Live imaging of megakaryocytes from cranial bone marrow using multiphoton microscopy

Platelet production is a tightly regulated process that occurs predominantly in the bone marrow. To study the biology of platelet precursor cells, megakaryocytes, we established a microscopy technique to visualize platelet production in live mice. This technique is now used to determine the role of Rap1, a small GTPase, in platelet production. Specifically, we monitor megakaryocyte proplatelet formation (PPF) in mutant mice for Rap1 and its regulator, Rasa3, in real time. With the videos collected, it is evident that the developed protocol was successful at capturing PPF in cranial BM of mice. The preliminary qualitative observations made from acquisition of these events in mutant mice help to support data made ex-vivo and better understand the role of Rap1 in platelet production. Compared to control, mice lacking both isoforms of Rap1, Rap1a and Rap1b, exhibited periods of proplatelet extension and retraction, suggesting a difficulty in proplatelet release from Mk cell body. Rasa3 mutant mice exhibited mutant Mk morphology, however, were still capable of PPF in BM sinusoids.Bachelor of Scienc

Cost-Effectiveness Of Treatments For Mild-To-Moderate Obstructive Sleep Apnea In France

International audienceObjectives: Untreated obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with excessive daytime sleepiness, increased risk of cardiovascular (CV) disease, and road traffic accidents (RTAs), which impact survival and health-related quality of life. This study, funded by the French National Authority for Health (HAS), aimed to assess the cost-effectiveness of different treatments (i.e., continuous positive airway pressure [CPAP], dental devices, lifestyle advice, and no treatment) in patients with mild-to-moderate OSAHS in France.Methods: A Markov model was developed to simulate the progression of two cohorts, stratified by CV risk, over a lifetime horizon. Daytime sleepiness and RTAs were taken into account for all patients while CV events were only considered for patients with high CV risk.Results: For patients with low CV risk, incremental cost-effectiveness ratio (ICER) of dental devices versus no treatment varied between 32,976 EUR (moderate OSAHS) and 45,579 EUR (mild OSAHS) per quality-adjusted life-year (QALY), and CPAP versus dental devices, above 256,000 EUR/QALY. For patients with high CV risk, CPAP was associated with a gain of 0.62 QALY compared with no treatment, resulting in an ICER of 10,128 EUR/QALY.Conclusion: The analysis suggests that it is efficient to treat all OSAHS patients with high CV risk with CPAP and that dental devices are more efficient than CPAP for mild-to-moderate OSAHS with low CV risk. However, out-of-pocket costs are currently much higher for dental devices than for CPAP (i.e., 3,326 EUR versus 2,430 EUR) as orthodontic treatment is mainly non-refundable in France

Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity

Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed

DSMM XI study: dose definition for intravenous cyclophosphamide in combination with bortezomib/dexamethasone for remission induction in patients with newly diagnosed myeloma

A clinical trial was initiated to evaluate the recommended dose of cyclophosphamide in combination with bortezomib and dexamethasone as induction treatment before stem cell transplantation for younger patients with newly diagnosed multiple myeloma (MM). Thirty patients were treated with three 21-day cycles of bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 plus dexamethasone 40 mg on the day of bortezomib injection and the day after plus cyclophosphamide at 900, 1,200, or 1,500 mg/m2 on day 1. The maximum tolerated dose of cyclophosphamide was defined as 900 mg/m2. At this dose level, 92% of patients achieved at least a partial response. The overall response rate [complete response (CR) plus partial response (PR)] across all dose levels was 77%, with a 10% CR rate. No patient experienced progressive disease. The most frequent adverse events were hematological and gastrointestinal toxicities as well as neuropathy. The results suggest that bortezomib in combination with cyclophosphamide at 900 mg/m2 and dexamethasone is an effective induction treatment for patients with newly diagnosed MM that warrants further investigation

Detection of t(11;14) using interphase molecular cytogenetics in mantle cell lymphoma and atypical chronic lymphocytic leukemia

