Simulation of receptor triggering by kinetic segregation shows role of oligomers and close contacts.

The activation of T cells, key players of the immune system, involves local evacuation of phosphatase CD45 from a region of the T cell's surface, segregating it from the T cell receptor. What drives this evacuation? In the presence of antigen, what ensures evacuation happens in the subsecond timescales necessary to initiate signaling? In the absence of antigen, what mechanisms ensure that evacuation does not happen spontaneously, which could cause signaling errors? Phenomena known to influence spatial organization of CD45 or similar surface molecules include diffusive motion in the lipid bilayer, oligomerization reactions, and mechanical compression against a nearby surface, such as that of the cell presenting the antigen. Computer simulations can investigate hypothesized spatiotemporal mechanisms of T cell signaling. The challenge to computational studies of evacuation is that the base process, spontaneous evacuation by simple diffusion, is in the extreme rare event limit, meaning direct stochastic simulation is unfeasible. Here, we combine particle-based spatial stochastic simulation with the weighted ensemble method for rare events to compute the mean first passage time for cell surface availability by surface reorganization of CD45. We confirm mathematical estimates that, at physiological concentrations, spontaneous evacuation is extremely rare, roughly 300 years. We find that dimerization decreases the time required for evacuation. A weak bimolecular interaction (dissociation constant estimate 460 µM) is sufficient for an order of magnitude reduction of spontaneous evacuation times, and oligomerization to hexamers reduces times to below 1 s. This introduces a mechanism whereby externally induced CD45 oligomerization could significantly modify T cell function. For large regions of close contact, such as those induced by large microvilli, molecular size and compressibility imply a nonzero reentry probability of 60%, decreasing evacuation times. Simulations show that these reduced evacuation times are still unrealistically long (even with a fourfold variation centered around previous estimates of parameters), suggesting that a yet-to-be-described mechanism, besides compressional exclusion at a close contact, drives evacuation.

Stochastic modeling reveals kinetic heterogeneity in post-replication DNA methylation.

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.

Plant silicon application alters leaf alkaloid concentrations and impacts parasitoids more adversely than their aphid hosts.

Grasses accumulate large amounts of silicon (Si) which acts as a highly effective physical defence against insect herbivory, however recent evidence shows that Si supplementation also modifies plant secondary metabolite concetrations. Changes in plant secondary metabolites concentrations can have cascading effects on higher trophic levels, such as parasitoids, as they are dependent on the host herbivore for growth and development. However, relatively little is known about how Si application affects higher trophic levels. We examined the effects of Si addition on alkaloid content in leaves of Phalaris aquatica (Poaceae) and the effect on interactions between an aphid (Rhopalosiphum padi) and its parasitoid (Aphidius colemani). Si supplementation had no effect on aphid abundance or parasitism rate. Adult aphids, aphid mummies (parasitised aphids) and the emergent parasitoids were, however, significantly smaller on Si+ plants. Parasitoid traits (size and emergence) were correlated with aphid mummy size. Si addition reduced parasitoid emergence rate and size due to reduced host mummy size, in addition, significantly fewer females emerged from mummies on Si+ plants. Aphid infestation significantly altered alkaloids concentrations, reducing gramine by 80% while increasing tryptamine by 91% in Si- plants. Si addition reduced aphid-induced tryptamine concentrations by 64% and increased 5-MeO-tryptamine by over 800% in control and 142% in aphid infested plants. Our results show that while Si addition has modest impacts on the herbivore, it significantly alters secondary metabolites and has stronger effects on the higher trophic level through changes in the quality of the parasitised host.

Solving Immunology?

Emergent responses of the immune system result from the integration of molecular and cellular networks over time and across multiple organs. High-content and high-throughput analysis technologies, concomitantly with data-driven and mechanistic modeling, hold promise for the systematic interrogation of these complex pathways. However, connecting genetic variation and molecular mechanisms to individual phenotypes and health outcomes has proven elusive. Gaps remain in data, and disagreements persist about the value of mechanistic modeling for immunology. Here, we present the perspectives that emerged from the National Institute of Allergy and Infectious Disease (NIAID) workshop 'Complex Systems Science, Modeling and Immunity' and subsequent discussions regarding the potential synergy of high-throughput data acquisition, data-driven modeling, and mechanistic modeling to define new mechanisms of immunological disease and to accelerate the translation of these insights into therapies.

Hydrodynamics of transient cell-cell contact: The role of membrane permeability and active protrusion length.

