VASP localization to lipid bilayers induces polymerization driven actin bundle formation.

Actin bundles constitute important cytoskeleton structures and enable a scaffold for force transmission inside cells. Actin bundles are formed by proteins, with multiple F-actin binding domains cross-linking actin filaments to each other. Vasodilator-stimulated phosphoprotein (VASP) has mostly been reported as an actin elongator, but it has been shown to be a bundling protein as well and is found in bundled actin structures at filopodia and adhesion sites. Based on in vitro experiments, it remains unclear when and how VASP can act as an actin bundler or elongator. Here we demonstrate that VASP bound to membranes facilitates the formation of large actin bundles during polymerization. The alignment by polymerization requires the fluidity of the lipid bilayers. The mobility within the bilayer enables VASP to bind to filaments and capture and track growing barbed ends. VASP itself phase separates into a protein-enriched phase on the bilayer. This VASP-rich phase nucleates and accumulates at bundles during polymerization, which in turn leads to a reorganization of the underlying lipid bilayer. Our findings demonstrate that the nature of VASP localization is decisive for its function. The up-concentration based on VASP's affinity to actin during polymerization enables it to simultaneously fulfill the function of an elongator and a bundler.

Intra-bundle contractions enable extensile properties of active actin networks.

The cellular cortex is a dynamic and contractile actomyosin network modulated by actin-binding proteins. We reconstituted a minimal cortex adhered to a model cell membrane mimicking two processes mediated by the motor protein myosin: contractility and high turnover of actin monomers. Myosin reorganized these networks by extensile intra­bundle contractions leading to an altered growth mechanism. Hereby, stress within tethered bundles induced nicking of filaments followed by repair via incorporation of free monomers. This mechanism was able to break the symmetry of the previously disordered network resulting in the generation of extensile clusters, reminiscent of structures found within cells.

The dynamics of actin network turnover is self-organized by a growth-depletion feedback.

The dynamics of actin networks is modulated by a machinery consisting of actin binding proteins that control the turnover of filaments in space and time. To study this complex orchestration, in vitro reconstitution approaches strive to project actin dynamics in ideal, minimal systems. To this extent we reconstitute a self-supplying, dense network of globally treadmilling filaments. In this system we analyze growth and intrinsic turnover by means of FRAP measurements and thereby demonstrate how the depletion of monomers and actin binding partners modulate the dynamics in active actin networks. The described effects occur only in dense networks, as single filament dynamics are unable to produce depletion effects to this extent. Furthermore, we demonstrate a synergistic relationship between the nucleators formin and Arp2/3 when branched networks and formin-induced networks are colocalized. As a result, the formin-enhanced filament turnover depletes cofilin at the surface and thus protects the dense, Arp2/3 polymerized network from debranching. Ultimately, these results may be key for understanding the maintenance of the two contradicting requirements of network stability and dynamics in cells.

Effect of PGG-glucan on the rate of serious postoperative infection or death observed after high-risk gastrointestinal operations. Betafectin Gastrointestinal Study Group.

BACKGROUND: Postoperative infections remain common after high-risk gastrointestinal procedures. PGG-glucan (Betafectin; Alpha Beta Technology Inc, Worcester, Mass), derived from yeast cell walls, promotes phagocytosis and intracellular killing of bacterial pathogens by leukocytes, prevents infection in an animal model of wound infection, and acts synergistically with antibiotics to reduce mortality in rat peritonitis. HYPOTHESIS: We hypothesized that infectious complications in these patients might be reduced by the administration of a nonspecific immune-enhancing agent. DESIGN: Multicenter, prospective, randomized, double-blind, placebo-controlled trial of 1249 patients prospectively stratified into colorectal or noncolorectal strata. SETTING: Thirty-nine medical centers throughout the United States. PATIENTS: Aged 18 years or older, scheduled for gastrointestinal procedure lasting 2 to 8 hours, with 2 or more defined risk factors. INTERVENTIONS: PGG-glucan, 0.5 mg/kg or 1.0 mg/kg, or placebo once preoperatively and 3 times postoperatively. All patients received standardized antibiotic prophylaxis. MAIN OUTCOME MEASURES: Serious infection or death within 30 days. RESULTS: All randomized patients revealed no difference in serious infections and deaths in the treated groups compared with placebo groups (15% vs 14%, P>.90). In the prospectively defined noncolorectal stratum (n = 391), PGG-glucan administration was associated with a statistically significant relative reduction (39%) in serious infections and death (placebo, 46 [36%] of 129 vs either PGG-glucan group, 29 [21%] of 132 and 28 [22%] of 130, P<.02). PGG-glucan reduced postoperative infection or death in malnourished patients having noncolorectal procedures (31 [44%] of 70, placebo group; 16 [24%] of 68, 0.5-mg/kg PGG-glucan group; 12 [17%] of 72, 1.0-mg/kg PGG-glucan group; P<.001). Study drug was stopped owing to adverse effects more frequently for patients receiving PGG-glucan than placebo (2%, 4%, and 7% for the placebo group, 0.5-mg/kg PGG-glucan group, and 1.0-mg/kg PGG-glucan group, respectively, P<.003). CONCLUSION: Perioperative administration of PGG-glucan reduced serious postoperative infections or death by 39% after high-risk noncolorectal operations.

