Spatial and Temporal Deposition of Adhesive Extracellular Polysaccharide Capsule and Fimbriae by Hyphomonas Strain MHS-3

Hyphomonas strain MHS-3, a member of a genus of primary colonizers of surfaces immersed in marine water, synthesizes two structures that mediate adhesion to solid substrata, namely, capsular exopolysaccharide and fimbriae. Specific stains, gold-labelled lectins, and monoclonal antibodies, along with transmission electron microscopy of synchronized populations, revealed that both structures are polarly and temporally expressed. The timed synthesis and placement of the fimbriae and capsule correlated with the timing and locus of MHS-3 adhesion.     

Effect on fertilization and localized binding of lectins in the ascidian Phallusia mammillata

The effect of concanavalin A (conA), fucose-binding protein (FBP), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) on fertilization of the ascidian Phallusia mammillata was investigated. ConA, FBP, and RCA had no influence on fertilization and did not bind to the chorion or sperm, as determined with FITC-conjugated conA and by electron microscopy with gold-labelled FBP. WGA (100 μg/ml) prevented fertilization of eggs by sperms in concentrations which gave 100% fertilization in controls (2 × 10⁷ sperm/ml). N-Acetyl-glucosamine (50 mM) abolished the effect of WGA, whereas an excess (100 mM) of this competitive sugar alone did not affect fertilization. FITC-conjugated and gold-labelled WGA revealed binding sites on the chorion, but not on follicle cells nor sperms. Electron microscopy showed that WGA gold-markers are bound to the fibrillar network forming the outer layer of the chorion and indicate that WGA inhibits fertilization by interfering with sperm binding to the chorion. Binding of WGA to the chorion may either mask sperm binding receptors or cause chorion resistance to sperm enzymes.     

Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease.The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells (¹⁴¹TGMAALEQYLGSGHAVIVSI¹⁶⁰) and other binding U937 monocyte-derived macrophages (²¹AVALGLASPADAAAGTMYGD⁴⁰). Such sequences’ ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure–function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine.     

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome

Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome.     

Evaluation of IgM,IgG, IgA,IgD, and IgE secretion by human peripheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aureus Cowan I

Sheep erythrocytes coated with staphylococcal protein A were used as target cells in a reverse hemolytic plaque assay. Monospecific antisera to human immunoglobulins G, M, A, D, and E were used to detect each of these classes in cultures of human peripheral blood mononuclear cells stimulated with the polyclonal B-cell activators pokeweed mitogen and Staphylococcus aureus (Cowan I). Both mitogens induced substantial increases in the numbers of cells actively secreting immunoglobulins; in time-course experiments, the peak response was seen on Day 5. The numbers of cell secreting IgM, IgE, and IgD were usually higher in cultures stimulated with S. aureus than with pokeweed mitogen.     

Schistosoma japonicum: Immunopathology of nephritis in Macaca fascicularis

Macaca monkeys experimentally infected with Schistosoma japonicum developed a chronic progressive kidney lesion characterized by an increase of mesangial matrix, local glomerular hypercellularity, and local thickening of glomerular basement membrane. Immunofluorescence studies revealed the localization of IgG, IgM, IgA, and IgE immunoglobulins mostly in the mesangial area of the glomeruli accompanied by the deposition of Schistosoma antigens. By electron microscopy, in addition to the local thickening of the glomerular basement membrane, dense homogeneous deposits and those with moth-eaten appearance were detected in the mesangial matrix. These findings suggest that worms in the bloodstream continuously release antigenic materials that stimulate host's antibody response belonging to various immunoglobulin classes including IgE. The produced antibodies and antigens would form immune complexes that deposited in the glomeruli. The increased vascular permeability caused by antigen-IgE antibody interaction may play an important role in the deposition of immune complexes and in the rapid development of kidney injury.     

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH₂) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc₁ (cytbc₁) complexes that oxidise QH₂ to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc₁ complexes with gold nanobeads, each attached by a Histidine₁₀ (His₁₀)-tag to the C-terminus of cytc₁. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc₁ complexes occur as dimers in the membrane. The cytbc₁ complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc₁ complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc₁ whereas LH2 complexes became detached, consistent with clusters of cytbc₁ complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc₁–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc₁, ATP synthase, cytaa₃ and cytcbb₃ membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc₁ dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.     

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using ¹²⁵I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (K_a) of about 5 · 10⁸?10 · 10⁸ M^?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (K_a about 45 · 10⁸ M^?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.     