In many biological settings, two or more cells come into physical contact to form a cell-cell interface. In some cases, the cell-cell contact must be transient, forming on timescales of seconds. One example is offered by the T cell, an immune cell which must attach to the surface of other cells in order to decipher information about disease. The aspect ratio of these interfaces (tens of nanometers thick and tens of micrometers in diameter) puts them into the thin-layer limit, or "lubrication limit", of fluid dynamics. A key question is how the receptors and ligands on opposing cells come into contact. What are the relative roles of thermal undulations of the plasma membrane and deterministic forces from active filopodia? We use a computational fluid dynamics algorithm capable of simulating 10-nanometer-scale fluid-structure interactions with thermal fluctuations up to seconds- and microns-scales. We use this to simulate two opposing membranes, variously including thermal fluctuations, active forces, and membrane permeability. In some regimes dominated by thermal fluctuations, proximity is a rare event, which we capture by computing mean first-passage times using a Weighted Ensemble rare-event computational method. Our results demonstrate a parameter regime in which the time it takes for an active force to drive local contact actually increases if the cells are being held closer together (e.g., by nonspecific adhesion), a phenomenon we attribute to the thin-layer effect. This leads to an optimal initial cell-cell separation for fastest receptor-ligand binding, which could have relevance for the role of cellular protrusions like microvilli. We reproduce a previous experimental observation that fluctuation spatial scales are largely unaffected, but timescales are dramatically slowed, by the thin-layer effect. We also find that membrane permeability would need to be above physiological levels to abrogate the thin-layer effect.

Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry.

Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC-HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure-activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.

Balint-style reflective practice groups in a year 4 undergraduate general practice attachment: experience of the first two years.

BACKGROUND AND AIMS: Many undergraduate medical curricula include reflective practice sessions based on traditional Balint-groups. Those sessions can help students to acknowledge that experiencing 'negative' feelings in relation to patients is normal and may contain important information about the clinical encounter. They may also help to protect students from some of the emotional challenges of studying medicine. The Edinburgh University scheme provides all students in their first clinical year with two dedicated reflective practice sessions. Here we report on experience of the first two years. METHODS: Students' attitudes to the sessions were ascertained using a questionnaire, and views of the group leaders were assessed using a questionnaire and through informal verbal and email discussions. Practical difficulties were recorded as they arose. RESULTS: Students generally rated the sessions positively with regard to exploring relationships and self-reflection, and they found the sessions interesting and helpful. The sessions did not seem to affect career choice. The free-text comments suggested four positive themes and four areas for future modification. CONCLUSION: We have succeeded in providing all undergraduate students with an opportunity to take part in a reflective practice. We have highlighted aspects which have been successful and suggested future improvements.

Rare-event sampling of epigenetic landscapes and phenotype transitions.

Stochastic simulation has been a powerful tool for studying the dynamics of gene regulatory networks, particularly in terms of understanding how cell-phenotype stability and fate-transitions are impacted by noisy gene expression. However, gene networks often have dynamics characterized by multiple attractors. Stochastic simulation is often inefficient for such systems, because most of the simulation time is spent waiting for rare, barrier-crossing events to occur. We present a rare-event simulation-based method for computing epigenetic landscapes and phenotype-transitions in metastable gene networks. Our computational pipeline was inspired by studies of metastability and barrier-crossing in protein folding, and provides an automated means of computing and visualizing essential stationary and dynamic information that is generally inaccessible to conventional simulation. Applied to a network model of pluripotency in Embryonic Stem Cells, our simulations revealed rare phenotypes and approximately Markovian transitions among phenotype-states, occurring with a broad range of timescales. The relative probabilities of phenotypes and the transition paths linking pluripotency and differentiation are sensitive to global kinetic parameters governing transcription factor-DNA binding kinetics. Our approach significantly expands the capability of stochastic simulation to investigate gene regulatory network dynamics, which may help guide rational cell reprogramming strategies. Our approach is also generalizable to other types of molecular networks and stochastic dynamics frameworks.

Preclinical studies for a phase 1 clinical trial of autologous hematopoietic stem cell gene therapy for sickle cell disease.

BACKGROUND AIMS: Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials. METHODS: Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/ßAS3-FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/ßAS3-FB LV in the context of a murine BM transplant. RESULTS: Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/ßAS3-FB vector in cells from primary and secondary transplanted mice. CONCLUSIONS: We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.

Effects of thymic selection of the T-cell repertoire on HLA class I-associated control of HIV infection.

