A multistage model for the action of cytotoxic T lymphocytes in multicellular conjugates

We propose a multistage stochastic model to explain data on the kinetics of target cell lysis by cytotoxic T lymphocytes in multicellular conjugates. A novel feature of our model is that we explicitly consider both the lethal hitting stage and the target cell disintegration stage of the cytolytic process. Further, we allow for the possibility that target cell disintegration is itself a complex process composed of many events. The comparison of our model with the data of other investigators suggests that cytotoxic T cells deliver lethal hits at random to undamaged target cells. Having received a lethal hit, the target cell disintegrates over a variable length of time. The disintegration times of target cells from different conjugates appear to be randomly distributed and to be consistent with a model in which disintegration occurs by at least two major, sequential, rate-limiting events. For conjugates containing one lymphocyte and multiple target cells, the mean rate at which a lethally hit target cell disintegrates is found to be independent of the total number of target cells in the conjugate. Our model predicts that in such multicellular conjugates, individual target cells lyse one by one, on average at approximately 30-min intervals, thus agreeing closely with previously reported experimental observations.     

Effects of radiation on host-tumor interactions using the multicellular tumor spheroid model

Summary Use of the multicellular tumor spheroid as a tumor model allows separate host or tumor treatment with ionizing radiation and examination of the effects on host-tumor immune interactions. Spheroids of EMT6/Ro, a BALB/c mammary tumor were implanted into the peritoneal cavity of syngeneic immunized mice, recovered, and dissociated into single cells. Cytolytic activity of mature spheroid associated cells and peritoneal cells was resistant to radiation doses as high as 1000 rads when irradiated directly prior to assay. Mice irradiated (200, 400, 700 rads) 24 h prior to spheroid injection had an increased number of tumor cells and decreased number of tumor infiltrating and peritoneal host cells upon spheroid recovery. This was paralleled by an increased colony forming efficiency per spheroid. Cytolytic activity of the spheroid associated cells against radiolabeled EMT6 cells was in many cases decreased with radiation although lysis was the same on a per cell basis. Cytolytic activity by peritoneal cells from these mice increased with dose as measured on a per cell basis. This activity from irradiated animals was carried out by a Thyl⁺ cell.     

Exploiting ligand-protein conjugates to monitor ligand-receptor interactions

We introduce three assays for analyzing ligand-receptor interactions based on the specific conjugation of ligands to SNAP-tag fusion proteins. Conjugation of ligands to different SNAP-tag fusions permits the validation of suspected interactions in cell extracts and fixed cells as well as the establishment of high-throughput assays. The different assays allow the analysis of strong and weak interactions. Conversion of ligands into SNAP-tag substrates thus provides access to a powerful toolbox for the analysis of their interactions with proteins.     

Modelling density-dependent resistance in insect-pathogen interactions.

We consider a mathematical model for a host-pathogen interaction where the host population is split into two categories: those susceptible to disease and those resistant to disease. Since the model was motivated by studies on insect populations, we consider a discrete-time model to reflect the discrete generations which are common among insect species. Whether an individual is born susceptible or resistant to disease depends on the local population levels at the start of each generation. In particular, we are interested in the case where the fraction of resistant individuals in the population increases as the total population increases. This may be seen as a positive feedback mechanism since disease is the only population control imposed upon the system. Moreover, it reflects recent experimental observations from noctuid moth-baculovirus interactions that pathogen resistance may increase with larval density. We find that the inclusion of a resistant class can stabilise unstable host-pathogen interactions but there is greatest regulation when the fraction born resistant is density independent. Nonetheless, inclusion of density dependence can still allow intrinsically unstable host-pathogen dynamics to be stabilised provided that this effect is sufficiently small. Moreover, inclusion of density-dependent resistance to disease allows the system to give rise to bistable dynamics in which the final outcome is dictated by the initial conditions for the model system. This has implications for the management of agricultural pests using biocontrol agents-in particular, it is suggested that the propensity for density-dependent resistance be determined prior to such a biocontrol attempt in order to be sure that this will result in the prevention of pest outbreaks, rather than their facilitation. Finally we consider how the cost of resistance to disease affects model outcomes and discover that when there is no cost to resistance, the model predicts stable periodic outbreaks of the insect population. The results are interpreted ecologically and future avenues for research to address the shortfalls in the present model system are discussed.     

Cell interactions of enkephalin/polypeptide conjugates.

Enkephalin molecules were bound to poly(Lys) (poly-K) or poly(Ala-Lys-Ala-Leu) (poly-A) and their interactions with NG108-15 cells, platelets, erythrocytes and fibroblast cells were investigated. A fluorescent probe, rhodamine, also was bound to the conjugates for monitoring interactions with these cells. Observations by fluorescence microscopy revealed that NG108-15 cells, platelets, and fibroblast cells were labelled by the conjugates, whereas erythrocytes were not. Since polypeptides without enkephalin moieties were only weakly adsorbed on the cells, it was concluded that the enkephalin/polypeptide conjugates were bound specifically to receptors on the cell membrane. Interestingly, when the enkephalin/poly-K conjugate was bound to NG108-15 and fibroblast cells, fluorescent patches appeared on the membrane. Such patch formation was not clearly observed with an enkephalin/rhodamine or enkephalin/poly-A conjugate. In the case of fibroblast cells, the fluorescence converged to a large cluster, which was ultimately internalized. The results suggest that clustering of the receptors in cell membranes is influenced by the carrier polymer presumably due to cross-linking of the receptors and/or the effect of the cationic polypeptides.     

