Vibronic coupling in molecular crystals: A Franck-Condon Herzberg-Teller model of H-aggregate fluorescence based on quantum chemical cluster calculations.

Here, we present a general approach to treating vibronic coupling in molecular crystals based on atomistic simulations of large clusters. Such clusters comprise model aggregates treated at the quantum chemical level embedded within a realistic environment treated at the molecular mechanics level. As we calculate ground and excited state equilibrium geometries and vibrational modes of model aggregates, our approach is able to capture effects arising from coupling to intermolecular degrees of freedom, absent from existing models relying on geometries and normal modes of single molecules. Using the geometries and vibrational modes of clusters, we are able to simulate the fluorescence spectra of aggregates for which the lowest excited state bears negligible oscillator strength (as is the case, e.g., ideal H-aggregates) by including both Franck-Condon (FC) and Herzberg-Teller (HT) vibronic transitions. The latter terms allow the adiabatic excited state of the cluster to couple with vibrations in a perturbative fashion via derivatives of the transition dipole moment along nuclear coordinates. While vibronic coupling simulations employing FC and HT terms are well established for single-molecules, to our knowledge this is the first time they are applied to molecular aggregates. Here, we apply this approach to the simulation of the low-temperature fluorescence spectrum of para-distyrylbenzene single-crystal H-aggregates and draw comparisons with coarse-grained Frenkel-Holstein approaches previously extensively applied to such systems.

The diene isomerization energies dataset: A difficult test for double-hybrid density functionals?

We have systematically analyzed the performance of some representative double-hybrid density functionals (including PBE0-DH, PBE-QIDH, PBE0-2, XYG3, XYGJ-OS, and xDH-PBE0) for a recently introduced database of diene isomerization energies. Double-hybrid models outperform their corresponding hybrid forms (for example, PBE0-DH, PBE0-2, and PBE-QIDH are more accurate than PBE0) and the XYG3, XYGJ-OS, and xDH-PBE0 functionals perform excellently, providing root mean square deviation values within "calibration accuracy." XYGJ-OS and xDH-PBE0 also rival the best performing post-Hartree-Fock methods at a substantially lower cost.

Dynamics of guest molecules in PHTP inclusion compounds as probed by solid-state NMR and fluorescence spectroscopy.

Partially deuterated 1,4-distyrylbenzene () is included into the pseudohexagonal nanochannels of perhydrotriphenylene (PHTP). The overall and intramolecular mobility of is investigated over a wide temperature range by (13)C, (2)H NMR as well as fluorescence spectroscopy. Simulations of the (2)H NMR spectral shapes reveal an overall wobble motion of in the channels with an amplitude of about 4 degrees at T = 220 K and 10 degrees at T = 410 K. Above T = 320 K the wobble motion is superimposed by localized 180 degrees flips of the terminal phenyl rings with a frequency of 10(6) Hz at T = 340 K. The activation energies of both types of motions are around 40 kJ mol(-1) which imply a strong sterical hindrance by the surrounding PHTP channels. The experimental vibrational structure of the fluorescence excitation spectra of is analyzed in terms of small amplitude ring torsional motions, which provide information about the spatial constraints on by the surrounding PHTP host matrix. Combining the results from NMR and fluorescence spectroscopy as well as of time-dependent density functional calculations yields the complete potential surfaces of the phenyl ring torsions. These results, which suggest that intramolecular mobility of is only reduced but not completely suppressed by the matrix, are corroborated by MD simulations. Unrealistically high potential barriers for phenyl ring flips are obtained from MD simulations using rigid PHTP matrices which demonstrate the importance of large amplitude motions of the PHTP host lattice for the mobility of the guest molecules.

Memory B cell responses and malaria.

Malaria is a serious cause of morbidity and mortality in millions of individuals each year. People living in endemic areas build up partial immunity only after repeated attacks of malaria over several years. At this meeting we discussed current knowledge about long-term protection and the challenges we face in the development of an effective malaria vaccine.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Regulation of CD40 function by its isoforms generated through alternative splicing.

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-kappa B, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

Isolation of dendritic cells from rat intestinal lymph and spleen.