Without therapy, most people infected with human immunodeficiency virus (HIV) ultimately progress to AIDS. Rare individuals ('elite controllers') maintain very low levels of HIV RNA without therapy, thereby making disease progression and transmission unlikely. Certain HLA class I alleles are markedly enriched in elite controllers, with the highest association observed for HLA-B57 (ref. 1). Because HLA molecules present viral peptides that activate CD8(+) T cells, an immune-mediated mechanism is probably responsible for superior control of HIV. Here we describe how the peptide-binding characteristics of HLA-B57 molecules affect thymic development such that, compared to other HLA-restricted T cells, a larger fraction of the naive repertoire of B57-restricted clones recognizes a viral epitope, and these T cells are more cross-reactive to mutants of targeted epitopes. Our calculations predict that such a T-cell repertoire imposes strong immune pressure on immunodominant HIV epitopes and emergent mutants, thereby promoting efficient control of the virus. Supporting these predictions, in a large cohort of HLA-typed individuals, our experiments show that the relative ability of HLA-B alleles to control HIV correlates with their peptide-binding characteristics that affect thymic development. Our results provide a conceptual framework that unifies diverse empirical observations, and have implications for vaccination strategies.

DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

Differences in the phenotype, cytokine gene expression profiles, and in vivo alloreactivity of T cells mobilized with plerixafor compared with G-CSF.

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Stochastic effects are important in intrahost HIV evolution even when viral loads are high.

Blood plasma viral loads and the time to progress to AIDS differ widely among untreated HIV-infected humans. Although people with certain HLA (HLA-I) alleles are more likely to control HIV infections without therapy, the majority of such untreated individuals exhibit high viral loads and progress to AIDS. Stochastic effects are considered unimportant for evolutionary dynamics in HIV-infected people when viral load is high or when selective forces strongly drive mutation. We describe a computational study of host-pathogen interaction demonstrating that stochastic effects can have a profound influence on disease dynamics, even in cases of high viral load and strong selective pressure. These stochastic effects are pronounced when the virus must traverse a fitness "barrier" in sequence space to escape the host's cytotoxic T-lymphocyte (CTL) response, as often occurs when a fitness defect imposed by a CTL-driven mutation must be compensated for by other mutations. These "barrier-crossing" events are infrequent and stochastic, resulting in divergent disease outcomes in genetically identical individuals infected by the same viral strain. Our results reveal how genetic determinants of the CTL response control the probability with which an individual is able to control HIV infection indefinitely, and thus provide clues for vaccine design.

Evidence for wavelike energy transfer through quantum coherence in photosynthetic systems.

Photosynthetic complexes are exquisitely tuned to capture solar light efficiently, and then transmit the excitation energy to reaction centres, where long term energy storage is initiated. The energy transfer mechanism is often described by semiclassical models that invoke 'hopping' of excited-state populations along discrete energy levels. Two-dimensional Fourier transform electronic spectroscopy has mapped these energy levels and their coupling in the Fenna-Matthews-Olson (FMO) bacteriochlorophyll complex, which is found in green sulphur bacteria and acts as an energy 'wire' connecting a large peripheral light-harvesting antenna, the chlorosome, to the reaction centre. The spectroscopic data clearly document the dependence of the dominant energy transport pathways on the spatial properties of the excited-state wavefunctions of the whole bacteriochlorophyll complex. But the intricate dynamics of quantum coherence, which has no classical analogue, was largely neglected in the analyses-even though electronic energy transfer involving oscillatory populations of donors and acceptors was first discussed more than 70 years ago, and electronic quantum beats arising from quantum coherence in photosynthetic complexes have been predicted and indirectly observed. Here we extend previous two-dimensional electronic spectroscopy investigations of the FMO bacteriochlorophyll complex, and obtain direct evidence for remarkably long-lived electronic quantum coherence playing an important part in energy transfer processes within this system. The quantum coherence manifests itself in characteristic, directly observable quantum beating signals among the excitons within the Chlorobium tepidum FMO complex at 77 K. This wavelike characteristic of the energy transfer within the photosynthetic complex can explain its extreme efficiency, in that it allows the complexes to sample vast areas of phase space to find the most efficient path.

Locally correlated kinetics of post-replication DNA methylation reveals processivity and region specificity in DNA methylation maintenance.

DNA methylation occurs predominantly on cytosine-phosphate-guanine (CpG) dinucleotides in the mammalian genome, and the methylation landscape is maintained over mitotic cell division. It has been posited that coupling of maintenance methylation activity among neighbouring CpGs is critical to stability over cellular generations; however, the mechanism is unclear. We used mathematical models and stochastic simulation to analyse data from experiments that probe genome-wide methylation of nascent DNA post-replication in cells. We find that DNA methylation maintenance rates on individual CpGs are locally correlated, and the degree of this correlation varies by genomic regional context. By using theory of protein diffusion along DNA, we show that exponential decay of methylation rate correlation with genomic distance is consistent with enzyme processivity. Our results provide quantitative evidence of genome-wide methyltransferase processivity in vivo. We further developed a method to disentangle different mechanistic sources of kinetic correlations. From the experimental data, we estimate that an individual methyltransferase methylates neighbour CpGs processively if they are 36 basepairs apart, on average. But other mechanisms of coupling dominate for longer inter-CpG distances. Our study demonstrates that quantitative insights into enzymatic mechanisms can be obtained from replication-associated, cell-based genome-wide measurements, by combining data-driven statistical analyses with hypothesis-driven mathematical modelling.

