Toward quantifying the thymic dysfunctional state in mouse models of inflammatory bowel disease.

Inflammatory bowel disease is characterized by a number of immunological alterations, not the least in the T-cell compartment. Numerous animal models of colitis have revealed aberrant thymocyte dynamics associated with skewed thymocyte development. The recent advancements in quantitative methods have proposed critical kinetic alterations in the thymocyte development during the progression of colitis. This review focuses on the aberrant thymocyte dynamics in Gαi2-deficient mice as this mouse model provides most quantitative data of the thymocyte development associated with colitis. Herein, we discuss several dynamic changes during the progression of colitis and propose a hypothesis for the underlying causes for the skewed proportions of the thymocyte populations seen in the Gαi2-deficient mice and in other mouse models of colitis.

Structure and interactions of the human programmed cell death 1 receptor.

PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

A qualitative study on caretakers' perceived need of bed-nets after reduced malaria transmission in Zanzibar, Tanzania.

BACKGROUND: The elimination of malaria in Zanzibar is highly dependent on sustained effective coverage of bed-nets to avoid malaria resurgence. The Health Belief Model (HBM) framework was used to explore the perceptions of malaria and bed-net use after a noticeable reduction in malaria incidence. METHODS: Nineteen in-depth interviews were conducted with female and male caretakers of children under five in North A district, Zanzibar. Deductive content analysis was used to identify meaning units that were condensed, coded and assigned to pre-determined elements of the HBM. RESULTS: Awareness of malaria among caretakers was high but the illness was now seen as easily curable and uncommon. In addition to the perceived advantage of providing protection against malaria, bed-nets were also thought to be useful for avoiding mosquito nuisance, especially during the rainy season when the malaria and mosquito burden is high. The discomfort of sleeping under a net during the hot season was the main barrier that interrupted consistent bed-net usage. The main cue to using a bed-net was high mosquito density, and children were prioritized when it came to bed-net usage. Caretakers had high perceived self-efficacy and did not find it difficult to use bed-nets. Indoor Residual Spraying (IRS), which was recognized as an additional means of mosquito prevention, was not identified as an alternative for bed-nets. A barrier to net ownership was the increasingly high cost of bed-nets. CONCLUSIONS: Despite the reduction in malaria incidence and the resulting low malaria risk perceptions among caretakers, the benefit of bed-nets as the most proficient protection against mosquito bites upholds their use. This, in combination with the perceived high self-efficacy of caretakers, supports bed-net usage, while seasonality interrupts consistent use. High effective coverage of bed-nets could be further improved by reinforcing the benefits of bed-nets, addressing the seasonal heat barrier by using nets with larger mesh sizes and ensuring high bed-net ownership rates through sustainable and affordable delivery mechanisms.

A quantitative study of the mechanisms behind thymic atrophy in Gαi2-deficient mice during colitis development.

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2(-/-) mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2(-/-) mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2(-/-) mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2(-/-) SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4(+) and CD8(+) thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC(+) T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2(-/-) mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis.

Dynamic modelling of cell death during biofilm development.

Biofilms are currently recognised as the predominant bacterial life-style and it has been suggested that biofilm development is influenced by a number of different processes such as adhesion, detachment, mass transport, quorum sensing, cell death and active dispersal. One of the least understood processes and its effects on biofilm development is cell death. However, experimental studies suggest that bacterial death is an important process during biofilm development and many studies show a relationship between cell death and dispersal in microbial biofilms. We present a model of the process of cell death during biofilm development, with a particular focus on the spatial localisation of cell death or cell damage. Three rules governing cell death or cell damage were evaluated which compared the effects of starvation, damage accumulation, and viability during biofilm development and were also used to design laboratory based experiments to test the model. Results from model simulations show that actively growing biofilms develop steep nutrient gradients within the interior of the biofilm that affect neighbouring microcolonies resulting in cell death and detachment. Two of the rules indicated that high substrate concentrations lead to accelerated cell death, in contrast to the third rule, based on the accumulation of damage, which predicted earlier cell death for biofilms grown with low substrate concentrations. Comparison of the modelling results with experimental results suggests that cell death is favoured under low nutrient conditions and that the accumulation of damage may be the main cause of cell death during biofilm development.

Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue.

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca(2+)-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca(2+)-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

Quantitative analysis predicts the relative therapeutic efficacy of different forms of CTLA4Ig.