Dendritic cells (DC) are rare cells in peripheral tissues, and their isolation from tissues is fraught with problems. Thus, the proportion of DC within a tissue that is extracted is unknown, isolation procedures may select for subpopulations, and the isolation procedure itself may affect their properties. As part of their life history, DC migrate from peripheral tissues, via peripheral, afferent lymph to lymph nodes, even in the absence of exogenous antigenic stimulation. They are extracted within the node and very few, if any, appear in efferent lymph. These lymph DC (L-DC) represent a population that has matured in the periphery, that may have acquired antigen (Ag), and that may be engaged in active Ag transport to lymph nodes. As such they are a physiologically relevant DC population. In large animals such as sheep and cattle, L-DC can be isolated by direct cannulation of peripheral lymphatics, but yields are relatively low. In rodents, direct cannulation of some peripheral lymphatics is possible (1), but yields of cells are minuscule. To get around this problem we and others (1 -7), have utilized lymphadenectomy as a means of collecting pseudo-afferent lymph. When lymph nodes are removed, over a period of weeks, the afferent and efferent lymphatics join as part of the healing process, leaving cells in peripheral lymph free to enter central lymph. Central lymphat ics are relatively easy to cannulate, and cannulation can be maintained for considerable periods of time (see Note 1).

Dendritic cell-B-cell interaction: dendritic cells provide B cells with CD40-independent proliferation signals and CD40-dependent survival signals.

Dendritic cells (DC) have recently been shown to play an important role in B-cell function. We have previously shown that DC can capture and retain unprocessed antigen in vitro and in vivo, and can transfer this antigen to naive B cells to initiate antigen-specific antibody responses. We also demonstrated that DC were providing B cells with isotype-switch signals independent of T cells but that T-cell help was essential for antibody production. In this study, using B cells and DC from wild type (WT) and CD40 knockout (CD40KO) mice we show that DC initiate proliferation of B cells independently of CD40, because WT or CD40KO DC could induce proliferation of WT or CD40KO B cells, but proliferation was greater in the absence of CD40. DC also provide B cells with survival signals as WT DC improved viability of B cells after a 5-day culture but survival was reduced in the absence of CD40 expression.

A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes.

This study identifies a dendritic cell (DC) subset that constitutively transports apoptotic intestinal epithelial cell remnants to T cell areas of mesenteric lymph nodes in vivo. Rat intestinal lymph contains two DC populations. Both populations have typical DC morphology, are major histocompatibility complex class II(hi), and express OX62, CD11c, and B7. CD4(+)/OX41(+) DCs are strong antigen-presenting cells (APCs). CD4(-)/OX41(-) DCs are weak APCs and contain cytoplasmic apoptotic DNA, epithelial cell-restricted cytokeratins, and nonspecific esterase (NSE)(+) inclusions, not seen in OX41(+) DCs. Identical patterns of NSE electrophoretic variants exist in CD4(-)/OX41(-) DCs, intestinal epithelial cells, and mesenteric node DCs but not in other DC populations, macrophages, or tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive DCs and strongly NSE(+) DCs are present in intestinal lamina propria. Peyer's patches and mesenteric but not other lymph nodes contain many strongly NSE(+) DCs in interfollicular and T cell areas. Similar DCs are seen in the ileum and in T cell areas of mesenteric nodes in gnotobiotic rats. These results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.

Dendritic cells, B cells and the regulation of antibody synthesis.

Dendritic cells (DC) are usually thought of as antigen-presenting cells for T cells. However, recent studies from our laboratory and those of others have shown that they have important roles in B-cell activation and regulation of antibody synthesis. Rat DC make short term interactions with resting B cells and these interactions can be stimulated by cross-linking molecules on either cell surface. These DC can retain antigen in native form for at least 36 h in vivo and in vitro and can subsequently release it for recognition by B cells. In vivo antibody responses induced by antigen-pulsed DC are skewed towards IgG. In vitro, naäive B cells incubated with antigen-pulsed DC subsequently secrete IgM and IgG when cultured with an antigen-specific CD4+ T-cell line, whereas if B cells are incubated with antigen without DC, only IgM is produced. These observations show that DC play an important role in the initiation of and regulation of antibody synthesis.

Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response.

