Survival of Patients with Cystic Fibrosis Depending on Mutation Type and Nutritional Status.

The purpose of the study was to evaluate the influence of nutrition and of the severity of mutation type on survival rate in cystic fibrosis (CF) patients. Data were longitudinally collected from 60 hospitalized adult CF patients, aged 18-50. The variables consisted of body mass index (BMI) ratio, Cole's BMI cut-off points, severity of mutation type, and survival rate of CF patients. We found that the mean BMI was strongly associated with the severity of mutation type and was significantly lower in patients with severe mutations of grade I and II. The mutation type significantly affected the patients' survival rate; survival was greater in patients with mild and undefined mutation types. The BMI and Cole's cut-off points also had a significant influence on survival rate. CF patients, who suffered from malnutrition and emaciation, had a shorter survival rate than those with proper nutritional status. In conclusion, the study findings confirmed a significant effect of nutritional status and of mutation type on survival rate of CF patients.

Characterization of Human CD8(+)TCR(-) Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγ(null) Mouse Model.

CD8(+)/TCR(-) facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8(+)/TCR(-)/CD56 negative (CD56(neg)); the remainder are CD8(+)/TCR(-)/CD56 bright (CD56(bright)). The CD56(neg) FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL-2 receptor γ-chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56(neg) FC subpopulation promotes rapid reconstitution of donor HSPCs without graft-versus-host disease (GVHD); recipients of CD56(bright) FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56(neg) or CD56(bright) FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56(bright) FCs with HSPCs upregulates cathelicidin and ß-defensin 2, factors that prime responsiveness of HSPCs to stromal cell-derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

Influence of Body Shape Composition on Respiratory Function in Adult Women.

The purpose of the study was to evaluate the influence of body size and shape, and of fat distribution on respiratory functions in adult women. The sample consisted of 107 women aged 17-82 years. Height, weight, chest, waist and hip circumferences, abdominal, and subscapular and triceps skinfolds were examined. The BMI and WHR were calculated. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), peak expiratory flow (PEF), maximal expiratory flow (MEF75), forced expiratory time (FET) were used as measures of respiratory function. Positive correlations were found between z-scores of height and VC, FEV1, FVC, between WHR and VC, and between circumference and FET. Negative correlations were found between z-scores of BMI and VC, FEV1, between subscapular skinfold and VC, FEV1, FVC and between abdominal skinfold and VC. Stepwise multiple regression analysis showed that traits of body size and shape mostly influenced VC (32 %) and FVC (31 %). Body height and WHR significantly affected VC, while height and subscapular skinfold affected FVC. A significant association between fat distribution described by BMI, WHR, and skinfold thickness and respiratory parameters was observed. These results confirm a complex effect of body size and shape, and of fat distribution on respiratory function.

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Dependence of cathodoluminescence on layer resistance applied for measurement of thin-layer sheet resistance.

The dependence of spatially and spectrally resolved cathodoluminescence in a scanning electron microscope on resistances in semiconductor structures, especially on the layer resistance, is reported. This previously unstudied dependence is utilized for thin-layer sheet-resistance measurement. The method is illustrated by an assessment of lateral confinements in semiconductor-laser heterostructures.

Depth measurements of etch-pits in GaN with shape reconstruction from SEM images.

The method, which allows shape reconstruction by reading the intensity from the scanning electron microscopy image, is presented and discussed in details. The method is applied to read the morphology of etch-pits, which were formed on the GaN surface by etching in molten KOH-NaOH eutectic mixture to delineate dislocations. The etch-pit depth distributions are obtained and used to determine densities of pits related to screw, mixed or edge-type dislocations. The results are compared with atomic force microscopy.

Identification of electron beam vibration sources by separation of magnetic distortion from electric distortion on scanning electron microscope imaging.

Different types of distortions in scanning electron microscopy require different methods of their elimination, and therefore influence of these types on particular elements of the SEM system should be known. The proposed method allows for separation of the direct influence of the magnetic field on the electron beam in the SEM chamber from its influence in the SEM column and from the distortions generated in the SEM scanning block. For this purpose, a series of distorted images is registered for several working distances (between the final aperture of the electron column and the specimen) and for several energies of the electrons. Magnitudes of the distortions are measured on these images. For each applied electron energy, the dependence of the results versus the working distance is approximated with the second-order polynomial function. The analysis of the polynomial coefficients allows for the separation of the above-mentioned kinds of distortions. The presented method enables a selection of the most efficient solution to the distortions reduction. It utilizes the SEM itself and does not need any additional equipment.

Transmission electron microscopy characterization of Au/Pt/Ti/Pt/GaAs ohmic contacts for high power GaAs/InGaAs semiconductor lasers.

