Mechanism for the activation of the anaplastic lymphoma kinase receptor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that regulates important functions in the central nervous system1,2. The ALK gene is a hotspot for chromosomal translocation events that result in several fusion proteins that cause a variety of human malignancies3. Somatic and germline gain-of-function mutations in ALK were identified in paediatric neuroblastoma4-7. ALK is composed of an extracellular region (ECR), a single transmembrane helix and an intracellular tyrosine kinase domain8,9. ALK is activated by the binding of ALKAL1 and ALKAL2 ligands10-14 to its ECR, but the lack of structural information for the ALK-ECR or for ALKAL ligands has limited our understanding of ALK activation. Here we used cryo-electron microscopy, nuclear magnetic resonance and X-ray crystallography to determine the atomic details of human ALK dimerization and activation by ALKAL1 and ALKAL2. Our data reveal a mechanism of RTK activation that allows dimerization by either dimeric (ALKAL2) or monomeric (ALKAL1) ligands. This mechanism is underpinned by an unusual architecture of the receptor-ligand complex. The ALK-ECR undergoes a pronounced ligand-induced rearrangement and adopts an orientation parallel to the membrane surface. This orientation is further stabilized by an interaction between the ligand and the membrane. Our findings highlight the diversity in RTK oligomerization and activation mechanisms.

Comparison of two different techniques used for the maintenance of peri-implant soft tissue health: a pilot randomized clinical trial.

OBJECTIVES: The aim of this pilot study was to compare the effectiveness of two different methods of debridement on maintaining and improving peri-implant soft tissue health over a period of 12 months. MATERIALS AND METHODS: Twenty adult patients (25 implants) were enrolled in a randomized, single-blinded, parallel group clinical trial. All implants included showed no signs of pathologic bone loss. Patients were scheduled to be reviewed every 3 months over a 12 months period. Nine patients (15 implants) were randomly allocated to a test group and treated with a low abrasive air polishing powder (Air-Flow® Perio, EMS) (AFP) and another nine (10 implants) to a control group and treated with titanium curettes (TC). Peri-implant crevicular fluid samples were analyzed to quantitatively measure the concentration of six interleukins (IL-6, IL-8, IL-1ß, TNF, IL-10 and IL-12). A multilevel analysis was used to test the comparison between the two treatments. The same analysis was used to study the relationship between clinical parameters and cytokines while controlling for confounding factors. RESULTS: There was no significant difference in bleeding on probing (BOP) between the two treatment methods (p = .35). Both debridement techniques resulted in a similar reduction of BOP (40.04% and 39.93%). IL-6 was the only cytokine of the six investigated that demonstrated a correlation with a clinical parameter (BOP) (p = .05). CONCLUSIONS: Both treatment methods were proven to be effective in reducing peri-implant inflammation and preventing further disease progression. Some cytokines may act as markers for peri-implant disease as the present study showed a significant relationship between IL-6 and BOP.

Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells.

BACKGROUND: Organelle remodeling processes are evolutionarily conserved and involved in cell functions during development, aging, and cell death. Some endogenous and exogenous molecules can modulate these processes. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has mainly been considered as a modulator of plasma membrane fluidity in brain development and aging, while DHA's role in organelle remodeling in specific neural cell types at the ultrastructural level remains largely unexplored. DHA is notably incorporated into dynamic organelles named lipid bodies (LBs). We hypothesized that DHA could attenuate the inflammatory response in lipopolysaccharide (LPS)-activated microglia by remodeling LBs and altering their functional interplay with mitochondria and other associated organelles. RESULTS: We used electron microscopy to analyze at high spatial resolution organelle changes in N9 microglial cells exposed to the proinflammogen LPS, with or without DHA supplementation. Our results revealed that DHA reverses several effects of LPS in organelles. In particular, a large number of very small and grouped LBs was exclusively found in microglial cells exposed to DHA. In contrast, LBs in LPS-stimulated cells in the absence of DHA were sparse and large. LBs formed in the presence of DHA were generally electron-dense, suggesting DHA incorporation into these organelles. The accumulation of LBs in microglial cells from mouse and human was confirmed in situ. In addition, DHA induced numerous contacts between LBs and mitochondria and reversed the frequent disruption of mitochondrial integrity observed upon LPS stimulation. Dilation of the endoplasmic reticulum lumen was also infrequent following DHA treatment, suggesting that DHA reduces oxidative stress and protein misfolding. Lipidomic analysis in N9 microglial cells treated with DHA revealed an increase in phosphatidylserine, indicating the role of this phospholipid in normalization and maintenance of physiological membrane functions. This finding was supported by a marked reduction of microglial filopodia and endosome number and significant reduction of LPS-induced phagocytosis. CONCLUSIONS: DHA attenuates the inflammatory response in LPS-stimulated microglial cells by remodeling LBs and altering their interplay with mitochondria and other associated organelles. Our findings point towards a mechanism by which omega-3 DHA participates in organelle reorganization and contributes to the maintenance of neural cell homeostasis.