Modelling the interactions of protein side-chains

The study of small model molecules containing the relevant functional groups can help us to understand the interactions between side-chains in proteins.Ab initio quantum chemical techniques allow the interactions between the model molecules to be studied with much greater accuracy than is possible for an entire protein, where the use of simple empirical potentials is the norm. In particular, the use ofab initio methods on model molecules permits us to incorporate the atom-atom anisotropic directionality of these interactions. We survey various methods of obtaining the components of theab initio interaction energy. These are then applied to three systems of biological interest. The first of these is the arginine/aspartate pair found in salt bridges, which involves hydrogen bonding between two charged species. Secondly, we look at the arginine/phosphotyrosine interaction found in complexes between SH2 domains and peptide ligands: here we find that the arginine/phosphate part of the interaction is energetically far more important than the arginine/aromatic part. Finally, we describe a detailed study of amino/aromatic interactions in proteins: unconventional hydrogen bonds are found to be remarkably uncommon relative to stacked geometries, and the reasons for this are examined.     

Modelling spatio-temporal interactions within the cell

Biological phenomena at the cellular level can be represented by various types of mathematical formulations. Such representations allow us to carry out numerical simulations that provide mechanistic insights into complex behaviours of biological systems and also generate hypotheses that can be experimentally tested. Currently, we are particularly interested in spatio-temporal representations of dynamic cellular phenomena and how such models can be used to understand biological specificity in functional responses. This review describes the capability and limitations of the approaches used to study spatio-temporal dynamics of cell signalling components.     

Understanding protein-protein interactions by genetic suppression

Protein-protein interactions influence many cellular processes and it is increasingly being felt that even a weak and remote interplay between two subunits of a protein or between two proteins in a complex may govern the fate of a particular biochemical pathway. In a bacterial system where the complete genome sequence is available, it is an arduous task to assign function to a large number of proteins. It is possible that many of them are peripherally associated with a cellular event and it is very difficult to probe such interaction. However, mutations in the genes that encode such proteins (primary mutations) are useful in these studies. Isolation of a suppressor or a second-site mutation that restores the phenotype abolished by the primary mutation could be an elegant yet simple way to follow a set of interacting proteins. Such a reversion site need not necessarily be geometrically close to the primary mutation site.     

A thermoplastic microfluidic microphysiological system to recapitulate hepatic function and multicellular interactions

Hepatic in vitro platforms ranging from multi-well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next-generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high-throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue-culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media-to-cell ratio in comparison with multi-well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte-Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic-based high-throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi-cellular interactions and therapeutic responses.     

Synthesis of deguelin-biotin conjugates and investigation into deguelin's interactions

Deguelin, a rotenoid, has emerged as an attractive pharmacophore for chemoprevention showing in vivo activity in several xenografts. Recently, several lines of evidence have suggested its mode of action may involve inhibition of HSP90, however binding in a different mode than known pharmacophores. To further probe the target of deguelin and related rotenoids, several biotin conjugates were prepared. None of the conjugates showed significant affinity for HSP90, however two conjugates showed a strong cellular co-localization with mitochondria, consistent with binding to mitochondrial complex 1. Contrarily to rotenone, deguelin and tephrosin were not found to inhibit tubulin polymerization demonstrating a dramatic pharmacological difference between these closely related rotenoids.     

Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

Multivalent poly(ethylene glycol)-containing conjugates for in vivo antibody suppression

Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of beta(2)GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-beta(2)GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with (125)I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress anti-domain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.     

Modelling molecular interactions with game networks' theory

We present a method to model biological systems, the theory of games networks. It extends game theory by multiplying the number of games, and by allowing agents to play several games simultaneously. Some important notions of biological systems, such as locality of interactions and modularity, can then be modelled.     

Regulatory T cells in pregnancy. VI. Evidence for T-cell-mediated suppression of CTL generation toward paternal alloantigens

The reactivity of spleen cells from allogeneically pregnant mice was assayed versus paternal strain target cells by a direct ⁵¹Cr-release assay. Despite multiple allogeneic parities, the lytic indexes of spleen cells were equivalent to those observed with nonpregnant controls. In view of previously obtained in vivo and in vitro results, spleen cells of allogeneically pregnant mice were added at the onset of MLC-CMLs³ of maternal strain responder cells versus paternal strain stimulator and target cells and studied for regulatory capacities. They did exert a suppressive effect, assessed by ⁵¹Cr release per culture. For the most, this effect was on the CTL induction, not the effector phase. The suppression of CTL generation was specific and mediated by a Thy 1⁺, Ly 2⁺ cell.     

Taste-smell interactions are tastant and odorant dependent

Four experiments were conducted to assess the nature of taste–smellinteractions. In the first experiment, the ability of strawberryodor to modify the sweetness of sucrose was investigated. Thiswas accomplished by having subjects rate the sweetness of whipped-creamstimuli with and without strawberry odor over time. The stimuliwere swallowed to augment retronasal stimulation of the olfactorysystem. It was found that strawberry odor tended to enhancethe maximal sweetness and total rating time of the stimuli.In the second experiment, it was found that peanut butter odordid not enhance sweetness, thus demonstrating that an odor'sability to enhance sweetness is odor-dependent, In the thirdexperiment, it was demonstrated that strawberry odor did notenhance the saltiness of sodium chloride indicating that anodor's ability to enhance taste is tastant-dependent. In thefourth experiment, it was shown that 85% of the strawberry odorant'sability to enhance sweetness was eliminated by pinching thenostrils. This suggests that the influence of the strawberryodorant on sweetness was olfactory rather than gustatory. Itwas concluded that an odor's influence on taste is both odorantand tastant dependent.