-négatives active la transcription de hTERT, alors que la répression de PAX5 dans les cellules lymphomateuses inhibe la transcription du gène. PAX5 est donc directement impliqué dans l'activation de l'expression de hTERT dans les lymphocytes B exprimant la télomérase. Ces résultats révèlent des différences entre les niveaux de méthylation de hTERT dans les cellules de carcinomes et les lymphocytes B exprimant la télomérase. La méthylation de hTERT en tant que biomarqueur de cancer a été évaluée, puis appliquée à la détection de métastases. Nous avons ainsi montré que la méthylation de hTERT est positivement corrélée au diagnostic cytologique dans les liquides céphalorachidiens. Nos résultats conduisent à un modèle de régulation de hTERT, qui aide à comprendre comment la transcription de ce gène est régulée par CTCF, avec un mécanisme lié ou non à la méthylation du gène hTERT. La méthylation de hTERT s'est aussi révélée être un nouveau et prometteur biomarqueur de cancer. SUMMARY Human telomerase is an "immortalizing" enzyme that enables cells to maintain telomere length, allowing unlimited replicative capacity to reproductive and cancer cells. Conversely, normal somatic cells that do not express telomerase have a finite replicative capacity. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors, and the role of DNA methylation and histone acetylation, an abundance of hTERT regulatory models have been suggested. The discovery of the implication of CTCF in the transcriptional regulation of hTERT in part explained the mechanism of silencing of telomerase in most somatic cells and its reactivation in neoplastic cells. In telomerase-positive cells, the inhibitory activity of CTCF is blocked by methylation-dependent and -independent mechanisms. In most carcinoma cells, hypermethylation of the hTERT 5' region has been shown to block the inhibitory effect of CTCF, while a short hypomethylated region allows a low transcription level of the gene. We have demonstrated that MBD2 protein specifically binds the methylated 5' region of hTERT in different cell lines and is therefore involved in the partial repression of hTERT transcription in methylated tumor cells. In contrast, we have shown that in normal and neoplastic B cells, hTERT regulation is methylation-independent. The PAX5 factor has been shown to bind to the hTERT 5'region downstream of the ATG translational start site. Ectopic expression of PAX5 in telomerase-negative cells or repression of PAX5 expression in B lymphoma cells respectively activated and repressed hTERT transcription. Thus, PAX5 is strongly implicated in hTERT expression activation in telomerase-positive B cells. These results reveal differences between the hTERT methylation patterns in telomerase-positive carcinoma cells and telomerase-positive normal B cells. The potential of hTERT methylation as a cancer biomarker was evaluated and applied to the detection of metastasis. We have shown that hTERT methylation correlates with the cytological diagnosis in cerebrospinal fluids. Our results suggest a model of hTERT gene regulation, which helps us to better understand how hTERT transcription is regulated by CTCF in methylation-dependant and independent mechanisms. Our data also indicate that hTERT methylation is a promising new cancer biomarker.

Inducible gene expression in fetal thymic epithelium: A new BAC transgenic model.

The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium. genesis 51:717-724. © 2013 Wiley Periodicals, Inc.