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PubMed1984

Identification of immunoreactive forms of human erythrocyte band 3 in nonerythroid cells.

Antibodies directed to the cytoplasmic domain of human erythrocyte band 3, the major integral protein of the erythrocyte membrane which is thought to be the main anchoring site of the membrane cytoskeleton, were demonstrated in the present study to react with the membrane of various nonerythroid cells, such as human leucocytes, fibroblasts or human umbilical mesenchyme cells, amniotic epithelium and vascular smooth muscle. In cultured fibroblasts staining was confined to small dots and streaks associated with both the dorsal and ventral cell membrane. In human lymphocytes band 3 antigen accompanied capping of concanavalin A binding surface receptors. The immunoreactive form of band 3 in fibroblasts was shown by immunoblotting studies to be a polypeptide of approximately 60 000 dalton. This polypeptide is immunologically and electrophoretically related to a major immunoreactive form of band 3 naturally occurring in the red blood cell membrane. Considering the recent identification in nonerythroid cells of immunoreactive forms of other major components of the erythrocyte membrane cytoskeleton, the present observation in nucleated cells of a polypeptide related to erythrocyte band 3 may indicate some of the features of erythrocyte membrane architecture are also present in nonerythroid cells.

Animals↗
PubMed2000

EXT 1 gene mutation induces chondrocyte cytoskeletal abnormalities and defective collagen expression in the exostoses.

Hereditary multiple exostoses (HME), an autosomal skeletal disorder characterized by cartilage-capped excrescences, has been ascribed to mutations in EXT 1 and EXT 2, two tumor suppressor-related genes encoding glycosyltransferases involved in the heparan sulfate proteoglycan (HSPG) biosynthesis. Taking advantage of the availability of three different exostoses from a patient with HME harboring a premature termination codon in the EXT 1 gene, morphological, immunologic, and biochemical analyses of the samples were carried out. The cartilaginous exostosis, when compared with control cartilage, exhibited alterations in the distribution and morphology of chondrocytes with abundant bundles of actin filaments indicative of cytoskeletal defects. Chondrocytes in the exostosis were surrounded by an extracellular matrix containing abnormally high amounts of collagen type X. The unexpected presence of collagen type I unevenly distributed in the cartilage matrix further suggested that some of the hypertrophic chondrocytes detected in the cartilaginous caps of the exostoses underwent accelerated differentiation. The two mineralized exostoses presented lamellar bone arrangement undergoing intense remodeling as evidenced by the presence of numerous reversal lines. The increased electrophoretic mobility of chondroitin sulfate and dermatan sulfate proteoglycans (PGs) extracted from the two bony exostoses was ascribed to an absence of the decorin core protein. Altogether, these data indicate that EXT mutations might induce a defective endochondral ossification process in exostoses by altering actin distribution and chondrocyte differentiation and by promoting primary calcification through decorin removal.

Adolescent↗
PubMed1987

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

Actin Cytoskeleton↗
PubMed2005

alpha2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells.

Placental protein 14 (PP14; glycodelin) is a pregnancy-associated immunoregulatory protein that is known to inhibit T cells via T-cell receptor desensitization. The recent demonstration of PP14 as lectin has provided insight into how it may mediate its CD45 glycoprotein-dependent T-cell inhibition. In this study, we have investigated PP14's lectin-binding properties in detail. Significantly, PP14 reacts with N-acetyllactosamine (LacNAc) as was also found for members of the galectin family, such as the potent immunoregulatory protein, galectin-1. However, in contrast to galectin-1, PP14's binding is significantly enhanced by alpha2,6-sialylation and also by the presence of cations. This was demonstrated by preferential binding to fetuin as compared with its desialylated variant asialofetuin (ASF) and by using free alpha2,6- versus alpha2,3-sialylated forms of LacNAc in competitive inhibition and direct solid-phase binding assays. Interestingly, from immunological point of view, PP14 also binds differentially to CD45 isoforms known to differ in their degree of sialylation. PP14 preferentially inhibits CD45RA+, as compared with CD45RO+ T cells, and preferentially co-capped this variant CD45 on the T-cell surface. Finally, we demonstrate that PP14 promotes CD45 dimerization and clustering, a phenomenon that may regulate CD45 activity.

