[Experiences with homoplastic osteocartilaginous transplantation of the femur head cap].
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Adolescent↗
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Blind evaluation of lymphocyte capping in Duchenne muscular dystrophy.
Recently, Pickard et al reported decreased "capping" in lymphocytes from patients with Duchenne type muscular dystrophy (DMD) as well as female carriers of the DMD trait. To resolve subsequent debate about the reproducibility of this finding, we carried out a "blinded" collaborative study designed to eliminate the possibility of observer bias. Blood samples from DMD patients, their mothers, and controls were obtained and coded at Johns Hopkins and transported to the Medical College of Virginia, where lymphocyte capping was tested using FITC-labeled polyvalent anti-human immunoglobulin. Diminished capping in lymphocytes was found in 12 of 13 DMD patients (17 of 18 blood samples) and in 14 of 17 mothers of DMD patients (19 of 23 blood samples), as compared with 8 of 21 control subjects (8 of 22 blood samples). The results in both the patient and the carrier groups differed significantly from those in the control group, confirming previous observations of diminished lymphocyte capping in DMD. The findings provide support for the concept of a systemic defect associated with cell membranes in this disorder. The relatively high incidence of false positive results limits the usefulness of lymphocyte capping as a diagnostic test for carriers under the conditions of this study.
Female↗
The role of central apparatus components in flagellar motility and microtubule assembly.
In order to generate the complex waveforms typical of beating cilia and flagella, the action of the dynein arms must be regulated. This regulation not only depends on the presence of multiple dynein isoforms, but also clearly involves other structures in the axoneme such as the radial spokes and central apparatus; mutants lacking these structures have paralyzed flagella. In this article, we review recent progress in identifying protein components of the central apparatus and discuss the role of these components in regulation of flagellar motility and central apparatus assembly. The central apparatus is composed of two single microtubules and their associated structures which include the central pair projections, the central pair bridges linking the two tubules, and the central pair caps which are attached to the distal or plus ends of the microtubules. To date, the genes encoding four components of the central apparatus have been cloned, PF15, PF16, PF20 and KLP1. PF16, PF20 and KLP1 have been sequenced and their gene products localized. Two additional components have been identified immunologically, a 110 kD polypeptide recognized by an antibody generated against highly conserved kinesin peptide sequence, and a 97 kD polypeptide recognized by CREST antisera. Based on a variety of data, one model that has emerged to explain the role of the central apparatus in flagellar motility is that the central apparatus ultimately regulates dynein through interactions with the radial spokes. The challenge now is to determine the precise mechanism by which the polypeptides comprising the central apparatus and the radial spokes interact to transduce a regulatory signal to the dynein arms. In terms of assembly, the central apparatus microtubules assemble with their plus ends distal to the cell body but, unlike the nine doublet microtubules, they are not nucleated from the basal bodies. Since some central apparatus defective mutants fail to assemble the entire central apparatus, their gene products may eventually prove to have microtubule nucleating or stabilizing properties. By continuing to identify the genes that encode central apparatus components, we will begin to understand the contribution of these microtubules to flagellar motility and gain insight into their nucleation, assembly, and stability.
Animals↗
Component-resolved diagnosis of house-dust mite allergy with purified natural and recombinant mite allergens.
BACKGROUND: Mites belong to the most frequent and potent allergen sources. Immunotherapy with mite allergen extracts is frequently performed if allergen avoidance is not possible or successful. However, highly controversial results have been reported for mite-specific immunotherapy. OBJECTIVE: The aim of this study was to develop diagnostic concepts that may contribute to an improved selection of patients for immunotherapy with Der p allergen extracts and that may be used for immunological monitoring of patients undergoing this treatment. METHODS: The IgE reactivity profiles to Der p extract were determined in a Middle European mite-allergic population by IgE immunoblotting and by using a panel of seven purified natural or recombinant Der p allergens (nDer p 1, nDer p 4, rDer p 2, rDer p 5, rDer p 7, rDer p 8, rDer p 10). Furthermore, we investigated the sensitization and cross-reactivity to house-dust- and storage-mite allergen extracts by CAP FEIA measurements and by IgE competition studies. RESULTS: More than 95% of the patients could be diagnosed with a combination of nDer p 1 and rDer p 2. With the methods used, we could discriminate mite-allergic patients who were mainly sensitized to the major Der p allergens (Der p 1, Der p 2) from patients with a broad sensitization profile, including highly cross-reactive allergens (e.g. Der p 10: tropomyosin) as well as reactivity to storage mites. CONCLUSIONS: Diagnostic tests containing the major mite allergens (i.e. Der p 1, Der p 2) and highly cross-reactive mite allergens (e.g. Der p 10) may improve the diagnostic selection of patients for immunotherapy with Der p extracts. These tests may also be used for the immunological monitoring of patients undergoing immunotherapy.
Adolescent↗
beta-Internexin, a ubiquitous intermediate filament-associated protein.
In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).
Animals↗
Correlation between movement of concanavalin A membrane receptors and cytolysis. A scanning electron microscopy study.
