Subpopulation Composition and Activation of T Lymphocytes during Coculturing with Mesenchymal Stromal Cells in Medium with Different O<Subscript>2</Subscript> Content

The concentration of O₂ during coculturing practically did not affect the subpopulation composition of T lymphocytes (CD3⁺/CD4⁺, CD3⁺/CD8⁺, CD3⁺/CD16⁺/CD56⁺ T cells) under conditions of PHA-induced activation. Coculturing with mesenchymal stromal cells (MSC) led to a significant decrease in the ratio of lymphocytes carrying activation markers (CD3⁺/CD25⁺ and CD3⁺/HLA-DR⁺) and increase in the number of CD3⁺/CD16⁺/CD56⁺ T cells. The percent of activated HLA-DR⁺ T cells in a heterotypic culture with MSC at 5% O₂ was much lower than that observed under normal conditions of culturing (20% O₂). Our results suggest that antigen presentation by T lymphocytes due to HLA-DR expression can be reduced in the target tissues at low concentration of O₂, while the interaction between allogeneic MSC probably contributes to more significant inhibition of the immune response.     

Ultrastructural Assessment of the Differentiation Potential of Human Multipotent Mesenchymal Stromal Cells

Abstract

Mesenchymal stromal (stem) cells (MSCs) are defined by plastic adherent growth, multiple phenotype expressions, and tripotential mesodermal capability. The authors report examples where electron microscopy (EM) plays a role in stem cell research. MSCs isolated from human arteries are ultrastructurally heterogeneous and become more homogenous after plastic adhesion. EM shows a moderate complement of organelles, mainly mitochondria, rough endoplasmic reticulum, and glycogen aggregates. Clear vacuoles and vesicles are prominent when cells are recovered from plates using an enzymatic method. Since the mesengenic plasticity is the single most important criterion to define a cell as mesenchymal stromal, the authors induced experimentally adipogenic, leiomyogenic, cardiomyogenic, osteo-chondrogenic differentiations. In no case did EM reveal the achievement of complete differentiation. The authors obtained multivacuolated pre-adipocytes and never univacuolated adipocytes typical of mature white fat; myofibroblast and rhabdomyoblast morphotypes, where contractile filaments were not organized to form functional complexes, i.e., dense bodies and sarcomeres. Chondrogenesis and osteogenesis assays resulted in extracellular matrix changes. Collagen and proteoglycan filament/particle deposition was seen when chondrogenesis was promoted. Hydroxyapatite crystals, psammoma bodies, and plaque-like calcified matrix deposits were found in the osteogenic matrix. EM provides detailed structural information on the degree of differentiation induced in stem cells and demonstrates that the methods so far developed are not able to promote complete cell differentiation. These observations contribute to explain why clinical applications with hMSCs have produced results far lower than initial expectations.     

Assessment of DNA Damage in Human Bone Marrow Cells and Multipotent Mesenchymal Stromal Cells

We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3–11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.     

Blood HbO<Subscript>2</Subscript> and HbCO<Subscript>2</Subscript> Dissociation Curves at Varied O<Subscript>2</Subscript>, CO<Subscript>2</Subscript>, pH, 2,3-DPG and Temperature Levels

New mathematical model equations for O₂ and CO₂ saturations of hemoglobin (S_HbO2 and S_HbCO2) are developed here from the equilibrium binding of O₂ and CO₂ with hemoglobin inside RBCs. They are in the form of an invertible Hill-type equation with the apparent Hill coefficients _KHbO2 and K_HbCO2 in the expressions for S_HbO2 and S_HbCO2 dependent on the levels of O₂ and CO₂ partial pressures (P_O2 and P_CO2), pH, 2,3-DPG concentration, and temperature in blood. The invertibility of these new equations allows P_O2 and P_CO2 to be computed efficiently from S_HbO2 and S_HbCO2 and vice-versa. The oxyhemoglobin (HbO₂) and carbamino-hemoglobin (HbCO₂) dissociation curves computed from these equations are in good agreement with the published experimental and theoretical curves in the literature. The model solutions describe that, at standard physiological conditions, the hemoglobin is about 97.2% saturated by O₂ and the amino group of hemoglobin is about 13.1% saturated by CO₂. The O₂ and CO₂ content in whole blood are also calculated here from the gas solubilities, hematocrits, and the new formulas for S_HbO2 and S_HbCO2. Because of the mathematical simplicity and invertibility, these new formulas can be conveniently used in the modeling of simultaneous transport and exchange of O₂ and CO₂ in the alveoli-blood and blood-tissue exchange systems.     