ABO Blood-Group System↗
PubMedDate not supplied

[Efficacy of specific immunotherapy in children with bronchial asthma, sensitised to dermatophagoides pteronyssinus].

The aim of study was the evaluation of efficacy of specific immunotherapy using Alavac S HDM for 2-4 years in children with atopic bronchial asthma induced by Deramtophagoides pteronyssinus. The study was carried out in a group of 33 children aged 5-18 years, from the Outpatient Department of Immunology at the National Research Institute of Mother and Child. The children demonstrated clinical symptoms of asthma and had a medical history typical for this disease. Diagnostic procedures including skin prick tests and estimation of specific IgE to Dermatophagoides pteronyssinus by Pharmacia-CAP system gave positive results. The efficacy of therapy was monitored by a clinical score of symptoms with 0-10-20-30 points, recording symptoms relating to intensity of dyspneaa, wheezing, cough, value of PEF and concomitant medications for example corticoids and beta-antagonists. In the study group, 31 children (94%) with asthma treated by specific immunotherapy (AlavacS HDM) improved. This was confirmed by a statistically significant difference. The results indicate that specific immunotherapy is effective in the treatment of asthma in children sesitized to Dermatophagoides pteronyssinus.

Adolescent↗
PubMed1979

Immunoregulatory properties of human esophageal tumor extract.

Noncytotoxic aqueous extracts of esophageal tumors (TEx) inhibit the spontaneous uptake of 3H-thymidine by human peripheral blood mononuclear cells (PBM). TEx also inhibits mitogen- and antigen-induced PBM blastogenesis, mitogenesis, 3H-thymidine uptake, and 3H-leucine incorporation. Inhibition is reversible and is mediated by a heat-labile protein with a m.w. of approximately 70 to 80,000 daltons. Inhibitory activity is not due to trivial binding of mitogen or neucleotide by the inhibitor, nor is it entirely tumor specific since similar but quantitatively less activity is extractable from adjacent normal esophageal mucosa. Assorted cell surface membrane phenomena such as contact inhibition of in vitro endothelial cell proliferation, lymphocyte E rosette formation, and cap formation are unaffected by TEx. In contrast, immunoglobulin synthesis by pokeweed mitogen-stimulated PBM is enhanced by TEx. The potential importance of the local production of immunoregulatory agents like TEx in the immunologic control of tumor cell growth is discussed.

Cell Membrane↗
PubMed2002

Calcium phosphate nanoparticles induce mucosal immunity and protection against herpes simplex virus type 2.

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.

Adjuvants, Immunologic↗
PubMedDate not supplied

The sperm acrosome: immunological analysis using specific polyclonal and monoclonal antibodies directed against the outer acrosomal membrane of boar spermatozoa.

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in female Balb/c mice and was characterized by means of an indirect ELISA. The hyperimmune serum reacted selectively with the acrosomal cap of the sperm head and showed an extremely good cross reactivity with bull and human spermatozoa when assayed by indirect immunofluorescence. Immunoelectron microscopy using the protein A-gold method further confirmed the specificity of the anti-OAM-antiserum for the OAM. In an effort to identify the OAM antigens recognized by the hyperimmune serum and to analyse the extent of cross reactivity on a molecular level, the SDS-extractable proteins were separated by SDS-PAGE, transblotted and immunoprinted using an 125J-conjugated anti-mouse-antibody. To facilitate functional and structural analysis of distinct OAM-proteins monoclonal antibodies were generated by hybridization of mouse myeloma cells with the splenocytes of female Balb/c mice immunized with the purified OAM. One fusion resulted in about 100 anti-OAM-antibodies secreting hybridoma cultures, of which about 30% showed cross reaction with human and bull spermatozoa. Four stable cell lines were selected for this study secreting antibodies directed against the outer acrosomal membrane of boar spermatozoa. Whereas the polyclonal immune mouse serum stained the entire acrosomal cap, the four hybridoma antibodies generated a patch-work-like immunofluorescence pattern over the acrosome. HPLC-ELISA of the solubilized OAM revealed first information on the nature of the corresponding membrane antigen.

