Molecular dynamics study of micelles properties according to their size.

Surfactants are molecules able to spontaneously self-assemble to form aggregates with well-defined properties, such as spherical micelles, planar bilayers, cylindrical micelles or vesicles. Micelles have notably several applications in many domains, such as drug delivery or membrane protein solubilization. In this context, the study of micelle formation in relation with the structural and physico-chemical properties of surfactants is of great interest to better control their use in the different application fields. In this work, we use the MD approach developed by Yoshii et al. and extend it to surfactants with different structures. We aim to systematically investigate different micellar properties as a function of the aggregates size by a molecular dynamics approach, to get an insight into the micellar organization and to collect some relevant descriptors about micelle formation. For this, we perform short MD simulations of preformed micelles of various sizes and analyze three parameters for each micelle size, namely the eccentricity of the micelles, the hydrophobic/hydrophilic surface ratio and the hydrophobic tails hydration. If these parameters are known descriptors of micelles, they were not yet studied in this way by MD. We show that eccentricity, used as "validator" parameter, exhibits minimal values when the aggregate size is close to the experimental aggregation number for surfactants that are known to form spherical micelles. This hence indicates that our methodology gives consistent results. The evolution of the two descriptors follows another scheme, with a sharp increase and decrease, respectively, followed by a leveling-off. The aggregate sizes at which this stabilization starts to occur are close to the respective aggregation number of each surfactant. In our approach, we validate the use of these descriptors to follow micelle formation by MD, from "simple" surfactants to more complex structures, like lipopeptides. Our calculations also suggest that some peculiar behavior, like that of TPC, can be highlighted by our approach. In the context of peptidic surfactants, our methodology could further help to improve computer simulations combined to molecular thermodynamic models to predict micellar properties of those more complex amphiphilic molecules.

[Role of T lymphocyte cyclic nucleotides and G protein in sarcoidosis]. / Quels rôles les anomalies de la voie des nucléotides cycliques et des protéines G jouent-elles dans le désordre biochimique des lymphocytes T dans la sarcoïdose ?

CD4+ T lymphocytes play a major role in the pathophysiology of sarcoidosis. Many studies have investigated the immunological and genetic abnormalities in this disease. There are few studies concerning the metabolic pathways. Essentially they concern the pathways: STAT1, MAPK38, NF-κB, Galphai, cAMP and cGMP PDE and PEMT1. Using studies in the literature and results of our own work concerning some metabolic aspects of T lymphocytes in sarcoidosis, we present a revue of the various hypotheses, which involve dysfunction of cAMP signaling pathways, such as RAS/RAF/MEK/ERK in T lymphocytes, leading to a disorder of immunity.

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8.

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.

[Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire]. / Vers une maîtrise industrielle du clonage des lymphocytes B humains pour la fabrication des anticorps monoclonaux issus du répertoire humain.

Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

Different roles of odontoblasts and fibroblasts in immunity.

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.

Molecular characterization of a human immunoglobulin G4 antibody specific for the major birch pollen allergen, Bet v 1.

BACKGROUND: Allergen-specific IgG4 antibodies induced by specific immunotherapy are thought to represent a protective immune response. Objective Our aim was the molecular characterization of a human IgG4 antibody (BAB5) specific for the major birch pollen allergen Bet v 1 that was derived from an immunotherapy-treated patient. METHODS: The cDNA coding for BAB5 was obtained by reverse transcriptase-PCR from the BAB5-producing cell line, compared with the germ line sequences and was expressed as a soluble antibody fragment in Escherichia coli. The epitope specificity and cross-reactivity of BAB5 were investigated with recombinant and synthetic Bet v 1 fragments and Bet v 1 homologous allergens from pollen. The ability of BAB5 to block allergic patients IgE was determined by competition experiments and sandwich ELISA. RESULTS: BAB5 is an affinity-matured Bet v 1-specific IgG4 antibody that reacts exclusively with Bet v 1 but not with Bet v 1-related allergens. Unlike an earlier-described monoclonal IgG1-blocking antibody, BAB1, which had been isolated from the same patient, BAB5 did not block allergic patients' IgE reactivity to Bet v 1. CONCLUSION: Our study demonstrates that not all allergen-specific IgG antibodies inhibit IgE recognition of allergens and can contribute to the success of immunotherapy. The epitope specificity and affinity of IgG antibodies but not their isotype are decisive for their protective activity.

