A comparison of blood gases and acid-base measurements in arterial, arterialized venous, and venous blood during short-term maximal exercise

The purpose of this study was to determine the relationship between blood gases and acid-base measurements in arterial, arterialized venous, and venous blood measured simultaneously during short-term maximal exercise. Ten well-trained male cyclists performed a graded maximal exercise test on a cycle ergometer to determine the power output corresponding to their peak oxygen consumption (test I), and a short-term maximal test on a cycle ergometer at peak power output (test II). During test II arterial, arterialized venous and venous blood were sampled simultaneously for determination of partial pressures of oxygen and carbon dioxide, pH, bicarbonate (HCO3-), base excess (BE), and lactate (La). Samples were taken at rest, the end of 1 min of exercise (1 ME), at the end of exercise (EE), and at 2 min of recovery (REC). During test II, subjects maintained a peak power output of 370.6 (62.1) W [mean (SD)] for 4.5, SD 1.6 min. Except at rest venous and arterialized venous measurements tended to be the same at all sampling intervals, but differed significantly from measurements in arterial blood (P less than 0.05). BE was the only variable that rendered consistently significant correlations between arterial and arterialized venous blood at each sampling interval. The pooled correlation coefficient between arterial and arterialized venous BE was r = 0.83 [regression equation: BEa = (0.84 BEav)-0.51]. Arterial La was significantly higher than venous La at 1 ME (2.8, 0.7 vs 0.8, 0.3 mmol.l-1) and higher than both venous and arterialized venous La at EE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.e. signaling) and inactive conformations of the receptor, but do not include the dynamics of G-protein activation or receptor desensitization. Yet understanding how these dynamic events effect response behavior is crucial to determining ligand efficacy. We developed a mathematical model for G-protein coupled receptor signaling that includes G-protein activation and receptor desensitization, and used it to predict how activation and desensitization would change if either the conformational selectivity (the effect of ligand binding on the distribution of active and inactive receptor states) or the desensitization rate constant was ligand-specific. In addition, the model was used to explore the implications of measuring responses far downstream from G-protein activation. By comparing the experimental data from the beta(2)-adrenergic, micro-opioid, D(1)dopamine, and neutrophil N -formyl peptide receptors with the predictions of our model, we found that the conformational selectivity is the predominant factor in determining the amounts of activation and desensitization caused by a particular ligand.     

Compensatory Hemagglutinin Mutations Alter Antigenic Properties of Influenza Viruses

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.     

Multiscale computational modeling reveals a critical role for TNF-α receptor 1 dynamics in tuberculosis granuloma formation

Multiple immune factors control host responses to Mycobacterium tuberculosis infection, including the formation of granulomas, which are aggregates of immune cells whose function may reflect success or failure of the host to contain infection. One such factor is TNF-α. TNF-α has been experimentally characterized to have the following activities in M. tuberculosis infection: macrophage activation, apoptosis, and chemokine and cytokine production. Availability of TNF-α within a granuloma has been proposed to play a critical role in immunity to M. tuberculosis. However, in vivo measurement of a TNF-α concentration gradient and activities within a granuloma are not experimentally feasible. Further, processes that control TNF-α concentration and activities in a granuloma remain unknown. We developed a multiscale computational model that includes molecular, cellular, and tissue scale events that occur during granuloma formation and maintenance in lung. We use our model to identify processes that regulate TNF-α concentration and cellular behaviors and thus influence the outcome of infection within a granuloma. Our model predicts that TNF-αR1 internalization kinetics play a critical role in infection control within a granuloma, controlling whether there is clearance of bacteria, excessive inflammation, containment of bacteria within a stable granuloma, or uncontrolled growth of bacteria. Our results suggest that there is an interplay between TNF-α and bacterial levels in a granuloma that is controlled by the combined effects of both molecular and cellular scale processes. Finally, our model elucidates processes involved in immunity to M. tuberculosis that may be new targets for therapy.     

Integrin organization: linking adhesion ligand nanopatterns with altered cell responses

This paper presents a modelling framework in which the mechanochemical properties of smooth muscle cells may be studied. The activation of smooth muscles is considered in a three-dimensional continuum model which is key to realistically capture the function of hollow organs such as blood vessels. On the basis of a general thermodynamical framework the mechanical and chemical phases are specialized in order to quantify the coupled mechanochemical process. A free-energy function is proposed as the sum of a mechanical energy stored in the passive tissue, a coupling between the mechanical and chemical kinetics and an energy related purely to the chemical kinetics and the calcium ion concentration. For the chemical phase it is shown that the cross-bridge model of Hai and Murphy [1988. Am. J. Physiol. Cell Physiol. 254, C99-C106] is included in the developed evolution law as a special case. In order to show the specific features and the potential of the proposed continuum model a uniaxial extension test of a tissue strip is analysed in detail and the related kinematics and stress-stretch relations are derived. Parameter studies point to coupling phenomena; in particular the tissue response is analysed in terms of the calcium ion level. The model for smooth muscle contraction may significantly contribute to current modelling efforts of smooth muscle tissue responses.     

A comparison of random vs. chemotaxis-driven contacts of T cells with dendritic cells during repertoire scanning

Generating adaptive immunity after infection or immunization requires physical interactions within a lymph node (LN) T-zone between antigen-bearing dendritic cells (DCs) that arrive from peripheral tissues and rare cognate T cells entering via high endothelial venules (HEVs). This interaction results in activation of cognate T cells, expansion of that T cell lineage and their exit from the LN T-zone via efferent lymphatics (ELs). How antigen-specific T cells locate DCs within this complex environment is controversial, and both random T cell migration and chemotaxis have been proposed. We developed an agent-based computational model of a LN that captures many features of T cell and DC dynamics observed by two-photon microscopy. Our simulations matched in vivo two-photon microscopy data regarding T cell speed, short-term directional persistence of motion and cell motility. We also obtained in vivo data regarding density of T cells and DCs within a LN and matched our model environment to measurements of the distance from HEVs to ELs. We used our model to compare chemotaxis with random motion and showed that chemotaxis increased total number of T cell DC contacts, but decreased unique contacts, producing fewer activated T cells. Our results suggest that, within a LN T-zone, a random search strategy is optimal for a rare cognate T cell to find its DC match and maximize production of activated T cells.     

Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: can a single model explain disparate results?

The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTPγS), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) >GTPγS-binding assays >cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data.     

Quantification of fibronectin adsorption to silicone-rubber cell culture substrates

As the role of mechanical force in cellular signaling gained recognition, investigators designed a number of devices to deliver controlled regimens of mechanical force to cultured cells. One type of device uses thin silicone-rubber membranes to support monolayer cell adhesion and to transmit mechanical force in the form of biaxial strain. We have observed that cell attachment and spreading are impaired on these membranes compared to polystyrene, even when both are passively coated with identical amounts of extracellular matrix. The purpose of these studies was to quantify the efficiency and stability of passive matrix adsorption onto commercially available elastic culture substrates. A theoretically saturating density (1 microg/cm2) of fibronectin was added to each well, and the initial efficiency of adsorption to the walls and elastic membranes was found to be 31 +/- 2% of the protein added. Strikingly, when the protein adsorbed specifically to the membranes was quantified after seven days, only 10-26 ng/cm2 fibronectin were present, revealing that most of the adsorption is to the sides of the wells. These results indicate that the adsorption of matrix proteins to silicone-rubber substrates is relatively inefficient and that investigators who use these systems must be aware of this fact and design their experiments accordingly.     

Untangling ligand induced activation and desensitization of G-protein-coupled receptors

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.