Acrosome↗
PubMed2005

Intracoronary thermography: does it help us in clinical decision making?

The concept of the "vulnerable" plaque has recently emerged to explain how quiescent atherosclerotic lesions evolve to cause clinical events. The morphologic and immunologic determinants specific for the vulnerable plaque have been reported: a large lipid core (>or=40% plaque volume) composed of free cholesterol crystals, cholesterol esters, and oxidized lipids impregnated with tissue factor; a thin fibrous cap depleted of smooth muscle cells and collagen; an outward (positive) remodeling; inflammatory cell infiltration of fibrous cap and adventitia (mostly monocyte-macrophages, some activated T cells, and mast cells); and increased neovascularity. Despite the large amount of information regarding the morphological characteristics of remote lesions, we lack studies with functional assessment of non-culprit lesions. Coronary thermography is a technique for functional assessment of coronary atherosclerotic plaques. Several catheter designs have been proposed. There are catheters with thermistor(s) and wires with thermal sensors at the distal tip. All designs have several advantages and disadvantages. Despite the current limitations of coronary thermography, we gained important pathophysiological and clinical information regarding the vulnerability of atheromatic plaques. It has been documented both experimentally and clinically that increased heat generation is associated with increased macrophage concentration within the plaque. The correlation between local inflammatory involvement and local heat generation has also been observed with the peripheral inflammatory markers such as C-reactive protein. Whether systemic treatment, with agents such as statins or interventional techniques, such as drug-eluting stents, will have an impact on stabilizing vulnerable plaques need to be determined in future studies. In conclusion, although there are several techniques for evaluating morphologically atheromatic plaques, thermography is a promising method for the functional assessment of vulnerable plaque and has been introduced into clinical practice, with a good predictive value for clinical events in patients with increased temperature in the atherosclerotic plaque.

Coronary Artery Disease↗
PubMedDate not supplied

[Efficacy of specific immunotherapy in children with seasonal allergic rhinitis and conjunctivitis, sensitized to grass pollen].

The aim of this study was the evaluation of efficacy of the specific immunotherapy using Pollinex for 3 years before the period of pollen dust in children with seasonal allergic rhinitis and conjunctivitis induced by grass pollen. The study was carried out in a group of 40 children aged 5-18 years, from the Outpatient Department of Immunology at the National Research Institute of Mother and Child. The children demonstrated clinical symptoms of seasonal allergic rhinitis with conjunctivitis and had a medical history typical for this disease. Diagnostic procedures including skin prick tests and estimation of specific IgE to grass pollen by Pharmacia - CAP system, gave positive results. The efficacy of therapy was monitored by a clinical score with 4-0 points, recording symptoms relating to intensity of rhinoedema, rhinorrhea, sneezing, itching, oedema, conjunctival congestion, pain and lacrimation. In the study group, 39 children (97%) with seasonal rhinitis and conjunctivitis treated by specific immunotherapy (Pollinex) improved. This was confirmed by a statistically significant difference. The results indicate that the specific immunotherapy is an effective treatment of seasonal allergic rhinitis with conjunctivitis in children sensitized to grass pollen.

Adolescent↗
PubMed1996

Immunological findings in groups of children after compensatory measures.

In 200 children residing permanently in a region with a high level of atmospheric pollution, indicators of humoral immunity, salivary immunity and specific IgE antibodies were determined before and after 14 days' attendance at an "outdoor school" in rural areas with a very low level of atmospheric pollution. A marked reduction of elevated albumine levels, which are characteristic of irritation or even inflammation in the oral cavity or respiratory tract, was found, as well as increasing levels of Slg A and lysozyme in saliva. The two-week stay in the outdoor school resulted in decreased IgE specific antibodies of the respiratory type of allergy (CAP Phadiatop). In the group of atopic children there were found high total IgE levels, with a trend of depression after 2 weeks in the outdoor school. Higher values of some immunoglobulins were observed, while low levels of IgA were found in some children with high frequency of respiratory diseases. The irritant effect of polluted atmosphere and the quality of indoor atmosphere influence some immunological parameters in children.