A common model for cytokine receptor activation: combined scissor-like rotation and self-rotation of receptor dimer induced by class I cytokine.

The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin) were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.

Preliminary crystallographic analysis of murine macrophage inflammatory protein 2.

Macrophage inflammatory protein 2 (MIP-2) has been crystallized by vapor diffusion of an 11 mg/ml protein solution in 100 mM-ammonium acetate against 30 to 40% polyethylene glycol (average molecular mass of 3350 Da). The crystals belong to space group P2(1)2(1)2(1) and have unit cell dimensions of a = 42.7 A, b = 59.3 A, and c = 100.3 A. The molecular mass of the protein and volume of the unit cell suggest that there are four monomers in the asymmetric unit. A data set to 2.3 A has been collected, and the self-rotation function identifies the presence of a non-crystallographic 2-fold axis.

Crystal structure of Epstein-Barr virus protein BCRF1, a homolog of cellular interleukin-10.

The crystal structure of Epstein-Barr virus protein BCRF1, an analog of cellular interleukin-10 (IL-10), has been determined at the resolution of 1.9 A and refined to an R-factor 0.191. The structure of this cytokine is similar to that of human IL-10 (hIL-10), forming an intercalated dimer of two 17 kDa polypeptides related by a crystallographic 2-fold symmetry axis. BCRF1 exhibits novel conformations of the N-terminal coil and of the loop between helices A and B compared to hIL-10. These regions are likely to be involved in binding of one or more components of the IL-10 receptor system, and thus the structural differences may account for the lower binding affinity and limited spectrum of biological activities of viral IL-10, compared to hIL-10.

Residue-specific immobilization of protein molecules by size-selected clusters.

The atomic force microscope (AFM), operating in contact mode, has been employed in buffer solution to study two proteins; (i) green fluorescent protein (GFP), from the hydromedusan jellyfish Aequorea victoria; and (ii) human oncostatin M (OSM), in the presence of size-selected gold nanoclusters pinned on to a highly oriented pyrolytic graphite substrate. The AFM images have revealed immobilization of single molecules of OSM, which are strongly bound to the gold nanoclusters. Conversely, no strong immobilization has been observed for the GFP, as these molecules were easily displaced by the scanning tip. The contrasting behaviour of the two proteins can be explained by the exposed molecular surface area of their cysteine residues as modelled on the basis of their respective X-ray crystallographic data structures. GFP contains two cysteine residues, but neither is readily available to chemisorb on the gold clusters, because the cysteines are largely inaccessible from the surface of the protein. In contrast, OSM has a total of five cysteine residues, with different degrees of accessibility, which make the protein amenable to anchoring on the nanoclusters. Statistical analysis of the height of the OSM molecules bound to the nanoclusters is in accordance with crystallographic data, and suggests various configurations of the proteins on the clusters, associated with the presence of different cysteine anchoring sites. These results suggest that the three-dimensional conformation of protein molecules is preserved when they are chemisorbed to size-selected gold clusters, thus opening a new route towards oriented immobilization of individual protein molecules.

High urine sIL-6R as a predictor of late graft failure in renal transplant recipients.

BACKGROUND: Chronic allograft nephropathy is an important cause of late renal transplant failure. Although numerous studies on cytokines have been carried out, the pathogenetic role of cytokines in chronic renal allograft nephropathy remains unclear. METHODS: In a retrospective study, the authors compared posttransplant plasma and urine cytokine levels (interleukin [IL]-1alpha, IL-1beta, soluble [s] IL-1 receptor [R] antagonist [A], IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-10, tumor necrosis factor-alpha, transforming growth factor-beta2, and interferon-gamma) in 34 matched pairs of patients with or without late graft failure and in 50 matched pairs with either normal or increased serum creatinine levels and continued stable graft function. RESULTS: Twelve and 6 months before late graft failure, urine levels of sIL-6R were significantly increased (P=0.003 and P=0.01, respectively). Serum creatinine levels were not associated with increased urine sIL-6R. CONCLUSION: High urine sIL-6R appears to be predictive of late graft failure in renal transplant recipients.

[Cytokines]. / Cytokines.

Cytokines are relatively small polypeptides that are essential components of inflammatory reactions and immunologic processes. They are produced by a great variety of cells. The molecular biology revolution of the seventies and eighties has enabled investigators to elucidate the structure of cytokines and to unravel their spectrum of activity. Cytokines play a central role in the pathogenesis of infectious diseases and in the regulation of the immune system. Tumor necrosis factor (TNF) and interleukin-I (IL-I) were found to be responsible for most of the symptoms of infectious diseases, from fever to septic shock. In addition, TNF and IL-I are probably indispensable for repair of tissue damage. IL-2 is the pivotal cytokine for the regulation of the immune system; it is produced by CD4-positive T-cells and essential for the proliferation of T-lymphocytes in general and crucial for the induction of cytotoxic T-cells. Studies to define the therapeutic possibilities of cytokines are in progress.

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border.

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.

