Intralipid® may represent a new hope for patients with reproductive failures and simultaneously an over-immune endometrial activation.

PROBLEM: Continuous failures to achieve a pregnancy despite effective embryo transfers is extremely distressing for couples. In consequence, many adjuvant therapies to IVF have been proposed to achieve an "ideal" immune environment. We here focus on Intralipid® therapy (IL) reported to have immunosuppressive properties on NK cells. METHOD OF STUDY: 94 patients exhibited an immune profile of endometrial over-immune activation and an history of repeated implantation failures despite multiple embryos transfers (RIF). They received a slow perfusion of Intralipid®. We here report the live birth rate following the procedure at the next embryo transfer. To get new insight on its mechanism of action, a second immune profiling had been performed under Intralipid® before the embryo transfer. RESULTS: The live birth rate of the RIF cohort treated with Intralipid® reached 54% (51/94) at the next embryo transfer. In patients successfully pregnant under Intralipid® who benefitted of a test of sensibility before the embryo transfer, we observed a significant decrease of the three biomarkers used to diagnose the over-immune endometrial activation (CD56 cells; IL-18/TWEAK, IL-14/FN-14). CONCLUSIONS: Double blind placebo versus Intralipid® studies should be conducted. Intralipid® may be an option to explore in RIF patients who exhibit an over-immune activation of uNK cells.

Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?

INTRODUCTION: Corticotherapy is the leading medication worldwide for patients with history of repeated implantation failures (RIF) after IVF/ICSI. Nevertheless, we still do not know its local mechanism of action, hence its precise indication. Our objective is to document the impact of prednisone on the endometrial expression of immune biomarkers (CD56 cells count, IL-18/TWEAK, IL-15/Fn-14 mRNA ratio) at the time of uterine receptivity in a RIF population. MATERIALS AND METHOD: An endometrial biopsy was realized in the mid luteal phase for immune profiling: IL-15/Fn-14 and IL-18/TWEAK mRNA ratios were determined by quantitative RT-PCR and CD56 mobilization per IHC. Fifty-five patients with a RIF history were diagnosed to have local over-immune activation [high IL-18/TWEAK mRNA ratio, and/or high IL-15/Fn-14 mRNA ratio] likely to impair the implantation process. They underwent a second immune profiling with supplementation of prednisone. A paired comparison of the immune profile before and under prednisone was performed in the subset of patients subsequently pregnant under prednisone. FINDING: In 54.5% of the cases, both immune biomarkers were normalized and in 16.5%, only one was normalized under prednisone. In 29% we observed a paradoxical increase of both immune biomarkers. The IL-18/TWEAK mRNA ratio reflecting the Th-1/Th-2 local equilibrium was significantly reduced (0.29 versus 0.10, p = .004), through very significant increase of TWEAK expression, in patients who were subsequently pregnant under prednisone. CONCLUSION: Testing the response to prednisone in a RIF context may be very useful. Less than half of RIF patients with immune deregulation may be prednisone responders and would benefit from its administration.

Uterine immune profiling for increasing live birth rate: A one-to-one matched cohort study.

BACKGROUND: Embryo implantation remains the main limiting factor in IVF/ICSI program. Endometrial immune remodeling events begin before implantation and are a vital process for pregnancy, preparing future maternal immune tolerance and regulating the placentation process. METHODS: Between 2012 and 2014, 193 patients (analyzed group) enrolled in our IVF program benefitted of an endometrial immune profiling to determine if their uterus was immunologically ready to accept an embryo and, if not, the specific immune mechanisms involved. Subsequently, they had an effective embryo transfer (ET) with personalization of their treatments if an immune deregulation has been diagnosed. Each analyzed patient was paired to the closest patient included in the IVF program according to biological criteria (age, number of mature oocytes, stage and number of transferred embryo), which had no endometrial immune profiling (193 patients, non-analyzed group). FINDING: 78% of analyzed patients had a uterine immune dysregulation and therefore care personalization. Their corresponding live birth rate (LBR) was twice higher than observed in the matched control group with conventional cares (30.5% versus 16.6%, OR: 2.2 [1.27-3.83] p=0.004) with a simultaneous drastic reduction of miscarriages per initiated pregnancy (17.9% versus 43.2%, OR: 0.29 [0.12-0.71], p=0.005). 22% of analyzed patients had no dysregulation. They did not differ from their matched controls for LBR and miscarriages. CONCLUSION: Uterine immune profiling enables an integrated approach of infertility that includes endometrial immunity as a key factor in planning personalized IVF/ICSI treatments. Personalization of treatment according to the woman's uterine immune balance produced a very significantly higher LBR.