Modulating the activities of costimulatory molecules controlling immune responses holds considerable promise for immunotherapy. CTLA4Ig (abatacept), a soluble version of the T cell-expressed membrane receptor CTLA-4, is approved for the treatment of rheumatoid arthritis. Like natural CTLA-4 molecules, CTLA4Ig ligates B7-1 and B7-2 on antigen presenting cells, preventing CD28-mediated costimulation of T cells. However, CTLA4Ig can also prevent ligation of CTLA-4, potentially blocking vital inhibitory signals, thereby augmenting immunity. There have been no quantitative analyses of the likely effects of CTLA4Ig on costimulatory interactions at the immunological synapse. We present a mathematical model, based on rigorous biophysical and expression data, for simulating the effects of abatacept and a mutated derivative, LEA29Y, on the synaptic interactions of CD28 and CTLA-4. The simulations reveal an unexpectedly large window within which CD28, but not CTLA-4, ligation is blocked by CTLA4Ig, perhaps explaining the efficacy of abatacept at the recommended therapeutic dose (10mg/kg) and its relative safety. However, the simulations suggest that the present dosing regimen is close to the maximum theoretically safe dose. The simulations also show that, within the therapeutic window, LEA29Y enhances the interaction of CTLA-4 with the more potent of its two native ligands, B7-1. They also suggest that CTLA-4 ligation by B7-1 could, in principle, be enhanced by further decreasing the off-rate of CTLA4Ig for binding to B7-2. Our findings therefore offer molecular explanations for why LEA29Y might prove to be more effective than abatacept in a clinical setting, and suggest ways in which its therapeutic efficacy could be further optimised.

The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Kinetic proofreading and the search for nonself-peptides.

The T cells scan the surface of antigen-presenting cells with their T cell receptors (TCR) in order to find and respond to specific peptide-major histocompatibility complexes (pMHC). Since mainly self-peptides are expressed on antigen-presenting cells, the T cells must utilize sensitive mechanisms in order to quickly discriminate between self and nonself-peptides. A range of different models have been proposed to account for this process. Due to the experimental inconsistency of how T cells respond to altered peptides it has been difficult to validate the competing models. Recent models, based on the kinetic proofreading model, propose that a short life-time of the TCR/pMHC complexes may be compensated by fast rebinding of the individual molecules. Hence, both the on- and off-rate involved in the interaction between pMHCs and TCRs will determine the fate of the T cell discrimination. I here briefly review some of the proposed models on T cell discrimination and scanning, and discuss the significance of determining self-peptide kinetics to validate the different models.

A mathematical framework for analyzing T cell receptor scanning of peptides.

T cells continuously search for antigenic peptides presented on major histocompatibility complexes expressed on nearly all nucleated cells. Because only a few antigenic peptides are presented in a sea of thousands of self-peptides, the T cells have a critical task in discriminating between self- and nonself-peptides. This search process for antigens must be performed with sufficient speed in order to induce a fast response against invading pathogens. This study presents a mathematical framework for analyzing the scanning process of peptides. The framework includes analytic expressions for calculating the sampling rate as well as continuous-systems- and stochastic-agent-based models. The results show that the scanning of self-peptides is a very fast process due to fast off-rates. The simulations also predict the existence of an optimal sampling rate for a certain range of on-rates based on the recently proposed confinement time model. Calculations reveal that most of the self-peptides located within a microdomain are scanned within just a few seconds, and that the T cell receptors have kinetics for self-peptides, facilitating fast scanning. The derived mathematical expressions within this study provide conceptual calculations for further investigations of how the T cell discriminates between self- and nonself-peptides.

Biochemical modeling with Systems Biology Graphical Notation.

The Systems Biology Graphical Notation (SBGN) is an emerging standard for graphical notation developed by an international systems biology community. Standardized graphical notation is crucial for efficient and accurate communication of biological knowledge between researchers with various backgrounds in the expanding field of systems biology. Here, we highlight SBGN from a practical point of view and describe how the user can build and simulate SBGN models from a simple drag-and-drop graphical user interface in PathwayLab.

A kinetic overview of the receptors involved in 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 signaling: a systems biology approach.