Adolescent↗
PubMed2005

Immunological properties of surface proteins of Clostridium difficile.

Sera from patients with Clostridium difficile-associated disease (CDAD) and sera from a control group were analysed by an ELISA to detect antibodies directed against four surface proteins and toxins A and B of C. difficile. The surface proteins were the flagellar cap protein FliD, the flagellin FliC, the adhesin Cwp66 divided into two domains, Cwp66-Nterminal and Cwp66-Cterminal, and the fibronectin-binding protein Fbp68. For each antigen, antibody levels in the CDAD patient group and in the control group were compared. In the CDAD patient group, the mean of the antibody levels decreased from Cwp66-Cterminal to Fbp68, FliD, toxins B and A, Cwp66-Nterminal and finally FliC, suggesting different immunogenic properties among these adhesins. For Cwp66-Nterminal, FliC, FliD and Fbp68, the antibody level observed in the control group was higher than in the CDAD group with a statistically significant difference whereas the antibody level for toxins A and B was not statistically different. In conclusion, this study suggests that during the clinical course of disease, C. difficile adhesins are able to induce an immune response which could play a role in the defence mechanism of the host.

Adhesins, Bacterial↗
PubMed2002

Cord blood mononuclear cell cytokine responses in relation to maternal house dust mite allergen exposure.

BACKGROUND: Cord blood mononuclear cells have demonstrated specific immune responses to environmental allergens. OBJECTIVE: To establish whether the nature of this response is related to the level of maternal antenatal exposure to house dust mite (HDM) allergen and, hence, whether antenatal allergen avoidance may have a role in the prevention of allergic sensitization in children. METHODS: Children with a family history of asthma were recruited antenatally as subjects in a randomised controlled trial: the Childhood Asthma Prevention Study. HDM allergen (Der p 1) concentrations were measured in dust collected from the maternal bed at 36 weeks gestation. Cord blood mononuclear cells were stimulated in culture, separately, with phytohaemaglutinin (PHA) and HDM extract. Cytokine IL-4, IL-5, IL-10 and IFN-gamma concentrations in supernatant were measured by ELISA. mRNA signals for these cytokines were measured using RT-PCR. RESULTS: The median concentration of HDM allergen was 18.4 microg/g (interquartile range 7.3-35.3 microg/g). Median concentrations of IL-4, IL-5, IL-10 and IFN-gamma, after PHA stimulation were 4, 19, 401 and 1781 pg/mL, respectively. After HDM allergen stimulation the median concentrations were 0, 0, 20 and 14 pg/mL, respectively. The distribution of mRNA cytokine signals was similar. Neither cytokine protein concentrations nor cytokine mRNA signal levels were correlated with the concentration of HDM allergen in the mothers' beds at 36 weeks gestation. CONCLUSION: These findings do not support the view that the prevention of allergic disease in children requires the institution of HDM avoidance interventions during pregnancy.

Air Pollution, Indoor↗
PubMed1998

Inhalant allergy to egg yolk and egg white proteins.

BACKGROUND: Several egg white and egg yolk and avian proteins have been described as a cause of inhalant allergy. Sometimes inhalational type I hypersensitivity to these proteins is associated with food allergy to egg. OBJECTIVE: We studied two patients who experienced respiratory and food allergic symptoms upon exposure to egg or avian antigens through the inhalative or digestive routes. Clinical and immunological studies were carried out in order to identify individual allergens from these sources that could be responsible for crossreactivity reactions. RESULTS: Patient 1 showed IgE sensitization to egg yolk livetins, feathers, and chicken serum. Specific bronchial challenge with chicken albumin and livetin extracts elicited a positive early asthmatic response and an increase in serum eosinophil cationic protein. Immunoblot and CAP-inhibition studies in this patient supported that chicken albumin (alpha-livetin) was the crossreactive antigen present in egg yolk and chicken serum and feathers. Patient 2 showed sensitization to egg white, ovomucoid and lysozyme. However, SDS-PAGE and immunoblot studies demonstrated contaminating lysozyme in the ovomucoid extract and identified lysozyme as the main allergen causing egg sensitization in this patient. Conjunctival challenge test confirmed allergy to lysozyme. CONCLUSION: Egg yolk and egg white proteins may act not only as ingested allergens but also as aeroallergens. Immunological studies using highly purified preparations of egg proteins are useful for the accurate diagnosis of allergic reactions to egg proteins and to identify individual allergens that may be responsible for crossreactivity reactions.

