Effects of exercise countermeasure on myocardial contractility measured by 4D speckle tracking during a 21-day head-down bed rest.

OBJECTIVE: To evaluate functional myocardial contractility after 21 days of head-down bed rest (HDBR) in sedentary control (CON) or with a resistive vibration exercise (RVE) countermeasure (CM) applied, by using 4D echocardiographic (4D echo) imaging and speckle tracking strain quantification. METHODS: Twelve volunteers were enrolled in a crossover HDBR design, and 4D echo was performed in supine position (REST) at BDC-2 and at R + 2, and in - 6° HDT at day 18, and during the first and the last minute of the 80° head-up step of tilt test performed at both BDC-2 and R + 2. Radial (Rad-Str), longitudinal (Lg-Str) and twist (Tw-Str) strains were measured by 4D speckle tracking, as well as left ventricle diastolic volume (LVDV) and mass (LVmass). RESULTS: On HDT 18: in the CON group, LVDV and LVmass were reduced (p < 0.05), the Rad-Str decreased (p < 0.05) and Tw-Str showed a tendency to increase (p < 0.11), with no changes in Lg-Str. In RVE group, LVDV and LV mass, as well as all the strain parameters remained unchanged. On R + 2: in the CON group, LVDV and LVmass were not recovered in all subjects compared to pre-HDBR (p < 0.08) and Rad-Str was still decreased (p < 0.05), while Tw-Str tended to increase (p < 0.09). These parameters remained unchanged in the RVE group. Tilt 80°: Rad-Str and Lg-Str values at 80° tilt were similar post-HDT in both groups. CONCLUSION: The 4D echo and speckle tracking analysis showed that in the CON group, Rad-Str decreased concomitant with LVmass and LVDV with HDBR, but this observation did not allow concluding if HDBR induced a real remodeling or a muscle atrophy. RVE was able to preserve LVmass, LVDV and contractility during HDBR, thus proving its effectiveness to this aim. Nevertheless, the significant HDBR-induced changes observed in the CON group had only a limited effect on the cardiac contractile response as observed during post-HDBR tilt test. The level of contractility at 80° Tilt position was not affected either by HDBR or by RVE CM.

Lower body negative pressure identifies altered central vein characteristics without accompanying changes to baroreflexes in astronauts within hours of landing.

Cardiovascular deconditioning and altered baroreflexes predispose returning astronauts to Orthostatic Intolerance. We assessed 7 astronauts (1 female) before and following long-duration spaceflight (146 ± 43 days) with minimal upright posture prior to testing. We applied lower body negative pressure (LBNP) of up to - 30 mmHg to supine astronauts instrumented for continual synchronous measurements of cardiovascular variables, and intermittent imaging the Portal Vein (PV) and Inferior Vena Cava (IVC). During supine rest without LBNP, postflight elevations to total peripheral resistance (TPR; 15.8 ± 4.6 vs. 20.8 ± 7.1 mmHg min/l, p < 0.05) and reductions in stroke volume (SV; 104.4 ± 16.7 vs. 87.4 ± 11.5 ml, p < 0.05) were unaccompanied by changes to heart rate (HR) or estimated central venous pressure (CVP). Small increases to systolic blood pressure (SBP) and diastolic blood pressure (DBP) were not statistically significant. Autoregressive moving average modelling (ARMA) during LBNP did not identify differences to either arterial (DBP → TPR and SBP → HR) or cardiopulmonary (CVP → TPR) baroreflexes consistent with intact cardiovascular control. On the other hand, IVC and PV diameter-CVP relationships during LBNP revealed smaller diameter for a given CVP postflight consistent with altered postflight venous wall dynamics.

Bone marrow mesenchymal/fibroblastic stromal cells induce a distinctive EMT-like phenotype in AML cells.