Dendritic cells (DC) are thought to initiate Ab synthesis by activation of T cells, which then provide cytokine and cell-bound "help" to B cells. Here, we provide evidence that DC can capture and retain unprocessed Ag in vitro and in vivo, and can transfer this Ag to naive B cells to initiate a specific Ab response. The response is skewed with 4- to 13-fold higher titers of IgG than IgM, and the predominant subclasses of Ab produced in naive animals are those associated with Th2-type responses. Ag retention and the skew in class switching is a physiologic phenomenon because DC loaded with Ag in vivo and isolated 24 h later initiated a class-switched, Ag-specific Ab response in naive animals. In vitro studies confirmed that DC provide naive B cells with signals that are essential for the synthesis of class-switched Ab. Taken together, these observations show that DC have an important role in the initiation of Ab synthesis by direct interaction with B cells.

Regulation of cytoplasmic, surface and soluble forms of CD40 ligand in mouse B cells.

CD40 and CD40 ligand (CD40L) form one of most important receptor-ligand pairs that dock during T-B cell interactions as part of T-dependent antibody responses. It has been reported that among other cell types, B cells can express CD40L. Here we show that a large proportion of mouse B cells express CD40L in their cytoplasm, but not on the surface and that this is readily released as a soluble molecule. Thus, in their resting state up to 50% of mouse B cells express CD40L within their cytoplasm and both the proportion of cells expressing and the amount of CD40L is increased by signaling through immunoglobulin (Ig) or CD38. In contrast, T cell-derived signals such as CD40L (anti-CD40) or Th2-type cytokines cause a decrease in CD40L expression that is related to a release of a soluble form of the molecule from the cell. Supernatants from B cells activated with anti-Ig and anti-CD40 contain CD40L in a variety of forms (18 kDa, 33 kDa and 66 kDa) that are readily detectable by immunoprecipitation with CD40-Fc gamma fusion protein (CD40-Ig) followed by Western blotting with anti-CD40L antibody (MR1). The 33-kDa species is distinct from the 39-kDa membrane-bound molecule found in activated T cells or in resting B cells and appears to be a novel soluble form of CD40L. Inhibition of T cell-independent in vitro stimulation of B cells with CD40-Ig or anti-CD40L suggests that the B cell-derived soluble CD40L or CD40L expressed on the B cell surface can play a positive role in B cell proliferation.

Observations on memory B-cell development.

We dissect in this article the roles of CD40 and its ligand in memory B-cell formation. Our data indicate that CD40 ligation does not directly lead to GC formation but it plays an indirect role related to maturation of helper T cells; signalling is bidirectional, to B cells, via CD40, upregulating cytokine receptor expression and to T cells, via CD40L, causing secretion of cytokines necessary for GC initiation. Later in the GC, CD40 selects mutated B cells for entry into the memory pool. This second T-cell-mediated CD40 ligation has consequences distinct from the first (rescue versus proliferation) that arise from rewiring of CD40 signal transduction pathways.

Murine cytomegalovirus interacts with major histocompatibility complex class I molecules to establish cellular infection.

The expression of stable, correctly folded major histocompatibility complex class I molecules conferred susceptibility to murine cytomegalovirus (MCMV) in cells which were previously resistant to infection, demonstrating that these molecules interact critically with MCMV to initiate infection. All class I molecules could potentiate MCMV infection but H-2Dd and Kb molecules were most efficient. Monoclonal antibodies specific for the alpha 1 and/or alpha 2 domains of Dd and Kb inhibited infection. Infection of L cells transfected with hybrid major histocompatibility complex class I molecules demonstrated that allelic control of susceptibility to MCMV mapped to the alpha 1 domain of Dd when in correct configuration with the alpha 2 and alpha 3 domains. In MCMV-resistant RMA-S cells, an improvement in the conformation of class I molecules introduced susceptibility to infection.

The effects of beta-2-microglobulin on the infectivity of murine cytomegalovirus.

The role of beta-2-microglobulin (beta 2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2d) and resistant (H-2k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID50 titers. Infection of susceptible fibroblasts was significantly increased 1.6-4.7 fold by the addition of whole FCS, a less than 20 kDa FCS fraction, or purified human beta 2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID50 titers by 3.5-10 fold in susceptible MEF. In relatively resistant H-2k cells, the TCID50 titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by beta 2m or FCS, but plaque sizes were increased 2.5-3 fold in resistant BALB.K MEF. When human or murine beta 2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine beta 2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of beta 2m which were restricted to genetically susceptible cells, may be due to tegument-bound beta 2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous beta 2m, promoting MCMV binding to the target cell.