Suppressors of cytokine signaling inhibit effector T cell responses during Mycobacterium tuberculosis infection

Protective immune responses during Mycobacterium tuberculosis (M. tuberculosis) infection are regulated at multiple levels and critically dependent on the balance in the secretion of pro-inflammatory and regulatory cytokines. A key factor that governs this balance at the cellular level is suppressors of cytokine signaling (SOCS). We recently demonstrated that toll-like receptor 2 and dendritic cell (DC)-SIGNR1 differentially regulate SOCS1 expression in DCs during M. tuberculosis infection. This consecutively regulated IL-12 production and determined M. tuberculosis survival. In this study, we characterized the role of SOCS1 in regulating effector responses from CD4(+) and CD8(+) T cells during M. tuberculosis infection. Our data indicate that T cells from M. tuberculosis-infected mice show increased and differential association of SOCS1 with CD3 and CD28, when compared with uninfected mice. While SOCS1 displays increased association with CD3 than CD28 in CD4(+) T cells; SOCS1 is associated more with CD28 than CD3 in CD8(+) T cells. Further, SOCS1 shows increased association with IL-12 and IL-2 receptors in both CD4(+) and CD8(+) T cells from infected mice when compared with naive mice. Silencing SOCS1 in T cells increased signal transduction from T cell receptor (TCR) and CD28 with enhanced activation of key signaling molecules and proliferation. Significantly, SOCS1-silenced T cells mediated enhanced clearance of M. tuberculosis inside macrophages. Finally, adoptive transfer of SOCS1-silenced T cells in M. tuberculosis-infected mice mediated significant reduction in M. tuberculosis loads in spleen. These results exemplify the negative role played by SOCS1 during T cell priming and effector functions during M. tuberculosis infection.     

Thrombomodulin Contributes to Gamma Tocotrienol-Mediated Lethality Protection and Hematopoietic Cell Recovery in Irradiated Mice

Systemic administration of recombinant thrombomodulin (TM) confers radiation protection partly by accelerating hematopoietic recovery. The uniquely potent radioprotector gamma tocotrienol (GT3), in addition to being a strong antioxidant, inhibits the enzyme hydroxy-methyl-glutaryl-coenzyme A reductase (HMGCR) and thereby likely modulates the expression of TM. We hypothesized that the mechanism underlying the exceptional radioprotective properties of GT3 partly depends on the presence of endothelial TM. In vitro studies confirmed that ionizing radiation suppresses endothelial TM (about 40% at 4 hr after 5 Gy γ-irradiation) and that GT3 induces TM expression (about 2 fold at the mRNA level after 5 μM GT3 treatment for 4 hr). In vivo survival studies showed that GT3 was significantly more effective as a radioprotector in TM wild type (TM^+/+) mice than in mice with low TM function (TM^Pro/-). After exposure to 9 Gy TBI, GT3 pre-treatment conferred 85% survival in TM^+/+ mice compared to only 50% in TM^Pro/-. Thus, GT3-mediated radiation lethality protection is partly dependent on endothelial TM. Significant post-TBI recovery of hematopoietic cells, particularly leukocytes, was observed in TM^+/+ mice (p = 0.003), but not in TM^Pro/- mice, despite the fact that GT3 induced higher levels of granulocyte colony stimulating factor (G-CSF) in TM^Pro/- mice (p = 0.0001). These data demonstrate a critical, G-CSF-independent, role for endothelial TM in GT3-mediated lethality protection and hematopoietic recovery after exposure to TBI and may point to new strategies to enhance the efficacy of current medical countermeasures in radiological/nuclear emergencies.     