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty-eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL-type immunophenotype, i.e., CD19+, CD5+, CD23(-/low), CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual-color interphase FISH. We selected an IGH-specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH-CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH- CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)- positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities

Old and new immunophenotypic markers in multiple myeloma for discrimination of responding and relapsing patients: The importance of "normal" residual plasma cell analysis

Background Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (CPCs), which can be immunophenotypically distinguished from polyclonal plasma cells (PPCs) by multiparameter flow cytometry (MFC). The utility of PPCs analysis in detecting prognostic and predictive information is still a matter of debate. Methods: we tested the ability of 11 MFC markers in detecting differences in the immunophenotype of CPCs and PPCs among patients in various disease stages; we verified if these markers could be associated with disease stage/response to therapy despite the role of clinical parameters. Results: significant changes in the expression of markers occurred both in CPCs and PPCs. CD58 on PPCs of responding patients was downregulated compared with PPC of relapsing group. Fraction of CD200 expressing PCs was lower in control subjects than in PPCs from MGUS and myeloma groups. CD11a levels of expression on both CPCs and PPCs showed an upregulation in newly diagnosed and relapsing patients versus PCs of controls; CD20 was less expressed on control PCs than on MGUS CPCs and PPCs. CD49d revealed to be advantageous in discrimination of PPCs from CPCs. In our multiple regression model, CD19 and CD49d on CPCs, and CD45, CD58 and CD56 on PPCs maintained their association with groups of patients independently of other prognostic variables. Conclusions: we provide a feasible start point to put in order ranges of expression on PPCs in healthy and myeloma subjects; we propose a new approach based on PPC analysis to monitor the stages of the disease

Post-Transplant Outcomes in High-Risk Compared with Non-High-Risk Multiple Myeloma: A CIBMTR Analysis.

Conventional cytogenetics and interphase fluorescence in situ hybridization (FISH) identify high-risk multiple myeloma (HRM) populations characterized by poor outcomes. We analyzed these differences among HRM versus non-HRM populations after upfront autologous hematopoietic cell transplantation (autoHCT). Between 2008 and 2012, 715 patients with multiple myeloma identified by FISH and/or cytogenetic data with upfront autoHCT were identified in the Center for International Blood and Marrow Transplant Research database. HRM was defined as del17p, t(4;14), t(14;16), hypodiploidy (-Y) or chromosome 1 p and 1q abnormalities; all others were non-HRM. Among 125 HRM patients (17.5%), induction with bortezomib and immunomodulatory agents (imids) was higher compared with non-HRM (56% versus 43%, P \u3c .001) with similar pretransplant complete response (CR) rates (14% versus 16%, P .1). At day 100 post-transplant, at least a very good partial response was 59% in HRM and 61% in non-HRM (P = .6). More HRM patients received post-transplant therapy with bortezomib and imids (26% versus 12%, P = .004). Three-year post-transplant progression-free (PFS) and overall survival (OS) rates in HRM versus non-HRM were 37% versus 49% (P \u3c .001) and 72% versus 85% (P \u3c .001), respectively. At 3 years, PFS for HRM patients with and without post-transplant therapy was 46% (95% confidence interval [CI], 33 to 59) versus 14% (95% CI, 4 to 29) and in non-HRM patients with and without post-transplant therapy 55% (95% CI, 49 to 62) versus 39% (95% CI, 32 to 47); rates of OS for HRM patients with and without post-transplant therapy were 81% (95% CI, 70 to 90) versus 48% (95% CI, 30 to 65) compared with 88% (95% CI, 84 to 92) and 79% (95% CI, 73 to 85) in non-HRM patients with and without post-transplant therapy, respectively. Among patients receiving post-transplant therapy, there was no difference in OS between HRM and non-HRM (P = .08). In addition to HRM, higher stage, less than a CR pretransplant, lack of post-transplant therapy, and African American race were associated with worse OS. In conclusion, we show HRM patients achieve similar day 100 post-transplant responses compared with non-HRM patients, but these responses are not sustained. Post-transplant therapy appeared to improve the poor outcomes of HRM