New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data.

Cheese microbiota and metabolites and their inter-relationships that underpin specific cheese quality attributes remain poorly understood. Here we report that multi-omics and integrative data analysis (multiple co-inertia analysis, MCIA) can be used to gain deeper insights into these relationships and identify microbiota and metabolite fingerprints that could be used to monitor product quality and authenticity. Our study into different brands of artisanal and industrial cheddar cheeses showed that Streptococcus, Lactococcus and Lactobacillus were the dominant taxa with overall microbial community structures differing not only between industrial and artisanal cheeses but also among different cheese brands. Metabolome analysis also revealed qualitative and semi-quantitative differences in metabolites between different cheeses. This also included the presence of two compounds (3-hydroxy propanoic acid and O-methoxycatechol-O-sulphate) in artisanal cheese that have not been previously reported in any type of cheese. Integrative analysis of multi-omics datasets revealed that highly similar cheeses, identical in age and appearance, could be distinctively clustered according to cheese type and brand. Furthermore, the analysis detected strong relationships, some previously unknown, which existed between the cheese microbiota and metabolome, and uncovered specific taxa and metabolites that contributed to these relationships. These results highlight the potential of this approach for identifying product specific microbe/metabolite signatures that could be used to monitor and control cheese quality and product authenticity.

Photon echo studies of photosynthetic light harvesting.

The broad linewidths in absorption spectra of photosynthetic complexes obscure information related to their structure and function. Photon echo techniques represent a powerful class of time-resolved electronic spectroscopy that allow researchers to probe the interactions normally hidden under broad linewidths with sufficient time resolution to follow the fastest energy transfer events in light harvesting. Here, we outline the technical approach and applications of two types of photon echo experiments: the photon echo peak shift and two-dimensional (2D) Fourier transform photon echo spectroscopy. We review several extensions of these techniques to photosynthetic complexes. Photon echo peak shift spectroscopy can be used to determine the strength of coupling between a pigment and its surrounding environment including neighboring pigments and to quantify timescales of energy transfer. Two-dimensional spectroscopy yields a frequency-resolved map of absorption and emission processes, allowing coupling interactions and energy transfer pathways to be viewed directly. Furthermore, 2D spectroscopy reveals structural information such as the relative orientations of coupled transitions. Both classes of experiments can be used to probe the quantum mechanical nature of photosynthetic light-harvesting: peak shift experiments allow quantification of correlated energetic fluctuations between pigments, while 2D techniques measure quantum beating directly, both of which indicate the extent of quantum coherence over multiple pigment sites in the protein complex. The mechanistic and structural information obtained by these techniques reveals valuable insights into the design principles of photosynthetic light-harvesting complexes, and a multitude of variations on the methods outlined here.

Detection of migration of locally implanted AC133+ stem cells by cellular magnetic resonance imaging with histological findings.

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB), platelet-derived growth factor (PDGF), hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1 (SDF-1), matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells, both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1, MMP-2, and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells.

Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes.

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product. STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-gamma (IFN-gamma). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated. RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-gamma and soluble CD40L/IFN-gamma were used. CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.

Pigment organization and energy level structure in light-harvesting complex 4: insights from two-dimensional electronic spectroscopy.

Photosynthetic light-harvesting antennae direct energy collected from sunlight to reaction centers with remarkable efficiency and rapidity. Despite their common function, the pigment-protein complexes that make up antenna systems in different types of photosynthetic organisms exhibit a wide variety of structural forms. Some individual organisms express different types of complexes depending on growth conditions. For example, purple photosynthetic bacteria Rp. palustris preferentially synthesize light-harvesting complex 4 (LH4), a structural variant of the more common and widely studied LH2, when grown under low-light conditions. Here, we investigate the ultrafast dynamics and energy level structure of LH4 using two-dimensional (2D) electronic spectroscopy in combination with theoretical simulations. The experimental data reveal dynamics on two distinct time scales, consistent with coherent dephasing within approximately the first 100 fs, followed by relaxation of population into lower-energy states on a picosecond time scale. We observe excited state absorption (ESA) features marking the existence of high-energy dark states, which suggest that the strongest dipole-dipole coupling in the complex occurs between bacteriochlorophyll transition dipole moments in an in-line geometry. The results help to refine the current understanding of the pigment organization in the LH4 complex, for which a high-resolution crystal structure is not yet available.