Influence of arterial wall compliance on the pressure drop across coronary artery stenoses under hyperemic flow condition

Hemodynamic endpoints such as flow and pressure drop are often measured during angioplasty procedures to determine the functional severity of a coronary artery stenosis. There is a lack of knowledge regarding the influence of compliance of the arterial wall-stenosis on the pressure drop under hyperemic flows across coronary lesions. This study evaluates the influence in flow and pressure drop caused by variation in arterial-stenosis compliance for a wide range of stenosis severities. The flow and pressure drop were evaluated for three different severities of stenosis and tested for limiting scenarios of compliant models. The Mooney-Rivlin model defined the non-linear material properties of the arterial wall and the plaque regions. The non-Newtonian Carreau model was used to model the blood flow viscosity. The fluid (blood)-structure (arterial wall) interaction equations were solved numerically using the finite element method. Irrespective of the stenosis severity, the compliant models produced a lower pressure drop than the rigid artery due to compliance of the plaque region. A wide variation in the pressure drop was observed between different compliant models for significant (90% area occlusion) stenosis with 41.0, 32.1, and 29.8 mmHg for the rigid artery, compliant artery with calcified plaque, and compliant artery with smooth muscle cell proliferation, respectively. When compared with the rigid artery for significant stenosis the pressure drop decreased by 27.7% and 37.6% for the calcified plaque and for the smooth muscle cell proliferation case, respectively. These significant variations in pressure drop for the higher stenosis may lead to misinterpretation and misdiagnosis of the stenosis severity.     

Oxygen transport and consumption by suspended cells in microgravity: a multiphase analysis

A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 microm; 12 rpm for 300 microm; 14 rpm for 400 microm; 18 rpm for 600 microm. As the rotating speed and the bead size increase from 10 rpm/200 microm to 18 rpm/600 microm, the mean oxygen concentration in the 80% midzone of the vessel is increased by approximately 85% after 1-h rotation due to the high convective flow for 18 rpm/600 microm case as compared to 10 rpm/200 microm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.     

Temperature distribution during ICG-dye-enhanced laser photocoagulation of feeder vessels in treatment of AMD-related choroidal neovascularization

Laser photocoagulation of the feeder vessels of age-related macula degeneration-related choroidal neovascularization (CNV) membranes is a compelling treatment modality, one important reason being that the treatment site is removed from the fovea in cases of sub- or juxtafoveal CNV. To enhance the energy absorption in a target feeder vessel, an indocyanine green dye bolus is injected intravenously, and the 805 nm wavelength diode laser beam is applied when the dye bolus transits the feeder vessel; this tends to reduce concomitant damage to adjacent tissue. A 3D theoretical simulation, using the Pennes bioheat equation, was performed to study the temperature distribution in the choroidal feeder vessel and its vicinity during laser photocoagulation. The results indicate that temperature elevation in the target feeder vessel increases by 20% in dye-enhanced photocoagulation, compared to just photocoagulation alone. The dye bolus not only increases the laser energy absorption in the feeder vessel but also shifts the epicenter of maximum temperature away from the sensitive sensory retina and retinal pigment epithelial layers and toward the feeder vessel. Two dominant factors in temperature elevation of the feeder vessel are location of the feeder vessel and blood flow velocity through it. Feeder vessel temperature elevation becomes smaller as distance between it and the choriocapillaris layer increases. The cooling effect of blood flow through the feeder vessel can reduce the temperature elevation by up to 21% of the maximum that could be produced. Calculations were also performed to examine the effect of the size of the laser spot. To achieve the same temperature elevation in the feeder vessel when the laser spot diameter is doubled, the laser power level has to be increased by only 60%. In addition, our results have suggested that more studies are needed to measure the constants in the Arrhenius integral for assessing thermal damage in various tissues.     