Application of the Laurell immunoelectrophoresis technique to the study of serological relationships between granulosis viruses

The Laurell technique of two-dimensional immunoelectrophoresis was used to distinguish between isolates of granulosis virus (GV) from Plodia interpunctella (GV strains A and B), Ephestia cautella, Spodoptera littoralis (GV strain 65), Pieris brassicae, and Cydia pomonella. Granules, alkali-soluble proteins, and virus particles of P. interpunctella GV strain A and granules of P. interpunctella GV strain B were used as sources of antigens. They were reacted with the immunoglobulins of antisera prepared against whole granules of each strain of virus. Peaks of precipitation were most clearly defined when antigens were pretreated with 0.1 m Na₂CO₃, 2% Triton X, and succinic anhydride, Granules and alkali-soluble proteins of P. interpunctella GV strain A treated in this way exhibited at least one peak of precipitation when reacted with each of the antisera studied. Four peaks were observed in both the homologous reaction and in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B. Four different peaks were present in the homologous reaction between immunoglobulins and virus particles of P. interpunctella GV strain A. Two peaks were present in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B and one in that with the antiserum prepared against granules of S. littoralis GV strain 65.     

Production of humanized F(ab’)<Subscript>2</Subscript> fragment of rabies blocking antibodies in <Emphasis Type="Italic">Pichia pastoris</Emphasis> yeast

The yeast strain Pichia pastoris, a producer of humanized F(ab’)₂ fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab’)₂ fragments of the shared pool of secreted immunoglobulins up to 75%.     

Electron cytochemical study of carbohydrate components in cultured nerve and glial cells of the snail Helix pomatia

• 1.1. A variety of colloidal gold-labelled lectins with different sugar specificities to determine whether different nerve and glial cells of the snail Helix pomatia cultured in vitro, can be distinguished by the carbohydrates that they express was screened. The analysis of lectin binding has shown substantial differences in the carbohydrate pattern between nerve and glial cells and between the soma of monoaminergic and peptidergic neurons.
• 2.2. The surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine and N-acetyllactosamine determinants, and does not exhibit neuraminic acid and complex branched N-glycosyl chains. Moreover, N-acetylgalactosamine can be detected on peptidergic neuron membranes only.
• 3.3. N-Acetylglucosamine residues are not present on the surface of the glial cells, and the density of the N-acetyllactosamine and/or terminal β-galactose residues is much higher here than on the surface of the nerve cells.
• 4.4. These results suggest that nerve cells in the snail brain can be distinguished from glial cells by the presence of a cell-surface glycoconjugate containing terminal N-acetyl-d-glucosamine residues, whereas peptidergic neurons can be distinguished from monoaminergic neurons by the presence of a surface glycoconjugate containing terminal α-linked N-acetyl-d-galactosamine residues.

     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Effect of Anti-immunoglobulin Antisera on Homograft Rejection in Mice

HETEROLOGOUS antisera against immunoglobulins or their component protein chains have been shown to inhibit the immune response in a variety of systems. Antibodies against mouse immunoglobulins, for example, inhibit the response of mouse spleen cells cultured in vitro^1–3. Antibodies against the heavy chains of chicken IgM (anti-μ), administered during embryonation and again at hatching, have produced agamma-globulinaemia in bursectomized chickens⁴, apparently by plasma cell line elimination⁵. Graft-versus-host (GVH) and delayed hypersensitivity reactions have been suppressed in neonatal mice by in vitro pretreatment of injected lymphoid cells with antiserum against light chains⁶. Similar pretreatment with univalent fragments (Fab) of anti-immunoglobulin antibodies has diminished the GVH reaction in adult mice⁷.     

Experimental trichinosis-II. Quantitative and qualitative variations of serum in CBA mouse and Wistar rat (author's transl)]

Variations of serum globuline and immunoglobulins were measured by electrophoresis on cellulose acétate and radial immunodiffusion during the course of infection in inbred CBA mice and Wistar rats.In the two animal species, electrophoresis showed a significant decrease of serum albumin attending an increase of γ globulins. This phenomenon was earlier and greater in mice than in rats. This difference might be explained by suggesting that Trichinella is better adapted to rats than to mice.Beginning in the second week of infection, all classes of immunoglobulins were increased in the mouse with IgG_2a and IgA showing the greatest increase. The different classes of immunoglobulins continued to increase during the 4th month of infection but the rate of increase diminished after the second month except for IgA which increased between the 60th and 120th day.     

Large-Scale In Vitro Expansion of Polyclonal Human Switched-Memory B Lymphocytes

Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients’ accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10⁶-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG₁, IgG₂, IgG₃ and IgG₄ is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.