The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization.

LABELED PROBLEM: Embryo implantation remains the main limiting factor in assisted reproductive medicine (20% success rate). METHODS OF STUDY: An endometrial immune profiling was performed among 394 women with the previous history of repeated embryo implantation failures (RIF). The endometrial immune profile documented the ratio of IL-15/Fn-14 mRNA as a biomarker of uNK cell activation/maturation (together with the uNK cell count) and the IL-18/TWEAK mRNA ratio as a biomarker of both angiogenesis and the Th1/Th2 balance. According to their profile, we recommended personalized care to counteract the documented dysregulation and assessed its effects by the live birth rate (LBR) for the next embryo transfer. RESULTS: Endometrial immune profiles appeared to be dysregulated in 81.7% of the RIF patients compared to control. Overactivation was diagnosed in 56.6% and low activation in 25%. The LBR among these dysregulated/treated patients at the first subsequent embryo transfer was 39.8%. CONCLUSION: Endometrial immune profiling may improve our understanding of RIF and subsequent LBR if treated.

Prognostic Value of 25-hydroxyvitamin D3 Levels at Diagnosis and During Follow-up in Melanoma Patients.

BACKGROUND: A low 25-hydroxyvitamin D3 (25(OH)D3) serum concentration at melanoma diagnosis might be associated with worse survival. We prospectively studied the prognostic value of 25(OH)D3 at diagnosis and during follow-up. METHODS: MelanCohort is a cohort of invasive melanoma patients. Serum 25(OH)D3 was measured by mass spectrometry and standardized on month of blood drawn, age, sex, and body mass index (BMI). Role of 25(OH)D3 levels and risk of relapse was analyzed in a Cox proportional hazards model adjusting for age, sex, BMI, and American Joint Committee on Cancer (AJCC) stage. All statistical tests were two-sided. RESULTS: One thousand one hundred seventy-one patients were included. 25(OH)D3 levels at diagnosis (median = 49.0 nmol/L) were inversely correlated with prognostic factors such as AJCC stage (P < .001 Kruskal-Wallis), Breslow's thickness (P < .001 Spearman correlation), and ulceration (P < .001 Kruskal-Wallis), but not with risk of relapse. Changes in 25(OH)D3 levels during follow-up were associated with worse prognosis: With a third quartile Q3 of average change per year (-0.30 to 4.60 nmol/L/Y) used as reference, hazard ratios for the first, second, and fourth quarters were 1.94 (95% confidence interval [CI] = 1.36 to 2.76), 1.23 (95% CI = 0.85 to 1.78), and 1.61 (95% CI = 1.14 to 2.28), respectively. In sensitivity analyses, no changes were observed either by AJCC stage, number of 25(OH)D3 measures performed, or by 25(OH)D3 level at baseline. No evidence of reverse causation was identified. Analyses performed on overall survival yielded similar results. CONCLUSIONS: We show that 25(OH)D3 variation during follow-up is an independent melanoma prognostic marker, but not its level at diagnosis. Previously reported associations between low 25(OH)D3 level at diagnosis and poor prognosis seem to be due to insufficient adjustment for prognostic factors.

Multicentric evaluation of eight glucose and four ketone blood meters.