Elevated MMP-12 protein levels in induced sputum from patients with COPD.

BACKGROUND: Several matrix metalloproteinases (MMPs) are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). In mice, MMP-12 plays a crucial role in the development of cigarette smoke induced emphysema. A study was undertaken to investigate the role of MMP-12 in the development of COPD in human smokers. METHODS: Induced sputum samples were collected from patients with stable COPD (n = 28), healthy smokers (n = 14), never smokers (n = 20), and former smokers (n = 14). MMP-12 protein levels in induced sputum were determined by ELISA and compared between the four study groups. MMP-12 enzymatic activity in induced sputum was evaluated by casein zymography and by cleaving of a fluorescence quenched substrate. RESULTS: Median (IQR) MMP-12 levels were significantly higher in COPD patients than in healthy smokers, never smokers, and former smokers (17.5 (7.1-42.1) v 6.7 (3.9-10.4) v 4.2 (2.4-11.3) v 6.1 (4.5-7.6) ng/ml, p = 0.0002). MMP-12 enzymatic activity was significantly higher in patients with COPD than in controls (4.11 (1.4-8.0) v 0.14 (0.1-0.2) microg/microl, p = 0.0002). CONCLUSION: MMP-12 is markedly increased in induced sputum from patients with stable COPD compared with controls, suggesting a role for MMP-12 in the development of COPD in smokers.

What's new in the field of cancer vaccines?

The observation that in some cases tumors undergo spontaneous regression concomitantly with autoimmune manifestations has been interpreted as an indication of the involvement of the immune system in tumor rejection. This raised the conceptual possibility that the immune system could be used against the tumor. However, since tumor cells are poorly immunogenic by themselves, early attempts to develop immune-based approaches for cancer therapy saw the use of tumor cells transduced with genes coding for cytokines or costimulatory molecules to enhance in vivo immunity. The identification of cytotoxic T lymphocyte (CTL)-defined tumor-associated antigens has allowed the development of new strategies for cancer immunotherapy. Novel adjuvants have been identified, and different modes of antigen delivery were devised which aim at inducing efficient CTL responses in patients. This review will discuss some of what is currently considered as relevant aspects of antitumor immunization.

TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer.

TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents is suspected to influence dental pulp response, including the modulation of cell migration. To determine the consequences of TGF-beta1 action on pulp immune cells, we analyzed, by immunohistochemistry, the effect of transdentinally diffusing TGF-beta1 on their localization in a human tooth slice culture model. TGF-beta1 induced an accumulation of HLA-DR-positive cells in both odontoblast and subodontoblast layers of the stimulated zone. Together with HLA-DR, these cells co-expressed Factor XIIIa and CD68, two features of immature antigen-presenting dendritic cells (DC), as well as the TGF-beta1 specific receptor TbetaRII. In contrast, no effect could be detected on the localization of either mature DC-LAMP-positive DC or of T- and B-lymphocytes. Analysis of these data suggests that TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents could be involved in the immune response of the dental pulp resulting from tooth injury.

[Langerin: a new lectin specific for Langerhans cells induces the formation of Birbeck granules]. / Langerin: une nouvelle lectine spécifique des cellules de Langerhans induit la formation de granules de Birbeck.

Generation of monoclonal antibodies restricted to human dendritic cells generated from CD34+ hematopoietic precursors has enabled the identification of Langerin, a Ca(++)-dependent type II lectin. Only expressed by Langerhans cells, Langerin is responsible for Birbeck granule formation by membrane superimposition and zippering. Furthermore, cell-surface Langerin is rapidly internalized into Birbeck granules, and does not colocalize with MHC class II rich compartments. Langerin gene transfected into mouse fibroblasts induces the formation of Birbeck granule-like structures, that would permit a better understanding of the function of Birbeck granules.