The development of epithelial-to-mesenchymal transition (EMT) like features is emerging as a critical factor involved in the pathogenesis of acute myeloid leukaemia (AML). However, the extracellular signals and the signalling pathways in AML that may regulate EMT remain largely unstudied. We found that the bone marrow (BM) mesenchymal/fibroblastic cell line HS5 induces an EMT-like migratory phenotype in AML cells. AML cells underwent a strong increase of vimentin (VIM) levels that was not mirrored to the same extent by changes of expression of the other EMT core proteins SNAI1 and SNAI2. We validated these particular pattern of co-expression of core-EMT markers in AML cells by performing an in silico analysis using datasets of human tumours. Our data showed that in AML the expression levels of VIM does not completely correlate with the co-expression of core EMT markers observed in epithelial tumours. We also found that vs epithelial tumours, AML cells display a distinct patterns of co-expression of VIM and the actin binding and adhesion regulatory proteins that regulate F-actin dynamics and integrin-mediated adhesions involved in the invasive migration in cells undergoing EMT. We conclude that the BM stroma induces an EMT related pattern of migration in AML cells in a process involving a distinctive regulation of EMT markers and of regulators of cell adhesion and actin dynamics that should be further investigated. Understanding the tumour specific signalling pathways associated with the EMT process may contribute to the development of new tailored therapies for AML as well as in different types of cancers.

Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

Impaired cerebrovascular autoregulation and reduced CO2 reactivity after long duration spaceflight.

Long duration habitation on the International Space Station (ISS) is associated with chronic elevations in arterial blood pressure in the brain compared with normal upright posture on Earth and elevated inspired CO(2). Although results from short-duration spaceflights suggested possibly improved cerebrovascular autoregulation, animal models provided evidence of structural and functional changes in cerebral vessels that might negatively impact autoregulation with longer periods in microgravity. Seven astronauts (1 woman) spent 147 ± 49 days on ISS. Preflight testing (30-60 days before launch) was compared with postflight testing on landing day (n = 4) or the morning 1 (n = 2) or 2 days (n = 1) after return to Earth. Arterial blood pressure at the level of the middle cerebral artery (BP(MCA)) and expired CO(2) were monitored along with transcranial Doppler ultrasound assessment of middle cerebral artery (MCA) blood flow velocity (CBFV). Cerebrovascular resistance index was calculated as (CVRi = BP(MCA)/CBFV). Cerebrovascular autoregulation and CO(2) reactivity were assessed in a supine position from an autoregressive moving average (ARMA) model of data obtained during a test where two breaths of 10% CO(2) were given four times during a 5-min period. CBFV and Doppler pulsatility index were reduced during -20 mmHg lower body negative pressure, with no differences pre- to postflight. The postflight indicator of dynamic autoregulation from the ARMA model revealed reduced gain for the CVRi response to BP(MCA) (P = 0.017). The postflight responses to CO(2) were reduced for CBFV (P = 0.056) and CVRi (P = 0.047). These results indicate that long duration missions on the ISS impaired dynamic cerebrovascular autoregulation and reduced cerebrovascular CO(2) reactivity.

CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.

Long-Chain Omega-3 Fatty Acid Intake Is Associated with Age but not Cognitive Performance in an Older Australian Sample.

BACKGROUND: Long-chain omega-3 polyunsaturated fatty acids (LCn-3 PUFA) are essential nutrients and may be capable of delaying age-related cognitive decline. However, previous studies indicate that Australians are not meeting recommendations for LCn-3 PUFA intake. The current study therefore examined LCn-3 PUFA intake in an older Australia sample, as well as associations between LCn-3 PUFA intake and cognitive function. METHODS: Cross-sectional data were collected from 90 adults aged 50 to 80 years. LCn-3 PUFA intake was assessed using a food frequency questionnaire and red blood cell fatty acid profiles were used to calculate the Omega-3 Index (RBC n-3 index). Cognitive function was measured using Addenbrooke's Cognitive Examination-III. RESULTS: Positive associations were observed between age and RBC n-3 index (b=0.06, 95% CI: 0.01 - 0.10, P=0.01), and age and LCn-3 PUFA intake from fish oil capsules (b=17.5, 95% CI: 2.4 - 32.5 mg/day, P=0.02). When adjusting for LCn-3 PUFA from fish oil capsules, the association between age and RBC n-3 index was no longer significant. No associations were observed between LCn-3 PUFA intake and cognitive function. CONCLUSION: LCn-3 PUFA and fish oil consumption increased with age in this sample of older Australians, particularly due to supplement intake. However, LCn-3 PUFA intake was not associated with cognitive function.

Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition.

CC-chemokines are important mediators in the pathogenesis of atherosclerosis. Atherosclerosis progression is reduced by high-level, short-term inhibition of CC-chemokine activity, for example by adenoviral gene transfer. However, atherosclerosis is a chronic condition where short-term effects, while demonstrating proof-of-principle, are unlikely to provide maximum therapeutic benefit. Accordingly, we generated a recombinant lentivirus, lenti35K, encoding the broad-spectrum CC chemokine inhibitor, 35K, derived from the vaccinia virus. To investigate the effects of prolonged broad-spectrum chemokine inhibition on atherosclerosis, lenti35K, or lentiGFP or PBS were delivered to 6-week-old ApoE knockout (ApoE-KO) mice by hydrodynamic injection. Sustained lentiviral transduction and transgene expression were demonstrated by 35K mRNA and viral DNA in liver tissue, and recombinant 35K protein circulating in the plasma, 3 months after gene transfer. Plasma from lenti35K animals had reduced chemokine activity compared with plasma from lentiGFP or PBS-treated animals. Histologic analysis of aortic sinus sections revealed that atherosclerotic plaque area in lenti35K mice was significantly reduced compared with both lentiGFP and PBS controls. Furthermore, plaque macrophage content was substantially reduced in lenti35K mice. Lentiviral 35K gene transfer is a promising experimental strategy to reduce atherosclerosis progression, and demonstrates the potential of long-term CC-chemokine inhibition as a potential therapeutic target in atherosclerosis.

Programmed gene rearrangements altering gene expression.

Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens). Two major mechanisms for expression control are DNA inversion and DNA transposition. In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression. In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion. Gene rearrangements altering gene expression have mainly been found in some unicellular organisms. They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus. The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way.

Molecular immunobiology of macrophages: recent progress.

Macrophage molecules involved in adhesion, signalling, secretion and interactions with pathogens and T-helper lymphocytes have been cloned and defined using monoclonal antibodies and genetic disruption. Studies of the regulation of macrophage differentiation and activation by cytokines have yielded insights into the heterogeneity of their functions in natural and acquired immunity, including immunity to infections by mycobacteria and HIV.

Heterogeneity of responses to orthostatic stress in homozygous twins.

Early analysis into the role of genetics on cardiovascular regulation has been accomplished by comparing blood pressure and heart rate in homozygous twins during unstressed, resting physiological conditions. However, many variables, including cognitive and environmental factors, contribute to the regulation of cardiovascular hemodynamics. Therefore, the purpose of this study was to determine the hemodynamic response of identical twins to an orthostatic stress, ranging from supine rest to presyncope. Heart rate, arterial blood pressure, middle cerebral artery blood velocity, an index of cerebrovascular resistance, cardiac output, total peripheral resistance, and end-tidal carbon dioxide were measured in 16 healthy monozygotic twin pairs. Five minutes of supine resting baseline data were collected, followed by 5 min of 60 degrees head-up tilt. After 5 min of head-up tilt, lower body negative pressure was applied in increments of 10 mmHg every 3 min until the onset of presyncope, at which time the subject was returned to the supine position for a 5-min recovery period. The data indicate that cardiovascular regulation under orthostatic stress demonstrates a significant degree of variance between identical twins, despite similar orthostatic tolerance. As the level of stress increases, so does the difference in the cardiovascular response within a twin pair. The elevated variance with increasing stress may be due to an increase in the role of environmental factors, as the influential role of genetics nears a functional limit. Therefore, although orthostatic tolerance times were very similar between identical twins, the mechanism involved in sustaining cardiovascular function during increasing stress was different.

Wave energy absorption by a submerged air bag connected to a rigid float.

A new wave energy device features a submerged ballasted air bag connected at the top to a rigid float. Under wave action, the bag expands and contracts, creating a reciprocating air flow through a turbine between the bag and another volume housed within the float. Laboratory measurements are generally in good agreement with numerical predictions. Both show that the trajectory of possible combinations of pressure and elevation at which the device is in static equilibrium takes the shape of an S. This means that statically the device can have three different draughts, and correspondingly three different bag shapes, for the same pressure. The behaviour in waves depends on where the mean pressure-elevation condition is on the static trajectory. The captured power is highest for a mean condition on the middle section.

