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Results for “Immunologic Capping”

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At least 811 records · Page 45Linked to original sources
PubMed1996

Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T cell activation.

Two binding sites for MHC class II have previously been identified on opposite sides of the superantigen, staphylococcal enterotoxin A (SEA). The sites mediate separate binding reactions with nonoverlapping regions of class II, and in solution cause SEA to complex with purified HLA-DR1 to form DR1.SEA2 trimers. Here, a set of complementary SEA class II-binding mutants was used to study the interaction of SEA with cell surface MHC class II. The results indicate that both class II binding sites are required on the same toxin molecule for maximal activity, demonstrating that simultaneous ligation of two MHC class II molecules on APCs by a single SEA is essential for effective superantigen function. Coalescence of MHC class II by SEA results in protein tyrosine kinase activation and contributes to the induction of cell:cell adhesion, pro-inflammatory cytokine gene transcription, and T cell proliferation.

Antigen Presentation↗
PubMed1995

Inhibitory effect of c-fos overexpression on B cell proliferative responses to membrane IGM cross-linking.

Constitutive expression of the c-fos gene perturbs the de novo synthesis of RNA and DNA in B cells stimulated by surface immunoglobulin (sIg) cross-linking. In order to examine kinetics of the regulatory effect on the activation process of B cells, we used splenic B cells from transgenic mice carrying the c-fos gene under the control of the interferon alpha/beta (IFN)-inducible Mx gene promoter (Mx-c-fos). In the absence of IFN, Mx-c-fos B cells proliferated well by anti-IgM stimulation. However, both RNA and DNA synthesis in the Mx-c-fos B cells were markedly reduced by the addition of IFN in the culture within 12 h after anti-IgM stimulation. These results suggest that this regulatory effect of c-Fos displays at the mid G1 phase of the cell cycle.

Animals↗
PubMed1997

Engagement of the Lewis X antigen (CD15) results in monocyte activation.

We previously reported that monocyte adhesion to tumor necrosis factor-alpha (TNF-alpha)-treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-alpha release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-alpha mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1 beta (IL-1 beta) mRNA, while no apparent change was observed in the levels of beta-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1 beta promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1 beta transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-kappa B, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.

Antibodies, Monoclonal↗
PubMed1997

Microphthalmia (mi) in murine mast cells: regulation of its stimuli-mediated expression on the translational level.

Mice harboring a mutation in the microphthalmia (mi) gene display a variety of abnormalities, including microphthalmia, depletion of skin melanocytes, deafness, a defect in osteoclasts, and a major decrease in mast cell number and function. However, despite the possible critical role played by this protein in mast cell development and function, characterization of its mRNA and protein synthesis in these cells has not yet been performed. In this study, we investigated the regulation of the synthesis of mi in murine mast cells activated by various physiologic stimuli. Using a specific rabbit polyclonal anti-mi antibody, we found that interleukin-3, interleukin-4, or aggregation of the mast cell high-affinity receptor for IgE (Fc epsilonRI) induced the synthesis of mi protein in these cells. None of these stimuli significantly affected the level of mi mRNA in the mast cells at any of the time points tested. Also, using this specific anti-mi antibody, an increase in mi protein synthesis was shown during differentiation of mast cells from their bone marrow cell precursors. Moreover, a complex containing mi bound to upstream stimulating factor 2 was detected only in activated mast cells. We conclude that the regulation of mi expression is on the translational level. Thus, stimulation of mast cells by a variety of stimuli elicits a signaling pathway that regulates mi expression.

Amino Acid Sequence↗
PubMed1998

Evidence for an upper affinity threshold for anti-IgM-induced apoptosis in a human B-cell lymphoma.