Effects of cleaning, chemical disinfection, and sterilization procedures on the mechanical properties of endodontic instruments.

The aim of this study was to test and compare the values of torsional moment, torsional angular deflection, bending moment, and permanent angular deflection of three designs of root canal files (Unifile, Flexofile, and H-File) before and after cross-infection treatment procedures, according to ANSI/ADA specification no. 28. An increase in value for all mechanical properties tested was observed after the treatment procedures, with the exception of Flexofile wherein a decrease in permanent angular deflection was evident. Unifile showed a decrease in torsional moment and bending moment. The changes in mechanical properties after treatment procedures ranged from 0.1 to 63% from the control groups. Generally, the changes in values observed were insignificant and still well within ANSI/ADA specification no. 28. Thus, they do not have any clinical significance.

Effects of cleaning, disinfection, and sterilization procedures on the cutting efficiency of endodontic files.

The effects of various cleaning, chemical disinfection, and sterilization procedures on the cutting efficiency of endodontic instruments Unifile (De Trey, Bois Colombes, France), (Flexofile Maillefer, Ballaigues, Switzerland), and H-File (Maillefer)) were investigated. The cross-infection control treatment procedures investigated were as follows: chemical disinfection--NaOCl (2.5%) for 12 and 48 h, and NH4 (5%) for 1 and 4 h; ultrasonic cleaning for 4 and 16 cycles of 15 min; and sterilization methods with chemiclave for 5 and 10 cycles of 20 min, Poupinel for 5 and 10 cycles of 120 min at 180 degrees C and glass beads for 10 and 40 cycles of 40 at 250 degrees C. Cutting efficiency was evaluated as the mass of Plexiglas cut per unit of energy expended by the instrument in microgram/Joule. The cutting efficiency decreased from 1 to 77%, depending on the file design and type of treatment procedures. Heat sterilization (Poupinel) did not modify the cutting efficiency of Unifile and Flexofile. The decrease in cutting efficiency was independent of frequency and duration of treatment procedures.

T-cell lines derived from lesional skin of lichen planus patients contain a distinctive population of T-cell receptor gamma delta-bearing cells.

Lichen planus is characterized by a dense infiltrate of T lymphocytes at the dermoepidermal junction. To determine the phenotypic and functional characteristics of the infiltrating lymphocytes, T-cell lines from normal and lesional skin from the same patients with lichen planus were established by culture with interleukin 2 followed by stimulation every 14 d with phytohemagglutinin and irradiated allogeneic feeder cells. Resultant T-cell lines were immunophenotyped by staining with monoclonal antibodies and their reactivity tested by determining their cytolytic activity to selected targets. T-cell lines from 13 lesional and nine normal biopsy specimens were studied. T-cell lines from normal skin were 61% CD4+ and 32% CD8+, whereas lines from lesional skin had significantly fewer CD4+ cells (13%) and more CD8+ cells (62%). T-cell lines from lesional skin contained a distinctive population of gamma delta T cells that was rarely present in lines derived from normal skin. We were able to culture gamma delta T cells out of the lesional skin of 12 of 13 patients. In these 12 patients, lesional T-cell lines were 17% gamma delta+ (range 2% to 47%). Only one T-cell line from normal skin contained significant numbers of gamma delta T cells. The gamma delta population from lesional skin was commonly V delta 1J delta 1+. These results suggest that CD8+ and TCR gamma delta+ T lymphocytes may be involved in the development or the maintenance of lichen planus.

CD1 gene expression in human skin.

In human epidermis, expression of CD1a is confined to Langerhans cells (LC), whereas CD1c expression has been observed in dendritic cells of the dermis, as well as the epidermis. In transfected fibroblasts, expression of CD1c at the cell surface appears to exclude expression of either CD1b or CD1a, despite continued transcription of the latter genes. In order to determine whether this mechanism might be operative in human skin, we have compared the expression of CD1a and CD1c on the surface of dermal and epidermal dendritic cells to their expression at the level of mRNA using a combination of dual-label immunofluorescence microscopy, northern blot hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR). By both immunofluorescence and Northern blotting, CD1c expression was observed in both dermal and epidermal cells, whereas expression of CD1a was confined largely to the epidermis. Moreover, as shown by immunomagnetic bead selection and RT-PCR, CD1a and CD1c were both expressed on epidermal LC, but were absent from other epidermal cell types. These results argue against cell surface exclusion as a mechanism for selective expression of CD1c in human dermis.

Most gamma delta T cells develop normally in the absence of MHC class II molecules.