OBJECTIVES: High precision meters for blood glycemia are mandatory for monitoring glucose status in patients, avoiding both hypo- and hyper-glycemia. Health care providers routinely used in both out- and in-patients point-of-care measurements of glucose and ketone. These measurements, frequently used for medical decisions, are known to be less accurate than those performed in laboratories. Our aim was to evaluate, within the frame of an Assistance Publique-Hôpitaux de Paris (AP-HP) multicentric study, the performances of eight glucose and four ketone meters, either connected or non-connected to a laboratory software. DESIGN AND METHODS: Glucose meter accuracy, precision, correlation with plasma glucose determined in central laboratories and hematocrit interferences were determined according to the ISO 15197:2003 norm. The same norm was applied for the determination of accuracy, precision and recovery of ketone meters for B-hydroxybutyrate measurements. RESULTS AND CONCLUSION: Among those meters, seven were considered as acceptable for glucose measurement and two for ketone measurement. Since all meters do not fit clinically relevant criteria, meters' performances have to be evaluated before use in clinical practice.

Highly sensitive multivariable assay detection of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes with melanoma micrometastases.

OBJECTIVES: To evaluate the prognostic value of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes (SLNs) with melanoma micrometastases. DESIGN: Prognostic study of an inception cohort. SETTING: Academic research. Patients Between July 1, 1999, and July 31, 2002, all patients who had primary cutaneous or mucosal melanomas that have a Breslow depth of 1.5 mm or greater, ulceration, or Clark level IV or V, or had SLN biopsies. MAIN OUTCOME MEASURES: By the use of quantitative reverse transcription-polymerase chain reaction, the expression of the following was analyzed in SLNs: 2 melanocytic differentiation antigens (tyrosinase [P17646] and melanoma antigen recognized by T cells [MART-1; Q16655]) and genes involved in angiogenesis (VEGF [NM_001025366] and VEGFR2 [AF035121]), lymphangiogenesis (VEGFC [NM_005429], VEGFR3 [X68203], LYVE1 [NM_016164], and PROX1 [002763]), and invasion (uPA [NM_002658], PAI1 [NM_00602], and EMMPRIN [L10240]). Outcome measures were the association of these melanocytic differentiation antigens and angiogenesis biomarkers with clinicopathologic characteristics of patients, and an evaluation of the prognostic value for relapse-free survival and overall survival. RESULTS: Ninety-one patients were included, with a median follow-up period of 41 months. Micrometastases were present in 15% (14 of 91) of patients. Tyrosinase (P < .001), MART-1 (P < .001), vascular endothelial growth factor 121 (VEGF(121)) (P = .007), and PAI1 (P = .02) expression was significantly associated with micrometastasis. In univariate analysis, histologic findings and tyrosinase and MART-1 expression were significantly associated with relapse-free survival. Tyrosinase and MART-1 expression was associated with overall survival. A multiple Cox proportional hazards regression model identified negative histologic findings and tyrosinase expression that exceeded 27 copies/copy of TATA box-binding protein (third quartile) as significantly associated with an increased risk of relapse or death. CONCLUSIONS: Quantitative assessment of melanocytic differentiation antigens in SLNs, which has prognostic value, is more specific than qualitative assessment. Prognosis may be more effectively predicted by the combination of quantitative assessment of melanocytic differentiation antigens in SLNs with histologic assessment. A significant association was found between the presence of micrometastases and the expression of angiogenesis biomarkers.

A coordinated phosphorylation cascade initiated by p38MAPK/MSK1 directs RARalpha to target promoters.

The nuclear retinoic acid (RA) receptor alpha (RARalpha) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARalpha target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARalpha at S369 located in the ligand-binding domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 controls the recruitment of RARalpha/TFIIH complexes to response elements and subsequently RARalpha target gene activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signalling.

Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation.

Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

Effect of low dose antioxidant vitamin and trace element supplementation on the urinary concentrations of thromboxane and prostacyclin metabolites.