We report on transmission electron microscopy studies of Au/Pt/Ti/Pt(10-30 nm) contact structures for high power GaAs/InGaAs semiconductor lasers. The studies showed that annealing at 450 degrees C of contact structures causes the reaction of whole Pt with substrate components (Ga and As) and the formation of Pt-GaAs interlayers with smooth interfaces as required for such structures. Annealing of the structures at 470 and 490 degrees C unfavourably affects the contact structure. At this condition, the strong downward diffusion of Au and Pt from the top layers causes a formation of Au-Pt pits, which break the Ti barrier. Transmission electron microscopy observation revealed that Au/Pt/Ti/Pt(10-30 nm) system annealed at 450 degrees C is appropriate for practical applications. The EDS technique used to identify the phase composition in the Pt(30 nm)/GaAs structure (specially produced for the EDS analysis) annealed at 450 degrees C showed that two layers were formed as a result of the reaction of the whole Pt layer with GaAs, and they consist of Ga, Pt and As. The top layer has the highest concentration of Ga. However, the bottom layer, which is close to the substrate, has the highest concentration of As.

Transmission electron microscopy study of erbium silicide formation from Ti/Er stack for Schottky contact applications.

In this paper, we present results of transmission electron microscopy studies on erbium silicide structures fabricated under various thermal conditions. A titanium cap has been used as a protective layer against oxidation during rapid thermal annealing of an erbium layer in a temperature range of 300-700 degrees C. Both layers (200 nm Ti and 25 nm Er) were deposited by electron-beam sputtering. The investigations have shown that the transformation of the 25-nm-thick erbium into erbium silicide is completed after annealing at 500 degrees C. At higher temperatures, the formation of a titanium silicide layer above erbium silicide is observed. The lowest Schottky barrier has been measured in the sample annealed at 700 degrees C.

Very small embryonic-like stem cells in adult tissues-potential implications for aging.

Recently our group identified in murine bone marrow (BM) and human cord blood (CB), a rare population of very small embryonic-like (VSEL) stem cells. We hypothesize that these cells are deposited during embryonic development in BM as a mobile pool of circulating pluripotent stem cells (PSC) that play a pivotal role in postnatal tissue turnover both of non-hematopoietic and hematopoietic tissues. During in vitro co-cultures with murine myoblastic C2C12 cells, VSELs form spheres that contain primitive stem cells. Cells isolated from these spheres may give rise to cells from all three germ layers when plated in tissue specific media. The number of murine VSELs and their ability to form spheres decreases with the age and is reduced in short-living murine strains. Thus, developmental deposition of VSELs in adult tissues may potentially play an underappreciated role in regulating the rejuvenation of senescent organs. We envision that the regenerative potential of these cells could be harnessed to decelerate aging processes.

New approach to cathodoluminescence studies in application to InGaN/GaN laser diode degradation.

Cathodoluminescence (CL) studies are widely applied in semi-conductor science and technology. However, for structures with a p-n junction the CL spatial distribution can be strongly affected by internal current flows of the electron beam induced current generated within the structure. This influence is the investigated in application to CL studies of degradation in aged laser diodes with InGaN multiquantum wells.

Selective etching of dislocations in GaN and quantitative SEM analysis with shape-reconstruction method.

Due to the differences in etch-pit morphologies, chemical etching offers a possibility to determine densities of dislocations in respect to their type. In the present paper we propose a method, which implements a simple shape-from-shading procedure, i.e. with results derived from image brightness dependence on surface slope. It allows estimation of etch-pit depth distributions from scanning electron microscopy micrographs. This method is used to obtain depth distributions from GaN surface after etching in molten KOH-NaOH eutectic mixture. Depth distributions are used to estimate densities of etch-pits related to a given dislocation type. The distributions are compared with dislocation densities determined with transmission electron microscopy.

Identification of very small embryonic like (VSEL) stem cells in bone marrow.

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 (G ai-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.

Cleavage fragments of the third complement component (C3) enhance stromal derived factor-1 (SDF-1)-mediated platelet production during reactive postbleeding thrombocytosis.

We hypothesized that the third complement component (C3) cleavage fragments (C3a and (des-Arg)C3a) are involved in stress/inflammation-related thrombocytosis, and investigated their potential role in reactive thrombocytosis induced by bleeding. We found that platelet counts are lower in C3-deficient mice in response to excessive bleeding as compared to normal littermates and that C3a and (des-Arg)C3a enhance stromal-derived factor-1 (SDF-1)-dependent megakaryocyte (Megs) migration, adhesion and platelet shedding. At the molecular level, C3a stimulates in Megs MAPKp42/44 phosphorylation, and enhances incorporation of CXCR4 into membrane lipid rafts increasing the responsiveness of Megs to SDF-1. We found that perturbation of lipid raft formation by statins decreases SDF-1/C3a-dependent platelet production in vitro and in an in vivo model statins ameliorated post-bleeding thrombocytosis. Thus, inhibition of lipid raft formation could find potential clinical application as a means of ameliorating some forms of thrombocytosis.