MHC class I and class II molecules are essential for intrathymic maturation of a normal repertoire of alpha beta T cells. The development of gamma delta T cells may similarly require exposure to conventional MHC or MHC-like molecules for appropriate negative and positive selection. The availability of mice that are devoid of cell surface expression of MHC class II molecules allowed us to test directly the hypothesis that conventional class II molecules play a role in the development of gamma delta T cells. The proportions of gamma delta cells in thymus, lymph nodes, and spleens were indistinguishable between class II-deficient mice and littermate controls by FACS analysis. gamma delta T cells from class II-deficient mice proliferated normally in response to anti-TCR mAb. Examination of epidermal sheets from ear, torso, and tail skin demonstrated that class II-deficient mice had the same population density and distribution of gamma delta+ dendritic epidermal T cells as that of control littermates. gamma delta Cells in the epidermis of class II-deficient mice expressed Thy-1 and CD3 and were predominantly V gamma 3+. There were no significant differences in the immunophenotype of class II-deficient mice and their normal littermates in two-color immunofluorescence analysis of intestinal intraepithelial lymphocytes. Class II-deficient mice and control mice had 29 to 39% gamma delta bearing intestinal intraepithelial lymphocytes, of which 16 to 21% expressed V delta 4. Dendritic cells that stained positively for Thy-1, CD3, and gamma delta were identified in the vaginal epithelium of class II-deficient mice and their normal littermates. Our results indicate that MHC class II expression is not essential for the development of most gamma delta T cells.

T-cell receptor V beta expression in normal human skin.

The skin-associated immune system is the first line of defense against pathogenic attack from the environment and is simultaneously tolerant to localized autoantigens and to antigens of the normal microbial flora. The T-cell receptor (TCR) repertoire of skin lymphocytes may therefore be influenced by the skin microenvironment. We studied the expression of TCR beta-chain variable region (V beta) genes in normal skin by a polymerase chain reaction-based comparative method. When comparing the amplification of V beta genes in peripheral blood and normal skin, we found that TCR V beta 1, -7, -14, and -16 were often highly expressed in skin relative to peripheral blood, whereas V beta 5.1 was often highly expressed in peripheral blood mononuclear cells but not in skin. These results demonstrate that the TCR repertoire of skin lymphocytes is not determined by random sampling of peripheral blood mononuclear cells but may be molded by the interaction with self antigens and/or the normal microbial flora in the microenvironment of the skin.

Cyclosporin increases granulocyte/macrophage colony-stimulating factor (GM-CSF) activity and gene expression in murine keratinocytes.

Keratinocytes produce multiple cytokines in response to a variety of stimuli. The release of interleukin 1 (IL-1) from keratinocytes may be significant in initiation of cutaneous inflammation, and the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) is thought to be important in the regulation of antigen-presenting function by epidermal Langerhans cells. Because cyclosporin inhibits interleukin 2 release from T cells, it has been suggested that cyclosporin may function as an anti-inflammatory agent within the epidermis through inhibition of keratinocyte cytokine release. This investigation examined the direct effect of cyclosporin on the production of GM-CSF by murine keratinocytes and the keratinocyte cell line PAM 212. GM-CSF bioactivity increased in cell supernatants from keratinocytes exposed in vitro to 1 microgram/ml cyclosporin for up to 24 h. GM-CSF and IL-1 mRNA levels in keratinocytes cultured under similar conditions or in the presence of lipopolysaccharide also increased. The lack of inhibition of GM-CSF expression following cyclosporin treatment is consistent with recent observations in T cells and is opposite to the effect of cyclosporin on interleukin 2.

Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells.

The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue.

Production of latent transforming growth factor-beta and other inhibitory factors by cultured murine iris and ciliary body cells.

Aqueous humor contains transforming growth factor-beta (TGF-beta) and other inhibitory factors for cellular proliferation. In the present study we investigated the possibility that these factors are produced locally by cells found within the iris and ciliary body. Iris and ciliary body (I/CB) cells or whole tissue explants from C57BL/6 mice produced soluble factors which inhibited both murine thymocyte and mink lung epithelial cell proliferation. Indomethacin partially blocked inhibition of thymocyte proliferation, but did not affect inhibition of Mv1 Lu proliferation. The inhibitory activity of culture supernatants was not blocked by neutralizing antibodies to TGF-beta 1 or TGF-beta 2. However, following acid activation of culture supernatants from both I/CB and corneal tissue, increased inhibitory activity consistent with activation of latent TGB-beta was detected. Antibody neutralization experiments demonstrated that this increase in activity was due primarily to TGF-beta 2 for I/CB tissue. Polymerase chain reaction (PCR) analysis of cDNA generated from I/CB tissue mRNA showed prominent fragments representing both TGF-beta 1 and TGF-beta 2 mRNA. Corneal tissue, however, showed a prominent fragment for TGF-beta 1 mRNA, but either no band or a barely detectable fragment for TGF-beta 2 mRNA. Therefore, it remains uncertain whether TGF-beta 2 mRNA is produced by the cornea in this strain. Overall, these results demonstrated that three distinct categories of substances inhibitory to proliferation may be locally produced by resident iris and ciliary body cells: 1) indomethacin sensitive products, 2) TGF-beta 2 in latent form, and 3) factors not blocked by indomethacin or anti-TGF-beta neutralizing antibodies. Products generated by these tissues may have important regulatory properties in the development of immune responses to antigens introduced into the anterior chamber.

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.

Expression of murine CD1 on gastrointestinal epithelium.

Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes.

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.