OBJECTIVE: This trial evaluated the effect of antioxidant supplementation on the urinary excretion of 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio, a marker of the pathogenesis of thrombosis and arteriosclerosis. METHODS: This study was a randomised, double-blind, placebo-controlled trial involving 186 presumably healthy volunteers. One hundred received a multi-antioxidant supplementation and 86 a placebo for two years. Blood zinc, selenium, beta-carotene, vitamin C and E and urinary excretion of 11-dehydro TXB(2) and 2,3 dinor 6 keto PGF(1alpha) were measured. RESULTS: Baseline subject characteristics did not differ between the two groups. Blood zinc, selenium, and beta-carotene concentrations significantly increased between baseline and two years in the multi-antioxidant supplementation group supporting subject compliance (p < 0.05). At two years, the median urinary 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio was significantly lower in the multi-antioxidant supplementation group (3.4 versus 2.78, p = 0.015). Serum selenium concentration was the only antioxidant studied that was significantly related to the urinary 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio. CONCLUSIONS: These results support the hypothesis that a low-dose multi-antioxidant supplementation may contributes to a reduction in platelet activation which is beneficial for cardiovascular function.

Effects of high doses of selenium, as sodium selenite, in septic shock: a placebo-controlled, randomized, double-blind, phase II study.

INTRODUCTION: Sepsis is associated with the generation of oxygen free radicals and (lacking) decreased selenium plasma concentrations. High doses of sodium selenite might reduce inflammation by a direct pro-oxidative effect and may increase antioxidant cell capacities by selenium incorporation into selenoenzymes. We investigated the effects of a continuous administration of high doses of selenium in septic shock patients. METHODS: A prospective, multicentre, placebo-controlled, randomized, double-blind study was performed with an intention-to-treat analysis in severe septic shock patients with documented infection. Patients received, for 10 days, selenium as sodium selenite (4,000 microg on the first day, 1,000 microg/day on the nine following days) or matching placebo using continuous intravenous infusion. The primary endpoint was the time to vasopressor therapy withdrawal. The duration of mechanical ventilation, the mortality rates in the intensive care unit, at hospital discharge, and at 7, 14, 28 and 180 days and 1 year after randomization, and adverse events were recorded. RESULTS: Sixty patients were included (placebo, n = 29; selenium, n = 31). The median time to vasopressor therapy withdrawal was 7 days in both groups (95% confidence interval = 5-8 and 6-9 in the placebo and selenium groups, respectively; log-rank, P = 0.713). The median duration of mechanical ventilation was 14 days and 19 days in the placebo and selenium groups, respectively (P = 0.762). Mortality rates did not significantly differ between groups at any time point. Rates of adverse events were similar in the two groups. CONCLUSION: Continuous infusion of selenium as sodium selenite (4,000 microg on the first day, 1,000 microg/day on the nine following days) had no obvious toxicity but did not improve the clinical outcome in septic shock patients. Trial Registration = NCT00207844.

RXR is an essential component of the oncogenic PML/RARA complex in vivo.

Although PML-enforced RARA homodimerization allows PML/RARA to bind DNA independently of its coreceptor RXR, the latter was identified within the PML/RARA complex. We demonstrate that a PML/RARA mutant defective for RXR binding fails to trigger APL development in transgenic mice, although it still transforms primary hematopoietic progenitors ex vivo. RXR enhances PML/RARA binding to DNA and is required for rexinoid-induced APL differentiation. In RA-treated PML/RARA-transformed cells, the absence of RXR binding results in monocytic, rather than granulocytic, differentiation. PML/RARA enhances posttranslational modifications of RXRA, including its sumoylation, suggesting that PML-bound sumoylation enzymes target RXRA and possibly other PML/RARA-bound chromatin proteins, further contributing to deregulated transcription. Thus, unexpectedly, RXR contributes to several critical aspects of in vivo transformation.

Acute promyelocytic leukemia: new issues on pathogenesis and treatment response.

Pathogenesis of acute promyelocytic leukemia appears to be one of the best understood among human malignancies. The ability of retinoic acid (RA) and arsenic trioxide to directly target the oncogenic promyelocytic leukemia-retinoic receptor A (PML-RARA) fusion protein also made this disease the first model for oncogene-targeted therapies. A set of recent data has significantly increased the complexity of our view of acute promyelocytic leukemia pathogenesis, as well as of therapeutic response. This review summarizes and discusses these findings, which yield novels questions and models.