Physiological flow analysis in significant human coronary artery stenoses

To evaluate the local hemodynamics in flow limiting coronary lesions, computational hemodynamics was applied to a group of patients previously reported by Wilson et al. (1988) with representative pre-angioplasty stenosis geometry (minimal lesion size d(m)=0.95 mm; 68% mean diameter stenosis) and with measured values of coronary flow reserve (CFR) in the abnormal range (2.3+/-0.1). The computations were at mean flow rates (Q) of 50, 75 and 100 ml/min (the limit of our converged calculations). Computed mean pressure drops Deltap were approximately 9 mmHg for basal flow (50 ml/min), approximately 27 mmHg for elevated flow (100 ml/min) and increased to an extrapolated value of approximately 34 mmHg for hyperemic flow (115 ml/min), which led to a distal mean coronary pressure p(rh) of approximately 55 mmHg, a level known to cause ischemia in the subendocardium (Brown et al., 1984), and consistent with the occurrence of angina in the patients. Relatively high levels of wall shear stress were computed in the narrow throat region and ranged from about 600 to 1500 dyn/cm(2), with periodic (phase shifted) peak systolic values of about 3500 dyn/cm(2). In the distal vessel, the interaction between the separated shear layer wave, convected downstream by the core flow, and the wall shear layer flow, led to the formation of vortical flow cells along the distal vessel wall during the systolic phase where Reynolds numbers Re(e)(t) were higher. During the phasic vortical mode observed at both basal and elevated mean flow rates, wide variations in distal wall shear stress occurred, distal transmural pressures were depressed below throat levels, and pressure recovery was larger farther along the distal vessel. Along the constriction (convergent) and throat segments of the lesion the pulsatile flow field was principally quasi-steady before flow separation occurred. The flow regimes were complex in the narrow mean flow Reynolds number range Re(e)=100-230 and a frequency parameter of alphae=2.25. The shear layer flow disturbances diminished in strength due to viscous damping along the distal vessel at these relatively low values of Re(e), typical of flow through diseased epicardial coronary vessels. The distal hyperemic flow field was likely to be in an early stage of turbulent flow development during the peak systolic phase.     

Small chromosomal integration site of classical CTX prophage in Mozambique <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1 biotype El Tor strain

An unusual strain of Vibrio cholerae O1 biotype El Tor harbouring multiple tandem copies of classical CTX prophage caused a cholera epidemic in Mozambique in 2004. However, the location of the classical CTX prophage in the genome of the Mozambique strain was unknown. In this study, pulsed field gel electrophoresis (PFGE) of the whole genome along with Southern hybridization experiments indicated that the classical CTX prophage present in the Mozambique strain is located in the small chromosome. To determine the CTX prophage integration site in the small chromosome of Mozambique strain, the 5'and 3' junctions of the prophage and small chromosome were PCR amplified, cloned and sequenced. Sequence analysis indicated that the prophage was integrated in the conserved dif site of the replication terminus region of the Mozambique strain. While using an O1 El Tor isolate VC44 as a control strain, which carries tandem copies of CTX prophage in its small chromosome like the Mozambique strain, it was unexpectedly detected that the strain VC44 also possesses classical cholera toxin B gene allele. Since the strain VC44 was isolated in India in the year 1992, it appears that the Mozambique strain has probably originated from a VC44-like strain.     

Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition

Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent thermal evaporation, and it absorbs visible light strongly. However, for a long time the record power conversion efficiency of SnS solar cells remained below 2%. Recently we demonstrated new certified record efficiencies of 4.36% using SnS deposited by atomic layer deposition, and 3.88% using thermal evaporation. Here the fabrication procedure for these record solar cells is described, and the statistical distribution of the fabrication process is reported. The standard deviation of efficiency measured on a single substrate is typically over 0.5%. All steps including substrate selection and cleaning, Mo sputtering for the rear contact (cathode), SnS deposition, annealing, surface passivation, Zn(O,S) buffer layer selection and deposition, transparent conductor (anode) deposition, and metallization are described. On each substrate we fabricate 11 individual devices, each with active area 0.25 cm². Further, a system for high throughput measurements of current-voltage curves under simulated solar light, and external quantum efficiency measurement with variable light bias is described. With this system we are able to measure full data sets on all 11 devices in an automated manner and in minimal time. These results illustrate the value of studying large sample sets, rather than focusing narrowly on the highest performing devices. Large data sets help us to distinguish and remedy individual loss mechanisms affecting our devices.