The present study was undertaken to test whether cytolysis induced by Concanavalin A (Con A) requires lateral mobility of membranal lectin receptor sites into caps. Treatment of interphase murine mastocytoma cells with 10(-4) M colchicine promoted cap formation by Con A in about 30% of the cells, followed by cytolysis. Pretreatment of the cells with NaN3, low temperature, or glutaraldehyde decreased the degree of capping and, to the same extent, the degree of cytolysis. The addition of antibodies to cells bound with Con A increased the appearance of capping and cytolysis. A linear relationship with a high correlation coefficient exists between the degree of capping and cytolysis, suggesting that lateral mobility of membrane Con A receptors is required for cytolysis by the lectin. The process of cap formation by Con A up to the stage of cytolysis was followed by scanning electron microscopy.
Animals↗
Localization of aggregated cell surface antigens of target cells bound to cytotoxic T lymphocytes.
The redistribution of aggregated cell surface antigens of target cells bound to cytotoxic T lymphocytes was investigated. It was found that cap formation induced by antibody always occurred toward the site of binding. It is suggested that the polar localization of capped target cell surface determinants is a consequence of cytotoxic T-lymphocyte target cell interaction.
Animals↗
Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.
Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.
Acrosome↗
Studies on the cytotoxic activity of human lymphocytes. II. Interactions between IgG and Fc receptors leading to inhibition of K cell function.
The inhibition of human K cell-mediated cytolysis by a variety of immunoglobulin preparations was studied. It was found that inhibition was not a simple function of IgG concentration but was dependent on the mode of immunoglobulin presentation. The relative inhibitory capacity of IgG preparations on a weight basis was as follows: cell associated Ig larger than or equal to immobilized immune complexes greater than insoluble immune complexes greater than soluble immune complexes greater than heat-aggregated Ig greater than monomeric Ig. These results imply that the inhibitory capacity of immunoglobulin is determined by ligand (Fc) multivalency. It was found that although interactions between immunoglobulin and Fc receptors occur at low temperatures, these interactions lead neither to lysis nor to K cell inactivation. A temperature-sensitive post-binding event was required both for lysis to occur and for K cells to be inactivated. These findings lead us to propose that an IgG-induced redistribution ("capping") of Fc receptors on the K cell surface may be required for cytolysis and for effector -ell inactivation.
Antigen-Antibody Complex↗
Assessment of immunological responsiveness in members of International Biomedical Expedition to the Antarctic 1980/81.
Analysis of immune responsiveness was performed in members of the IBEA during a controlled trial of acclimatization with cold baths and during the subsequent 71 day period of Antarctic isolation. Several parameters of humoral and cellular immunity were assessed. No differences were found for those individuals who underwent cold-acclimatization in the first phase of the expedition. On the ice-cap all members showed slight elevation of their immunoglobulins, the increase in IgA reaching significance (p less than 0.05). Values returned to normal on returning to Sydney. Enhanced reactivity to the mycobacterium antigen purified protein derivative (PPD) was found on the ice-cap with 4 individuals becoming positive and remaining so on returning to Sydney. There was no obvious exposure to mycobacterium during the expedition but it is recommended that in future expeditions all PPD negative individuals be vaccinated with BCG.
Adaptation, Physiological↗
Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement.
T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (alpha CD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead-cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to alpha CD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.
CD4 Antigens↗
Cross-reacting IgE antibodies recognizing latex allergens, including Hev b 1, as well as papain.
The cross-reactivity of IgE antibodies recognizing epitopes of latex allergens and papain was studied in sera of 36 latex-exposed subjects and 22 papain workers. Eight out of 24 latex-sensitized persons also showed a positive reaction to papain in the CAP assay (mostly of low or moderate degree). On the other hand, six out of the 12 sensitized papain workers also revealed IgE binding to latex allergen(s). Reciprocal inhibition experiments confirmed that groups monosensitized to one of the two allergens can be separated from a group showing partial or nearly complete immunologic cross-reactivity. Papain inhibited IgE binding by 20-33% in these subjects, whereas IgE binding to papain was strongly blocked in most cases. Comparison between the primary sequences of Hev b 1, a major latex allergen, and papain suggests that the cross-reactivity may be due to several identical trimers and tetramers.
Allergens↗
Surgical model for in vivo evaluation of cultured epidermal sheets in mice.
The objective of this study was to establish a novel surgical model for the grafting of cultured epidermal sheets in mice in order to optimize studies on the behavior of these grafts. Graft-related skin immunology and wound-healing studies also would benefit from this adapted surgical approach. Adapted tie-over surgical procedures were established for mice and promptly applied. Early-stage observation of grafts was made possible by replacing the cotton dressing with a saddle-like plastic tube dressing with a screw cap. We grafted normal Balb/c mice and athymic nude (nu/nu) mice with cultured human epidermis. Evaluation of graft rejection was carried out with the first group, whereas the second provided information on epidermal stratification and terminal differentiation. This innovation permitted direct evaluation of the grafted tissue at any time. Advances in applied transplantation research will certainly provide additional tools for human applications.