Central circulatory and peripheral O<Subscript>2</Subscript> extraction changes as interactive facilitators of pulmonary O<Subscript>2</Subscript> uptake during a repeated high-intensity exercise protocol in humans

It has frequently been demonstrated that prior high-intensity exercise facilitates pulmonary oxygen uptake response at the onset of subsequent identical exercise. To clarify the roles of central O₂ delivery and/or peripheral O₂ extraction in determining this phenomenon, we investigated the relative contributions of cardiac output (CO) and arteriovenous O₂ content difference to the transient during repeated bouts of high-intensity knee extension (KE) exercise. Nine healthy subjects volunteered to participate in this study. The protocol consisted of two consecutive 6-min KE exercise bouts in a supine position (work rate 70–75% of peak power) separated by 6 min of rest. Throughout the protocol, continuous-wave Doppler ultrasound was used to measure beat-by-beat CO (i.e., via simultaneous measurement of stroke volume and the diameter of the arterial aorta). The phase II response was significantly faster and the slow component (phase III) was significantly attenuated during the second KE bout compared to the first. This was a result of increased CO during the first 30 s of exercise: CO contributing to 100 and 56% of the speeding at 10 and 30 s, respectively. After this, the contribution of became increasingly more predominant: being responsible to an estimated 64% of the speeding at 90 s, which rose to 100% by 180 s. This suggests that, while both CO and clearly interact to determine the response, the speeding of kinetics by prior high-intensity KE exercise is predominantly attributable to increases in .     

Multipotent Mesenchymal Stromal Cells in Patients with Chronic Myeloid Leukemia before Discontinuation of Tyrosine Kinase Inhibitors

Bulletin of Experimental Biology and Medicine - We studied the influence of vascular prostheses made of polytetrafluoroethylene and polyethylene terephthalate on the proliferation, migration, and...     

Lipid bilayer coated Al<Subscript>2</Subscript>O<Subscript>3</Subscript> nanopore sensors: towards a hybrid biological solid-state nanopore

Solid-state nanopore sensors are highly versatile platforms for the rapid, label-free electrical detection and analysis of single molecules, applicable to next generation DNA sequencing. The versatility of this technology allows for both large scale device integration and interfacing with biological systems. Here we report on the development of a hybrid biological solid-state nanopore platform that incorporates a highly mobile lipid bilayer on a single solid-state Al₂O₃ nanopore sensor, for the potential reconstitution of ion channels and biological nanopores. Such a system seeks to combine the superior electrical, thermal, and mechanical stability of Al₂O₃ solid-state nanopores with the chemical specificity of biological nanopores. Bilayers on Al₂O₃ exhibit higher diffusivity than those formed on TiO₂ and SiO₂ substrates, attributed to the presence of a thick hydration layer on Al₂O₃, a key requirement to preserving the biological functionality of reconstituted membrane proteins. Molecular dynamics simulations demonstrate that the electrostatic repulsion between the dipole of the DOPC headgroup and the positively charged Al₂O₃ surface may be responsible for the enhanced thickness of this hydration layer. Lipid bilayer coated Al₂O₃ nanopore sensors exhibit excellent electrical properties and enhanced mechanical stability (GΩ seals for over 50 h), making this technology ideal for use in ion channel electrophysiology, the screening of ion channel active drugs and future integration with biological nanopores such as α-hemolysin and MspA for rapid single molecule DNA sequencing. This technology can find broad application in bio-nanotechnology.     

Effect of Nanodisperse Ferrite Cobalt (CoFe<Subscript>2</Subscript>O<Subscript>4</Subscript>) Particles on Contractile Reactions in Guinea Pigs Airways

The effect of nanopowder CoFe₂O₄ on contractile responses of smooth-muscle segments of guinea pigs airways was studied by mechanography. Both in vivo inhalation of nanopowder aerosol or in vitro application of nanopowder to isolated airway segments increased the amplitude of contractile responses to histamine and potentiated the dilatory reaction to adrenergic salbutamol.     

Anti-oxidative effects of the biennial flower of <Emphasis Type="Italic">Panax notoginseng</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity in cultured PC12 cells

Background  

Radix notoginseng is used in Chinese medicine to improve blood circulation and clotting; however, the pharmacological activities of other parts of Panax notoginseng have yet to be explored. The present study reports the anti-oxidative effects of various parts of Panax notoginseng.     

Analysis of Expression of Genes Involved in Immune Response Modulation in Silent Multipotent Mesenchymal Stromal Cells

The expression of some genes modulating the immune response was studied in multipotent mesenchymal stromal cells (MMSC) from the bone marrow of a healthy donor. Non-activated MMSC expressed IL-6 and IL-10, complement H factor, macrophage growth factor, prostaglandin E2 synthase, and indoleamine-2,3-dioxygenase. The expression of all these genes was higher in female MMSC. A close inverse relationship between IL-6 expression in MMSC and male donor age, close relationship between body weight index and fibroblast CFU concentration in female donor bone marrow and between indoleamine-2,3-dioxygenase and macrophage growth factor in MMSC from these donors were detected. The expression of the analyzed genes was higher in MMSC of donors who had no antibodies to cytomegalovirus, herpes simplex virus, and Epstein-Barr virus in the blood. The results demonstrate the MMSC regulation of immune reactions by MMSC at the cell and organism levels.     