A hypothesis for an embryonic origin of pluripotent Oct-4(+) stem cells in adult bone marrow and other tissues.

Accumulating evidence demonstrates that adult tissues contain a population of stem cells that express early developmental markers such as stage-specific embryonic antigen and transcription factors Oct-4 and Nanog. These are the markers characteristic for embryonic stem cells, epiblast stem cells and primordial germ cells. The presence of these stem cells in adult tissues including bone marrow, epidermis, bronchial epithelium, myocardium, pancreas and testes supports the concept that adult tissues contain some population of pluripotent stem cells that is deposited in embryogenesis during early gastrulation. In this review we will discuss these data and present a hypothesis that these cells could be direct descendants of the germ lineage. The germ lineage in order to pass genes on to the next generations creates soma and thus becomes a 'mother lineage' for all somatic cell lineages present in the adult body.

A reappraisal of the role of insulin-like growth factor I in the regulation of human hematopoiesis.

IGF-I has been reported to increase hematopoietic progenitor cell cloning efficiency. To investigate this phenomenon, we studied the IGF-I responsiveness of human marrow cells expressing IGF-I receptor (IGF-IR), a direct strategy not used previously. IGF-IR+ and control CD34+ marrow cells were isolated using immunoaffinity methods. Then, the cells were cloned in methylcellulose containing variable amounts of serum- and lineage-appropriate growth factors supplemented with recombinant human IGF-I. In contrast to CD34+ cells, IGF-IR+ cells never gave rise to CFU-Blast, CFU-Mix, CFU-GM, BFU-E, or CFU-E. To substantiate the suggestion that CD34+ and IGF-IR+ cells were distinct populations, we used reverse transcription PCR to detect IGF-I, EpO, and KIT receptor mRNAs in these cells. The mRNA phenotype of CD34+ cells was EpO (+), KIT (+), and IGF-IR (-), while IGF-IR+ cells were IGF-IR (+), EpO (-), and KIT (-). These results suggested that IGF-IR is either not expressed or expressed at low levels on normal hematopoietic progenitor cells. Functional significance of the latter possibility was tested by exposing CD34+ cells to IGF-IR antisense oligodeoxynucleotides. Colony formation was unaffected by oligodeoxynucleotide disruption of IGF-IR, suggesting that, even if expressed at low level, the receptor's functional significance was doubtful. Nevertheless, when cultured in the presence of IGF-I, IGF-IR+ cells elaborated an activity with mild BFU-E stimulatory effects. Accordingly, if IGF-I plays a role in hematopoietic colony formation, it is probably and results from stimulation of IGF-IR-positive ancillary cells to secrete growth factors. Studies carried out with human leukemia cells yielded similar results.

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of 'signaling complex', (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.

The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis.

Proper response of normal stem cells (NSC) to motomorphogens and chemoattractants plays a pivotal role in organ development and renewal/regeneration of damaged tissues. Similar chemoattractants may also regulate metastasis of cancer stem cells (CSC). Growing experimental evidence indicates that both NSC and CSC express G-protein-coupled seven-transmembrane span receptor CXCR4 and respond to its specific ligand alpha-chemokine stromal derived factor-1 (SDF-1), which is expressed by stroma cells from different tissues. In addition, a population of very small embryonic-like (VSEL) stem cells that express CXCR4 and respond robustly to an SDF-1 gradient was recently identified in adult tissues. VSELs express several markers of embryonic and primordial germ cells. It is proposed that these cells are deposited early in the development as a dormant pool of embryonic/pluripotent NSC. Expression of both CXCR4 and SDF-1 is upregulated in response to tissue hypoxia and damage signal attracting circulating NSC and CSC. Thus, pharmacological modulation of the SDF-1-CXCR4 axis may lead to the development of new therapeutic strategies to enhance mobilization of CXCR4+ NSC and their homing to damaged organs as well as inhibition of the metastasis of CXCR4+ cancer cells.

Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.

Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 microg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow.

By employing multiparameter sorting, we identified in murine bone marrow (BM) a homogenous population of rare (approximately 0.02% of BMMNC) Sca-1(+)lin(-)CD45- cells that express by RQ-PCR and immunohistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1. The direct electronmicroscopical analysis revealed that these cells are small (approximately 2-4 microm), posses large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. In vitro cultures these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in BM from young (approximately 1-month-old) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 animals. These cells in vitro respond strongly to SDF-1, HGF/SF and LIF and express CXCR4, c-met and LIF-R, respectively, and since they adhere to fibroblasts they may be coisolated with BM adherent cells. We hypothesize that this population of Sca-1(+)lin(-)CD45- very small embryonic-like (VSEL) stem cells is deposited early during development in BM and could be a source of pluripotent stem cells for tissue/organ regeneration.