Animals↗
The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis bacillus Calmette Guérin. Heterogeneity, structure, and role in the regulation of cytokine secretion.
Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune response; however, the molecular basis underlying the diversity of their immunological properties remain an open question. In this study a new extraction and purification approach was successfully applied to isolate ManLAMs (lipoarabinomannans with mannosyl extensions) from bacillus Calmette Guérin leading to the obtention of two types of ManLAMs namely parietal and cellular. Structurally, they were found to differ by the percentage of mannooligosaccharide caps, 76 and 48%, respectively, and also, thanks to a new analytical method, by the structure of the phosphatidyl-myo-inositol anchor lipid moiety. A novel fatty acid in the mycobacterium genus assigned to a 12-O-(methoxypropanoyl)-12-hydroxystearic acid was the only fatty acid esterifying C-1 of the glycerol residue of the parietal ManLAMs, while the phosphatidyl unit of the cellular ManLAMs showed a large heterogeneity due to a combination of palmitic and tuberculostearic acid. Finally, parietal and cellular ManLAMs were found to differentially affect interleukin-8 and tumor necrosis factor-alpha secretion from human dendritic cells. We show that parietal but not cellular ManLAMs were able to stimulate tumor necrosis factor-alpha secretion from dendritic cells. From these studies we propose that the 1-[12-O-(methoxypropanoyl)-12-hydroxystearoyl]-sn-glycerol part is the major cytokine-regulating component of the ManLAMs. It seems likely that modification of the ManLAM lipid part, which may occur in hostile environments, could regulate macrophagic mycobacterial survival by altering cytokine stimulation.
Antigens, Bacterial↗
Intermediate filament proteins in human sperm heads.
Monoclonal antibodies made against human sperm cells have been characterized with regard to binding patterns and molecular coordinates of the recognized antigens. Antibodies T5 and T6 gave uniform binding to the acrosomal cap in an intact cell, and decreased to equatorial segment binding in an 'acrosome-reacted' cell. Monoclonal antibody T15 gave the reverse: equatorial segment binding in intact cells and uniform acrosomal cap binding in reacted cells. From staining patterns on cultured cell lines, determination of molecular coordinates, immunoblots, and partial peptide analysis, we have determined that T15 is directed against the cytoskeletal protein, vimentin, while T5 and T6 recognize a keratin-like protein which may be unique to sperm cells. This is the first immunological and biochemical study to analyse both types of intermediate filament proteins in human sperm cells.
Antibodies, Monoclonal↗
Detection of root mucilage using an anti-fucose antibody.
Plant root mucilage is known to enhance soil quality by contributing towards the soil carbon pool, soil aggregation, detoxification of heavy metal ions and interactions with rhizospheric microflora. Mucilage consists of many monosaccharide units, including fucose which can be used as an indicator for plant root based polysaccharides. This is the first report of an immunological technique developed to use anti-fucose antibodies as markers for probing and localizing fucosyl residues in mucilage polysaccharide and, in turn, for localization of plant root mucilage. Fucose was complexed with bovine serum albumin to raise antibodies against fucose. A fucose-directed antibody was shown to cross-react with root cap mucilages from grasses. This antibody was used to localize root mucilage polysaccharide in maize and wheat root caps using immunogold electron microscopy. Abundant labelling could be localized on the cell wall, and in the intercellular matrix and vesicles of the peripheral root cap cells. Labelling was less intense in cells towards the centre of the root cap tissue. Control experiments confirmed that immunogold localization of fucose was specific and reliable.
Animals↗
Allergy diagnosis by Pharmacia Cap System.
From September 1995 to September 1998, sera from 959 suspected allergy patients have been tested by the new Pharmacia Cap System. Of these, 80 per cent were diagnosed to suffer from some allergy while 20 per cent reacted negative. It was found that the CAP system gave accurate and clear cut results to the satisfaction of the patient and referring physician.
Humans↗
Fate of antibodies bound to lymphocyte surface. I.--Study with complement-dependent cytotoxicity, indirect immunofluorescence and radiolabelled antibodies.
Anti-HL-A alloantiserum, heterologous antilymphocyte globulins and anti-beta2m antisera were allowed to react to human lymphocytes in vitro. The fate of the antigen-antibody complexes formed at the cell surface has been studied by means of the lymphocytoxicity test in the presence of rabbit complement, radiolabelled antibodies and the indirect immunofluorescence technique. Those techniques demonstrated that initially bound antibodies disappear from the cell surface. This disappearance requires optimal conditions; it does not result from a mere dissociation of antibodies but likely represents partial degradation, internalization and release of antigen-antibody complexes into the extracellular medium. This phenomenon can be regarded as a reflection of the dynamic state of the cell membrane, although it differs from the redistribution of antigen-antibody complexes on the lymphocyte surface (patching and capping). Possible implications of such events are discussed in relation to triggering or blocking of lymphocyte proliferation.
Antibodies↗