IKKalpha at the crossroads of inflammation and metastasis

The development of solid tumors is regulated by dynamic interactions between evolving neoplastic cells and their microenvironment. Luo et al. (2007) recently demonstrated that tumor-infiltrating immune cells expressing RANKL induce activation and nuclear localization of IKKalpha in prostatic epithelial tumor cells. This leads to repression of maspin, a critical suppressor of metastasis, and thus commits malignant prostatic epithelial cells to a metastatic fate.     

Fanconi anemia: contribution of molecular analyses to the identification of bone marrow graft donors and the study of chimerism in grafted patients

We report on the effectiveness of molecular studies regarding Fanconi anemia (FA) for a better selection of bone marrow graft donors and for post-transplant follow up. Ten unrelated FA patients and their families were analyzed by microsatellite markers. In 9 cases, the cytogenetic investigation of potential human leukocyte antigen (HLA)-identical related donors was normal, and the molecular analyses confirmed that they were also either normal or heterozygous carriers. For 1 patient, cytogenetic analysis of an HLA-identical sibling donor yielded ambiguous results with a relatively high number of chromosomal breakages using cross-linking agents. However, genotyping of this potential donor demonstrated his heterozygous state. Nine patients have received allogeneic bone marrow transplantation from HLA-matched related donors. Microsatellite analysis showed complete chimerism (CC) in all cases. The median follow up was 54 months (range 8-144 months). One patient out of 9 with CC rejected her graft without prior detection of a transitional mixed chimerism. Among these patients, 1 died 25 months after the transplantation of a chronic graft-versus-host-disease (GVHD). We conclude that, when the cytogenetic studies are not conclusive, molecular analyses are crucial to distinguish heterozygous carriers from asymptomatic FA Tunisian patients. Molecular analyses also allowed the evaluation of hematopoietic chimerism after allogeneic bone marrow transplantation and might be of value to identify patients with a high risk for graft rejection.     

Ambulatory measurement of 3D knee joint angle

Three-dimensional measurement of joint motion is a promising tool for clinical evaluation and therapeutic treatment comparisons. Although many devices exist for joints kinematics assessment, there is a need for a system that could be used in routine practice. Such a system should be accurate, ambulatory, and easy to use. The combination of gyroscopes and accelerometers (i.e., inertial measurement unit) has proven to be suitable for unrestrained measurement of orientation during a short period of time (i.e., few minutes). However, due to their inability to detect horizontal reference, inertial-based systems generally fail to measure differential orientation, a prerequisite for computing the three-dimentional knee joint angle recommended by the Internal Society of Biomechanics (ISB). A simple method based on a leg movement is proposed here to align two inertial measurement units fixed on the thigh and shank segments. Based on the combination of the former alignment and a fusion algorithm, the three-dimensional knee joint angle is measured and compared with a magnetic motion capture system during walking. The proposed system is suitable to measure the absolute knee flexion/extension and abduction/adduction angles with mean (SD) offset errors of -1 degree (1 degree ) and 0 degrees (0.6 degrees ) and mean (SD) root mean square (RMS) errors of 1.5 degrees (0.4 degrees ) and 1.7 degrees (0.5 degrees ). The system is also suitable for the relative measurement of knee internal/external rotation (mean (SD) offset error of 3.4 degrees (2.7 degrees )) with a mean (SD) RMS error of 1.6 degrees (0.5 degrees ). The method described in this paper can be easily adapted in order to measure other joint angular displacements such as elbow or ankle.     

Differentiation of Fanconi anemia and aplastic anemia using mitomycin C test in Tunisia

Fanconi anemia (FA) is a recessive chromosomal instability syndrome that is clinically characterized by multiple symptoms. Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for FA diagnosis. In this study, we provide a detailed laboratory protocol for accurate assessment of FA diagnosis based on mitomycin C (MMC) test. Induced chromosomal breakage study was successful in 171 out of 205 aplastic anemia (AA) patients. According to the sensitivity of MMC at 50 ng/ml, 38 patients (22.22%) were diagnosed as affected and 132 patients (77.17%) as unaffected. Somatic mosaicism was suspected in an 11-year-old patient with a FA phenotype. Twenty-six siblings of FA patients were also evaluated and five of them (19.23%) were diagnosed as FA. From this study, a standard protocol for diagnosis of FA was developed. It is routinely used as a diagnostic test of FA in Tunisia.     

Interplay of Oct4 with Sox2 and Sox17: a molecular switch from stem cell pluripotency to specifying a cardiac fate

Oct4 exerts a dose-dependent dual action, as both a gatekeeper for stem cell pluripotency and in driving cells toward specific lineages. Here, we identify the molecular mechanism underlying this dual function. BMP2- or transgene-induced Oct4 up-regulation drives human embryonic and induced pluripotent stem cells to become cardiac progenitors. When embryonic stem cell pluripotency is achieved, Oct4 switches from the Sox2 to the Sox17 promoter. This switch allows the cells to turn off the pluripotency Oct4-Sox2 loop and to turn on the Sox17 promoter. This powerful process generates a subset of endoderm-expressing Sox17 and Hex, both regulators of paracrine signals for cardiogenesis (i.e., Wnt, BMP2) released into the medium surrounding colonies of embryonic stem cells. Our data thus reveal a novel molecular Oct4- and Sox17-mediated mechanism that disrupts the stem cell microenvironment favoring pluripotency to provide a novel paracrine endodermal environment in which cell lineage is determined and commits the cells to a cardiogenic fate.     

Hyaluronan Disrupts Cardiomyocyte Organization within 3D Fibrin-Based Hydrogels

The extracellular matrix in vivo contains variable but often large amounts of glycosaminoglycans that influence cell and tissue function. Hyaluronan (HA) is an abundant glycosaminoglycan within the extracellular matrix of the myocardium during early development and in the aftermath of a myocardial infarction. Its flexible anionic structure has a strong influence on mechanical response and interstitial fluid flow within the matrix. Additionally, HA has a direct, biochemical effect on cells through an array of cell-surface receptors, including CD44, RHAMM/CD168, and other surface-exposed structures. Recent studies have shown that HA modulates the response of cardiomyocytes and other cell types to two-dimensional substrates of varying elastic moduli. This study investigates the force response to HA of cardiomyocytes and cardiac fibroblasts within three-dimensional matrices of variable composition and mechanical properties in vitro. HA significantly decreased the force exerted by the cell-matrix constructs in a tensiometer testing platform and within microfabricated tissue gauges. However, its effect was no different from that of alginate, an anionic polysaccharide with the same charge density but no specific transmembrane receptors. Therefore, these results establish that HA exerts a generic physical-chemical effect within three-dimensional hydrogels that must be accounted for when interrogating cell-matrix interactions.     

Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (-36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA+polyunsaturated FA):(saturated FA+TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Immunophenotyping of hematopoietic progenitor cells: Comparison between cord blood and adult mobilized blood grafts

AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.