The influence of ligand:receptor affinity on B-cell antigen receptor (BCR)-induced apoptosis in the IgM+ Burkitt lymphoma line, Ramos, was evaluated with a group of affinity-diverse murine monoclonal antibodies (MoAbs) specific for human B-cell IgM. The studies showed not only a minimal affinity threshold for the induction of apoptosis, but, interestingly, also a maximal affinity threshold above which increases in affinity were associated with diminished apoptosis. The lesser capacity of high-affinity MoAb to induce apoptosis was paralleled by a lesser capacity to induce receptor cross-linking. At high ligand concentration, high MoAb affinity was also associated with a diminished capacity to induce early protein tyrosine phosphorylation. The compromised capacity of two high-affinity MoAbs to trigger apoptosis may be, at least in part, explained by two separate phenomena that can impair the formation of mIgM cross-links: (1) more stable univalent binding and (2) a tendency for monogamous binding of both MoAb Fab to two Fab epitopes on mIgM. These in vitro studies suggest that the use of the highest affinity MoAbs for antireceptor immunotherapies that depend on receptor cross-linking might, on occasion, be contraindicated.

Animals↗
PubMed1995

[CAP system methods versus FAST methods in immunologic monitoring of specific immunotherapy in pollen allergy].

The aim was to study the value of the CAP system (Pharmacia) in comparison with the FAST method (Bio Whittaker) in determination of total IgE, specific IgE and IgG4 during specific immunotherapy (sIT). The studies were carried out on a group of 67 patients with pollinosis treated with various pollen allergen extracts. The immunological studies were performed before and after several courses of sIT. A higher reproducibility and sensitivity of the CAP System was shown. The values of specific IgE were somewhat higher on using the CAP system than using the FAST method. The CAP system seems to be of great value in diagnostics and monitoring sIT, especially in birch pollen allergy.

Adolescent↗
PubMed1979

Capping of variable antigen on Trypanosoma brucei, and its immunological and biological significance.

Pathogenic trypanosomes undergo antigenic variation, whereby the glycoprotein molecules constituting the cell coat are changed, the parasite thus evading the host's immune response. On application of homologous antiserum in indirect immunofluorescence to a given variable antigen type of Trypanosoma brucei, the surface variable antigen moves to the flagellar pocket region, which overlies the Golgi apparatus. This redistribution, or capping, is temperature-dependent, occurring at 37 degrees C but not at 0-4 degree C. Patching does not occur at either temperature. Immediately after capping no homologous or heterologous variable antigen, or host plasma or blood cell antigens, can be detected by immunofluorescence on the cell surface outside the cap; only trypanosome membrane common antigens can be found. It seems unlikely for two reasons that this antibody-induced redistribution is relevant to antigenic variation. Capping of the coat requires the indirect, rather than the direct, immunofluorescent method; a single layer of antibody, in nature, would appear to be ineffective. Also, capping of variable antigen of one type is followed within 3 h by appearance of antigen of the same, and not another, type. The necessity for 2 antibody layers is usually thought of as meaning that the individual molecules of the cell surface antigen are spaced further apart than the binding sites of an individual antibody molecule, so that the necessary cross-linked lattice cannot be formed, but on T. brucei the surface variable antigen molecules are very closely packed. It is proposed that one layer of antibody is ineffective for steric reasons; the dimensions of the exposed face of each variable antigen molecule may not permit the binding of more than one molecule of immunoglobulin, or perhaps the antigen molecules are so closely packed that most of the antigenic determinants are hidden from antibodies. To test this hypothesis, an attempt was made to cap variable antigen on trypanosomes transforming in vitro from the bloodstream to the procyclic (insect midgut) stage; such forms have a much less densely packed surface coat. Patching was observed, indicative of lattice formation, but these trypanosomes did not survive the in vitro manipulation long enough to permit any possible capping. T. brucei differs structurally from most other eukaryotic cells. It has no detectable microfilaments under the plasma membrane, except at the desmosomes in the region of flagellar binding, and it also has a pellicular cortex of microtubules. Capping of its surface antigen would appear then to differ from that on mammalian cells, either in the cellular components involved or in that specialized areas of the plasma membrane are involved.

Animals↗
PubMed1985

Immunological detection of the messenger RNA cap-binding protein.

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.

Animals↗
PubMed2002

Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Adult↗
PubMed1983

Immunological effects of amplitude-modulated radio frequency radiation: B lymphocyte capping.