Concentrações plasmáticas de citocinas e quimiocinas na infecção pelo HTLV-1 / Plasma concentrations of cytokines and chemokines in HTLV-1

INTRODUÇÃO: O vírus linfotrópico das células T humano tipo 1 (HTLV-1) é endêmico na Bahia e está associado com doenças graves, como a Paraparesia Espástica Tropical/Mielopatia associada ao HTLV-1 (HAM/TSP) e a Dermatite Infecciosa associada ao HTLV-1 (DIH). Escassos trabalhos tem sido reportados com a avaliação de citocinas e quimiocinas em indivíduos jovens infectados pelo HTLV-1 e não existem dados sobre a manifestação simultânea DIH e HAM/TSP na faixa infanto-juvenil. OBJETIVO: Avaliar as concentrações plasmáticas de citocinas e quimiocinas na infecção pelo HTLV-1 em indivíduos infanto-juvenis. MÉTODO: Foram incluídos 61 indivíduos portadores do HTLV-1 distribuídos nos grupos Portadores assintomáticos, pacientes com a DIH, pacientes com DIH/HAM/TSP, pacientes com a HAM/TSP e 20 indivíduos saudáveis sem a infecção pelo HTLV-1, todos na faixa infanto-juvenil. As concentrações plasmáticas foram comparadas através do método de Elisa e de Cytometric Bead Array (CBA)...

INTRODUCTION: The lymphotropic virus of cells T human type 1 (HTLV ) is endemic in Bahia and it is associated with serious diseases such as Tropical Spastic Paraparesis/associated myelopathy with HTLV-1 and Infectious Dermatitis associated with HTLV -1 (IDH). Very little work has been reported with the evaluation of cytokines and chemokines in the IDH and there has been no data on the manifestation simultaneous IDH and HAM/TSP in children and youth range. OBJECTIVE: To evaluate the plasma concentrations of cytokines and chemokines in HTLV-1 infection in children and young individuals. METHOD: We included 61 individuals HTLV-1 spread in groups Asymptomatic Carriers, patients with IDH, patients with IDH/HAM/TSP, patients with HAM/TSP and 20 healthy individuals without HTLV-1, all in children's range. Plasma concentrations were compared using the ELISA method and Cytometric Bead Array (CBA)...

[Quantification of mRNA using the polymerase chain reaction--cytokines in rheumatoid arthritis]. / Quantifizierung von mRNA mittels der Polymerase-Ketten-Reaktion--Zytokine in der rheumatoiden Arthritis.

A method for quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are co-amplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequence of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid method of quantifying the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species in parallel. This technique affords a very sensitive means of measuring important regulatory cytokines in the local inflammatory tissue of rheumatoid arthritis (RA).

Common structural patterns of cytokine outer surfaces.

Schemes of the four-helix bundle surfaces of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, interferon-beta, -gamma and growth hormone were designed. All cytokines appeared to have the structurally similar "holes" on the surfaces. They were suggested to serve as a part of the main receptor-binding sites.

Immunohistochemical localization of cytokines and receptor-associated molecules in human tonsils and skin.

Human tonsilla palatina and skin were investigated by means of light microscopical and electron microscopical immunohistochemistry using monoclonal antibodies (Mab) against some cytokines. In both tonsils and skin we found intracellular immunoreactivity for interleukin-1-beta in macrophages and interdigitating cells. Also some, but not all crypt-epithelial cells were positive, while keratinocytes in the skin were negative. Interleukin-2-immunoreactivity was found in a subpopulation of lymphocytes (probably T-cells) and unexpectedly also in some antigen-presenting cells (APCs). A Mab against interleukin-4 revealed weak labelling of lymphatic cells in the T-cell area of tonsils. The human skin was negative. A Mab that recognizes a molecule associated with the interleukin-4-receptor gave strong surface labelling in tonsils and skin on APCs and weak immunoreactivity on lymphoid cells. Frequently these APCs formed rosettes with weakly labeled lymphocytes. Mabs against interleukin-8 stained starry sky macrophages in the germinal centers of the tonsil and different APCs in the T-cell region. IL-8 is stored in keratinocytes of normal skin, but becomes mobilized under inflammatory conditions. Our results expand the understanding of cell cell-interactions under normal and inflammatory conditions in tonsil and skin.

Principios básicos de la respuesta inmunológica / Basic principles of immunologyc response

La protección del ataque de microorganismos invasores incluye mecanismos de resistencia específicos y no específicos. Los primeros son los responsables de impedir la mayoría de las infecciones, mientras que los segundos participan una vez que los microorganismos o sus productos han entrado a los tejidos. Los mecanismos de resistencia específicos, también conocidos como respuesta inmunológica, incluyen la participación de células y moléculas con capacidad de reconocer y reaccionar específicamente en contra del microorganismo invasor. En la activación de estos mecanismos participan linfocitos y células accesorias que se comunican a través de una compleja red de señales que incluyen moléculas asociadas a la membrana y moléculas solubles. Del tipo de interacciones establecidas de producirá una respuesta mediada por anticuerpos o por células inmunes, en algunos casos estas interacciones también generan una falta de respuesta (anergia) específica