The mouse and human IGSF6 (DORA) genes map to the inflammatory bowel disease 1 locus and are embedded in an intron of a gene of unknown function.

We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse.

The human anti-bullous pemphigoid monoclonal autoantibody P22 is encoded by genes of the IGHV4 and IGLV4 families.

We identified, cloned, and biochemically characterized the full-length cDNAs encoding the heavy and light chains of a human monoclonal antibody (mAb) from the Epstein-Barr virus (EBV)-cell line P22. The cell line P22, which originated from a patient with bullous pemphigoid (an autoimmune disease causing skin blistering) expressed immunoglobulin-G (IgG) with a lambda light chain. Although the variable heavy (IGHV) chain gene family could not be assigned by IGHV repertoire analysis, the determination of its nucleotide sequence demonstrated that the heavy chain of P22 belonged to the IGHV4 family. The limited IGHV4 gene usage by memory IgG, IGA and IgE-expressing cells supports the notion of the autoreactivity-associated IGHV4 genes and stresses the strong selection pressure within germinal centres towards IGHV4 family. Alignment of P22 IGHV4 cDNA sequence to germline sequences from gene databases, revealed a remarkable divergence, suggesting that the heavy chain of the P22 mAb encodes a distinct IGHV4 gene. The variable light chain (IGLV) encodes a IGLV4 gene and is 98% similar to a previously reported IGLV gene. Furthermore, fluorescent staining with the recombinant mAb showed the same reactivity to that of the native antibody. The data reported herein, (a) reveal an autoantibody encoding a distinct IGHV4 gene, (b) confirm the notion that autoantibodies preferentially use IGHV4 genes, and (c) hypothesize that somatic hypermutation within GC may be a mechanism by which autoreactive B lymphocytes escape negative selection.

Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors.

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3alpha plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3alpha was the most potent chemokine inducing the selective migration of in vitro-generated CD34(+) hematopoietic progenitor cell-derived LC precursors and skin LCs in accordance with the restricted MIP-3alpha receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3alpha was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3alpha. (c) In vivo, MIP-3alpha was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3alpha upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3alpha was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1beta plus tumor necrosis factor alpha) or T cell signals. Results of this study suggest a major role of MIP-3alpha in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.

Immunobiology of dendritic cells.

Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell-mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis.

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.

Human dendritic cells in the severe combined immunodeficiency mouse model: their potentiating role in the allergic reaction.

Dendritic cells (DCs) are present in the lungs and airways of healthy and allergic subjects where they are exposed to inhaled antigens. After the uptake of antigens, DCs migrate to lymphoid organs where T cells initiate and control the immune response. The migratory properties of DCs are an essential component of their function but remain unclear in the situation of allergic diseases. To better understand the role of DCs in response to allergens, we first investigated their presence in an original experimental model of allergic asthma: the humanized severe combined immunodeficiency (SCID) mouse reconstituted with peripheral blood mononuclear cells from patients sensitive to Dermatophagoides pteronyssinus (Dpt). Human DCs were detected in lungs of mice developing an inflammatory pulmonary infiltrate and appeared to be mainly located in the alveolar spaces. In a second step, human DCs were generated in vitro from monocytes and injected into naive SCID mice exposed or not exposed to Dpt aerosols. Their migratory behavior was explored, as well as their potential role in modulating the IgE production after exposure to Dpt. After exposure to Dpt, the number of DCs present in airways decreased, while it increased into the spleen and thymus of the mice. The IgE production increased in the presence of DCs as compared with mice not injected with DCs. These results suggest that DCs may play a role in the pulmonary allergic reaction developed in response to Dpt in SCID mice.

Antigen receptors and dendritic cells.

Several age-related alterations occurring in the immune system, especially in T cells and in B cells, may account for an increased susceptibility to infections, autoimmune diseases, and malignancies. In particular, the adaptive immune response has been shown to lose part of its diversity and its memory in old mice. However, whether dendritic cells, which play a central role in the initiation of the cellular immunity but are also involved in humoral immunity, participate qualitatively or quantitatively in immunosenescence remains to be determined.

Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.

We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.