B lymphocytes collected from normal ICR Swiss mouse spleens were exposed in vitro in a Crawford cell to 147-MHz radiofrequency (RF) radiation, amplitude modulated by a 9-, 16-, or 60-Hz sine wave. The power densities ranged between 0.11 and 48 mW/cm2. The irradiated samples and the controls were maintained at 37 degrees C or 42 degrees C, with temperature variations less than 0.1 degrees C. Immediately after a 30-minute exposure, the distribution of antigen-antibody (Ag-Ab) complexes on the cell surface was evaluated at 37 degrees C by immunofluorescence. Under normal conditions (37 degrees C, no RF), Ag-Ab complexes are regrouped into a polar cap by an energy-dependent process. Our results demonstrate that the irradiated cells and the nonirradiated controls capped Ag-Ab complexes equally well after exposure at 37 degrees C. Capping was equally inhibited at 42 degrees C in both the controls and irradiated cells. No statistically significant differences in capping were observed between the RF-exposed and control samples at any of the modulation frequencies and power densities employed as long as both preparations were maintained at the same temperature.

Animals↗
PubMed2003

Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum).

BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food. OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins. METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA. RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin. CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.

Adolescent↗
PubMed1998

Identification of complement activators and elucidation of the fate of complement activation products during extracorporeal plasma purification therapy.

It has been known for many years that the complement system is activated during extracorporeal plasma purification (ECCP) therapy. In a previous study, we showed that high concentrations of complement activation products (CAPs) are returned to the patient during immunoadsorption treatment. In the present study, we investigated the question of where complement activation takes place with different forms of ECPP equipments as well as the fate of the CAPs. Eleven patients (8 men and 3 women), mean age 52 +/- 18 years, were included in the study. They were treated either with plasmapheresis (PP), immunoadsorption, bilirubin adsorption, or low density lipoprotein (LDL) apheresis. It was found that during all ECPP treatments and after the plasma separation filter, the plasma concentrations of CAPs were increased, and that high concentrations of CAPs were returned to the patients, except with PP. The plasma levels of individual CAPs varied between different types of ECPP. These variations were due to several factors: (1) complement activation (CA) on the plasma separator and a secondary device, e.g., column or membrane; (2) adsorption of specific CAPs to separation columns; and (3) reduction of CAPs due to separation and waste. Since CAPs have inflammatory and immunological effects, it is possible that high serum concentration of CAPs in the treated patients may influence the clinical outcome of the treatment. In conclusion, complement activation is a fact that should not be ignored during performance of any form of an ECPP. It is the plasma separator that plays a key role in the process of complement activation. Different ECPP treatments may have different effects regarding the levels of individual CAPs.

Adult↗
PubMed1987

Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method.

Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffin-embedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on non-motile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.

Avidin↗
PubMed1980

Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

We have analyzed the assembly of microtubules and the distribution of concanavalin A(Con A)-receptor complexes in the same populations of human peripheral blood T and B lymphocytes. We hoped to resolve the prolonged controversy over the relationship of microtubules to Con A cap formation in lymphocytes and to explain the abnormally high spontaneous and colchicine-induced Con A capping that was observed recently in lymphocytes from a patient with an inherited form of severe combined immunodeficiency disease (SCID) characterized by total immunologic dysfunction despite normal numbers and distribution of T and B cells. The data establish that (i) microtubule disassembly is correlated with enhanced Con A cap formation on normal human lymphocytes; (ii) T and B cells differ significantly from each other and from circulating polymorphonuclear leukocytes with respect to their capping responses after exposure to colchicine; and (iii) there is an abnormal relationship of microtubule assembly to surface topography in the functionally defective SCID cells.

B-Lymphocytes↗
PubMed1980

The distribution of selected hematology measurements in the CAP survey.

Investigation of the 1978 CAP Hematology Survey results for hemoglobin, hematocrit, erythrocyte count, leukocyte count, prothrombin time, and partial thromboplastin time with respect to the assumption of normality and the method for detecting outliers was performed. The findings indicate that the assumption of normality, while not exactly valid, is reasonable for the purposes of the Survey, but that the method of determining outliers may be too stringent in the case of hemoglobin, hematocrit, erythrocyte count, and leukocyte count, and not appropriate for prothrombin time and partial thromboplastin time. These findings are similar to those reported earlier for selected chemistry and immunology constituents in the CAP Survey.

Erythrocyte Count↗