<Emphasis Type="Italic">V</Emphasis>O<Subscript>2</Subscript>max during successive maximal efforts

The concept of VO₂max has been a defining paradigm in exercise physiology for >75 years. Within the last decade, this concept has been both challenged and defended. The purpose of this study was to test the concept of VO₂max by comparing VO₂ during a second exercise bout following a preliminary maximal effort exercise bout. The study had two parts. In Study #1, physically active non-athletes performed incremental cycle exercise. After 1-min recovery, a second bout was performed at a higher power output. In Study #2, competitive runners performed incremental treadmill exercise and, after 3-min recovery, a second bout at a higher speed. In Study #1 the highest VO₂ (bout 1 vs. bout 2) was not significantly different (3.95 ± 0.75 vs. 4.06 ± 0.75 l min⁻¹). Maximal heart rate was not different (179 ± 14 vs. 180 ± 13 bpm) although maximal V _E was higher in the second bout (141 ± 36 vs. 151 ± 34 l min⁻¹). In Study #2 the highest VO₂ (bout 1 vs. bout 2) was not significantly different (4.09 ± 0.97 vs. 4.03 ± 1.16 l min⁻¹), nor was maximal heart rate (184 + 6 vs. 181 ± 10 bpm) or maximal V _E (126 ± 29 vs. 126 ± 34 l min⁻¹). The results support the concept that the highest VO₂ during a maximal incremental exercise bout is unlikely to change during a subsequent exercise bout, despite higher muscular power output. As such, the results support the “classical” view of VO₂max.     

Human Multipotent Mesenchymal Stromal Cells from Distinct Sources Show Different In Vivo Potential to Differentiate into Muscle Cells When Injected in Dystrophic Mice

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4–6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.     

Validity of criteria for establishing maximal O<Subscript>2</Subscript> uptake during ramp exercise tests

The incremental or ramp exercise test to the limit of tolerance has become a popular test for determination of maximal O₂ uptake However, many subjects do not evidence a definitive plateau of the -work rate relationship on this test and secondary criteria based upon respiratory exchange ratio (RER), maximal heart rate (HR_max) or blood [lactate] have been adopted to provide confidence in the measured We hypothesized that verification of using these variables is fundamentally flawed in that their use could either allow underestimation of (if, for any reason, a test were ended at a sub-maximal ), or alternatively preclude subjects from recording a valid Eight healthy male subjects completed a ramp exercise test (at 20 W/min) to the limit of tolerance on an electrically braked cycle ergometer during which pulmonary gas exchange was measured breath-by-breath and blood [lactate] was determined every 90 s. Using the most widely used criterion values of RER (1.10 and 1.15), as determined during the ramp test (4.03 ± 0.10 l/min) could be undermeasured by 27% (2.97 ± 0.24 l/min) and 16% (3.41 ± 0.15 l/min), respectively (both P < 0.05). The criteria of HR_max (age predicted HR_max ± 10 b/min) and blood [lactate] (≥8 mM) were untenable because they resulted in rejection of 3/8 and 6/8 of the subjects, most of whom (5/8) had demonstrated a plateau of at These findings provide a clear mandate for rejecting these secondary criteria as a means of validating on ramp exercise tests.     

CD<Subscript>4</Subscript><Superscript>+</Superscript>CD<Subscript>25</Subscript><Superscript>+</Superscript> Regulatory T Cells Decreased the Antitumor Activity of Cytokine-Induced Killer (CIK) Cells of Lung Cancer Patients

CD₄ ⁺CD₂₅ ⁺ regulatory T cells (Tregs) have been shown to inhibit cytotoxic lymphocytes-mediated immune responses. Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches. Therefore, the purpose of this report was to determine the effect of Tregs on CIK cell growth and CIK-induced cytotoxicity for inhibition of tumor growth in vivo as well as in vitro. After depletion of CD₄ ⁺CD₂₅ ⁺ cells before culture, the proliferation and cytotoxicity of CIK cells, which indicated in bromodeoxyuridine (BrdU) and lactic dehydrogenase (LDH) assays, were significantly increased. Depletion of CD₄ ⁺CD₂₅ ⁺ cells preculture also enhanced the suppression effect on the lung cancer cells inoculated in experimental animals. Blockage of glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor β1 (TGF-β1) by antibodies partially abrogated the suppressive effect of CD₄ ⁺CD₂₅ ⁺ cells on CIK. These results indicated that Tregs could inhibit the antitumor activity of CIK cells. The molecules TGF-β and GITR may contribute to the suppressive function of CD₄ ⁺CD₂₅ ⁺ cells.