Adenovirus-mediated gene transfer of a secreted form of human macrophage scavenger receptor inhibits modified low-density lipoprotein degradation and foam-cell formation in macrophages.

BACKGROUND: Macrophage scavenger receptors (MSRs) play an important role in the pathogenesis of atherosclerosis. Therefore, local modulation of MSR activity could have a beneficial effect on atherogenesis. METHODS AND RESULTS: We cloned a secreted "decoy" MSR (sMSR) that contains an extracellular portion of the human MSR type AI and constructed an adenoviral vector that directs high-level expression of sMSR in macrophages under the control of the human CD68 promoter. Expression of the sMSR protein inhibited the degradation of (125)I-labeled acetylated LDL and oxidized LDL by murine macrophages up to 90%. sMSRs also reduced acetylated LDL degradation in MSR knockout mouse peritoneal macrophages by 60% to 80%, which suggests that the decoy construct can compete for the uptake mediated via other related scavenger receptors. In addition, sMSRs inhibited foam-cell formation in murine macrophages in the presence of cytochalasin D. The mechanism of inhibition is through ligand binding to the sMSRs, which prevents the ligand binding to MSRs on cell membranes. CONCLUSIONS: The demonstration that recombinant adenovirus-mediated gene transfer of decoy sMSRs can block foam-cell formation suggests a possible new strategy for gene therapy of atherosclerosis and for the treatment of lipid accumulation after arterial manipulations.

Trypanosoma brucei variant-specific glycoprotein gene chromatin is sensitive to single-strand-specific endonuclease digestion.

Active variant surface glycoprotein (VSG) gene chromatin is preferentially digested by the restriction enzyme HinfI in nuclei of bloodstream variants of Trypanosoma brucei. HinfI sensitivity of VSG gene chromatin is not observed in nuclei of relapse variants in which the VSG gene has been inactivated in situ. Active VSG gene chromatin is preferentially degraded by the single-strand-specific endonucleases S1 and Bal31. This sensitivity is not the result of pre-existing single-strand breaks or a detectably altered nucleosomal organization. Trypanosome nuclei in which the run-on transcription of VSG genes has been specifically shut down have been used to show that Hinfl and Bal31 sensitivity is not dependent upon continued transcription of the VSG gene. The presence of single-stranded DNA regions within VSG gene chromatin is consistent with a model in which VSG genes are activated by increased torsional stress.

Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene.

We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.

5' structural motifs and Xenopus beta globin gene activation.

We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.

Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

Globin gene switching: a paradigm or what?

The delineation of the beta-globin locus control region has led to a new understanding of the developmental regulation of the beta-globin gene cluster. It now seems that globin gene switching is effected through the sequential and mutually exclusive interaction of the locus control region with the embryonic, fetal and adult stage specific globin genes.

Femoral asymmetry in the Thoroughbred racehorse.

OBJECTIVE: To investigate the occurrence of geometrical asymmetries in the macro-architecture of left and right femurs from Thoroughbred racehorses previously used in competitive training and racing in New South Wales, Australia. METHODS: Detailed postmortem measurements were made of 37 characteristics of left and right femurs from eleven Thoroughbred racehorses euthanased for reasons unrelated to the study. Measurements focused on articulating surfaces and sites of attachment of muscles and ligaments known to be associated with hindlimb locomotion. RESULTS: Five measurements were significantly larger in left compared to right femurs (P < 0.05). The regions showing significant differences between left and right limbs were proximal cranial and overhead medio-lateral widths, greater trochanter depth, depth of the fovea in the femoral head and distal inter-epicondylar width. CONCLUSION: The left-right differences in femoral morphology were associated with sites of muscle and ligament attachment known to be involved with hindlimb function in negotiating turns. These differences may be the result of selection pressure for racing performance on curved race tracks and/or adaptations related to asymmetrical loading of the outside hindlimb associated with repeated negotiation of turns on such tracks.