Adult↗
PubMed1988

Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin.

In this study we have used complementary biochemical and immunological techniques to establish that the lymphoma GP85 membrane glycoprotein is a transmembrane protein with a cytoplasmic domain that binds directly to ankyrin, a molecule known to link the membrane to the cytoskeleton. The evidence supporting our conclusion that the GP85 is a transmembrane glycoprotein is as follows: (a) GP85 can be surface-labeled with Na 125I and contains wheat germ agglutinin-binding sites, indicating that it has an extracellular domain; (b) GP85 can be phosphorylated by intracellular kinases, indicating that it has an intracellular domain; and (c) GP85 can be successfully incorporated into phospholipid vesicles, indicating the existence of a hydrophobic domain in the molecule. Further studies show that GP85 displays immunological cross-reactivity with the lymphocyte Pgp-1 (differentiation-specific) membrane glycoprotein, and with the erythrocyte anion transport membrane protein, band 3. Immunocytochemical studies indicate that an ankyrin-like protein accumulates underneath the lymphoma GP85 cap structure, suggesting an association of the ankyrin-like protein and GP85. This relationship has been further confirmed by the following results of binding and reconstitution experiments: (a) purified GP85 binds directly to an ankyrin-Sepharose column; (b) purified GP85 inserts into phospholipid vesicles in both the normal (right side out) and reversed (inside out) orientation (and with only the reversed configuration permits binding of ankyrin to GP85); and (c) cleavage of GP85 with trypsin yields a 40-kD peptide fragment that is part of the cytoplasmic domain and contains the ankyrin binding site(s). Based on these findings, we suggest that the lymphoma GP85 transmembrane glycoprotein contains a cytoplasmic domain that is directly involved in linking ankyrin to the cytoskeleton. This transmembrane linkage may play a pivotal role in receptor capping and cell activation in lymphocytes.

Animals↗
PubMed1982

Tumor-associated surface membrane antigens on CFUs: characterization using the fluorescent activated cell sorter.

Antiserum raised in rabbits against the FMP1.1 mouse tumor cell line is known to cross-react with GM-CFC and BFUE from normal marrow using the complement-dependent cytoxicity assay. It has been found that this antiserum is cytotoxic to CFUs, either with or without complement addition in vitro. Immunofluorescent analysis using the fluorescent activated cell sorter (FACS) has confirmed this immunological cross-reactivity with CFUs. Recovery of antibody-coated CFUs posed a potential problem in FACS experiments due to in vivo destruction by either opsinization or cytotoxic lysis. Experiments involving enzyme digestion (papain) of antibody and preincubation of bone marrow cells to allow antibody capping did not show improved recovery of CFUs. However, incubation of anti-FMP1.1 coated bone marrow cells with specific anti-rabbit IgG serum resulted in up to 67% of CFUs being detected. Dual parameter sorting with FACS, involving forward light scatter and fluorescence, gave about 26 and 19 times enrichment of GM-CFC and BFUE, respectively, whereas CFUs were enriched by only 6 times. These colony-forming cells were found in the cell population with high intensity forward light scatter and with relatively low fluorescence. Bone marrow cells undergoing regeneration 7 days after 5-fluorouracil (5-FU) treatment of mice exhibited more intense immunofluorescence than normal marrow cells. In particular, a distinct population of lymphocytes in 5-FU treated marrow reacted with anti-FMP1.1 serum, which was not observed with normal marrow.

Animals↗
PubMed2001

Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer.

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

Animals↗