The vitamin D endocrine system modulates an arsenal of important biological functions in more than 30 different tissues in short- and long-term perspectives. Two membrane receptors and one nuclear receptor are suggested to be involved in the vitamin D signaling system, but the function and physiological relevance of the receptors are debated. The complexity of the vitamin D endocrine system makes it necessary to combine experimental data with in silico simulations to get a holistic view of vitamin D-dependent regulation of tissue and cell physiology. This review focus on binding characteristics for the three putative vitamin D receptors and proposes a future systems biology approach including mathematical modeling that will be helpful together with experimental methods in depicting antitumoral and other biological effects promoted by the vitamin D endocrine system.

An individual-based network model to evaluate interventions for controlling pneumococcal transmission.

BACKGROUND: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, but also a common colonizer of the upper respiratory tract. The emergence and spread of antibiotic resistant pneumococcal strains has threatened effective therapy. The long-term effects of measures aiming to limit pneumococcal spread are poorly understood. Computational modeling makes it possible to conduct virtual experiments that are impractical to perform in real life and thereby allows a more full understanding of pneumococcal epidemiology and control efforts. METHODS: We have developed a contact network model to evaluate the efficacy of interventions aiming to control pneumococcal transmission. Demographic data from Sweden during the mid-2000s were employed. Analyses of the model's parameters were conducted to elucidate key determinants of pneumococcal spread. Also, scenario simulations were performed to assess candidate control measures. RESULTS: The model made good predictions of previous findings where a correlation has been found between age and pneumococcal carriage. Of the parameters tested, group size in day-care centers was shown to be one of the most important factors for pneumococcal transmission. Consistent results were generated from the scenario simulations. CONCLUSION: We recommend, based on the model predictions, that strategies to control pneumococcal disease and organism transmission should include reducing the group size in day-care centers.

3D computation modelling of the influence of cytokine secretion on Th-cell development suggests that negative selection (inhibition of Th1 cells) is more effective than positive selection by IL-4 for Th2 cell dominance.

Th-cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL-4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon-gamma (IFN-gamma) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL-4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL-4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL-4 ligand and its IL-4 receptors expressed on discrete IL-4 secreting cells. The simulations, based on existing experimental data on the IL-4 receptor-ligand system, illustrate how Th-cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 microm and that an activated Th2 cell manages to fill most of its own IL-4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL-4 for promoting Th2 dominance.

In silico simulations suggest that Th-cell development is regulated by both selective and instructive mechanisms.

Th-cell differentiation is highly influenced by the local cytokine environment. Although cytokines such as IL-12 and IL-4 are known to polarize the Th-cell response towards Th1 or Th2, respectively, it is not known whether these cytokines instruct the developmental fate of uncommitted Th cells or select cells that have already been committed through a stochastic process. We present an individual based model that accommodates both stochastic and deterministic processes to simulate the dynamic behaviour of selective versus instructive Th-cell development. The predictions made by each model show distinct behaviours, which are compared with experimental observations. The simulations show that the instructive model generates an exclusive Th1 or Th2 response in the absence of an external cytokine source, whereas the selective model favours coexistence of the phenotypes. A hybrid model, including both instructive and selective development, shows behaviour similar to either the selective or the instructive model dependent on the strength of activation. The hybrid model shows the closest qualitative agreement with a number of well-established experimental observations. The predictions by each model suggest that neither pure selective nor instructive Th development is likely to be functional as exclusive mechanisms in Th1/Th2 development.

A theoretical framework for quantitative analysis of the molecular basis of costimulation.

We present a theoretical framework for simulating the synaptic accumulation of the costimulatory molecules CD28, CTLA-4, B7-1, and B7-2, based on a system of mean-field, ordinary differential equations, and rigorous biophysical and expression data. The simulations show that binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects all profoundly influence complex formation at cellular interfaces. B7-2 engages 33-fold more CD28 than CTLA-4 at the synapse in contrast to B7-1, which ligates approximately 7-fold more CTLA-4 than CD28. Although B7-1 completely dominates interactions with CTLA-4, forming linear arrays of 7-18 receptor-ligand pairs, CTLA-4 is fully engaged by B7-2 when B7-1 is absent. Additional simulations reveal the sensitivity of CD28 interactions to modeled transport processes. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be due to the differential binding properties of B7-1 and B7-2 only. We discuss the apparent redundancy of B7-1 in the context of a potentially dynamic synaptic microenvironment, and in light of functions other than the direct enhancement of T cell inhibition by CTLA-4.