Rexinoid-triggered differentiation and tumor-selective apoptosis of acute myeloid leukemia by protein kinase A-mediated desubordination of retinoid X receptor.

Apart from PML-retinoic acid receptor-alpha (RARalpha) acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists ("rexinoids") the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cells and patients' blasts. Protein kinase A activation leads to corepressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in "desubordination" of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARbeta is blunted in mouse embryo fibroblasts lacking RARs, but reintroduction of exogenous RARalpha reestablishes responsiveness, thus confirming that the RARalpha-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, tumor necrosis factor-related apoptosis inducing ligand, both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of tumor necrosis factor-related apoptosis inducing ligand and DR5 in AML patient blasts cultured ex vivo. AML patients' blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and French-American-British classification status. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Our results suggest that despite the genetic, morphologic, and clinical variability of this disease, the combination of rexinoids and cAMP-elevating drugs, such as phosphodiesterase inhibitors, might lead to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

PML-RARA-RXR oligomers mediate retinoid and rexinoid/cAMP cross-talk in acute promyelocytic leukemia cell differentiation.

PML-RARA was proposed to initiate acute promyelocytic leukemia (APL) through PML-RARA homodimer-triggered repression. Here, we examined the nature of the PML-RARA protein complex and of its DNA targets in APL cells. Using a selection/amplification approach, we demonstrate that PML-RARA targets consist of two AGGTCA elements in an astonishing variety of orientations and spacings, pointing to highly relaxed structural constrains for DNA binding and identifying a major gain of function of this oncogene. PML-RARA-specific response elements were identified, which all conveyed a major transcriptional response to RA only in APL cells. In these cells, we demonstrate that PML-RARA oligomers are complexed to RXR. Directly probing PML-RARA function in APL cells, we found that the differentiation enhancer cyclic AMP (cAMP) boosted transcriptional activation by RA. cAMP also reversed the normal silencing (subordination) of the transactivating function of RXR when bound to RARA or PML-RARA, demonstrating that the alternate rexinoid/cAMP-triggered APL differentiation pathway also activates PML-RARA targets. Finally, cAMP restored both RA-triggered differentiation and PML-RARA transcriptional activation in mutant RA-resistant APL cells. Collectively, our findings directly demonstrate that APL cell differentiation parallels transcriptional activation through PML-RARA-RXR oligomers and that those are functionally targeted by cAMP, identifying this agent as another oncogene-targeted therapy.

In vivo activation of cAMP signaling induces growth arrest and differentiation in acute promyelocytic leukemia.

Differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells. The most relevant clinical example is acute promyelocytic leukemia (APL), which responds dramatically to either retinoic acid (RA) or arsenic trioxide (As(2)O(3)). In many myeloid leukemia cell lines, cyclic adenosine monophosphate (cAMP) triggers growth arrest, cell death, or differentiation, often in synergy with RA. Nevertheless, the toxicity of cAMP derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations. We show that, in an APL cell line, cAMP analogs blocked cell growth and unraveled As(2)O(3)-triggered differentiation. Similarly, in RA-sensitive or RA-resistant mouse models of APL, continuous infusions of 8-chloro-cyclic adenosine monophosphate (8-Cl-cAMP) triggered major growth arrest, greatly enhanced both spontaneous and RA- or As(2)O(3)-induced differentiation and accelerated the restoration of normal hematopoiesis. Theophylline, a well-tolerated phosphodiesterase inhibitor which stabilizes endogenous cAMP, also impaired APL growth and enhanced spontaneous or As(2)O(3)-triggered cell differentiation in vivo. Accordingly, in an APL patient resistant to combined RA-As(2)O(3) therapy, theophylline induced blast clearance and restored normal hematopoiesis. Taken together, these results demonstrate that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach.