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K J Turner

Publications and source records attributed to K J Turner.

At least 55 records · Page 3Linked to original sources
PubMed1995

Purified rat peritoneal mast cells: characteristics of the removal and binding of IgE.

Mast cells were purified by discontinuous density gradient centrifugation to greater than 90% and subsequently exposed to lactic acid in order to dissociate endogenous IgE. Unfortunately no removal of this IgE was achieved, despite previous reports of lactic acid exposure dissociating IgE from human basophils. Only a minimal level of endogenous IgE was found on the surface of mast cells, however, which allowed investigations of IgE binding to proceed without the initial removal of this endogenous IgE. Conditions for binding of normal rat IgE and myeloma IgE were established while efforts to bind human IgE to purified rat mast cells were unsuccessful. Our results suggest that while purified rat mast cells have the potential to play an important role in the study of the IgE requirement of various stimuli including histamine-releasing factor, this role is limited at present.

Animals↗
PubMed1995

Failure to detect interleukin (IL)-3 mRNA or protein in human keratinocytes: antibodies to granulocyte macrophage-colony-stimulating factor or IL-6 (but not IL-3) neutralize "IL-3" bioactivity.

Interleukin (IL)-3-like bioactivity has been found in culture supernatants from human and murine keratinocytes. However, there is controversy as to the presence of IL-3 mRNA in human keratinocytes. Using highly sensitive reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay techniques, we examined human keratinocytes from four different donors (neonatal foreskins) and were unable to detect IL-3 mRNA or IL-3 protein. Despite successful amplification of DNA from an IL-3 cDNA, no product could be obtained by amplification of keratinocyte RNA treated with reverse transcription-polymerase chain reaction. Analysis of concentrated (up to 50-fold) supernatants failed to detect IL-3 protein by enzyme-linked immunosorbent assay. Because ultraviolet radiation up-regulates many cytokines, we irradiated human keratinocytes with 300 J/m2 ultraviolet B and collected supernatants 24 h post-irradiation. Supernatants concentrated 50-fold were also negative for IL-3 protein by enzyme-linked immunosorbent assay. When assayed on the IL-3-responsive M-07e cell line, unirradiated supernatants stimulated M-07e proliferation 22-fold over background levels. Irradiated supernatants stimulated M-07e proliferation 128-fold. Neither the unirradiated nor the irradiated supernatant activity could be neutralized with antibody to human IL-3. However, incubation of irradiated supernatants with antibody to granulocyte macrophage-colony-stimulating factor (GM-CSF) reduced the M-07e proliferation by 90%. Antibodies against GM-CSF and IL-6 completely abrogated proliferation. Reverse transcription-polymerase chain reaction confirmed a concomitant elevation of IL-6 (2.6- to 5.6-fold) and of GM-CSF mRNA (2.7- to 4.3-fold) at 6 and 24 h after ultraviolet B irradiation in keratinocytes, but no IL-3 amplification products could be detected. IL-3 mRNA was also not detected in adult keratinocytes. Even after stimulation by IL-1 alpha, tumor necrosis factor-alpha, or phobol myristate acetate, IL-3 mRNA was not detected in either neonatal or adult human keratinocytes. We have been unable to detect IL-3 mRNA or IL-3 protein in human keratinocytes. The IL-3-like activity in human keratinocytes is mainly due to GM-CSF, with a small contribution from IL-6.

Antibodies↗
PubMed1993

The mechanisms of IgE uptake by human alveolar macrophages and a human B-lymphoblastoid cell line (Wil-2wt).

Human alveolar macrophages (HAM) internalized more IgE (81%) than human Wil-2wt B-lymphoblastoid cells (28%) suggesting a difference in the metabolic processing of the specific IgE receptor (CD23) or, alternatively, the presence of another functionally distinct receptor. The mannose receptor (MR), demonstrated to be present on the AM, may fulfil this role as IgE is heavily mannosylated and binds to a greater extent to concanavalin A (Con A) (which has specificity for oligomannose oligosaccharide chains) than other antibody isotypes. The hypothesis of a second IgE receptor was tested using mannan which is a competitive inhibitor of ligand binding to the MR and mannosylated bovine serum albumin (MBSA) which binds with avidity to the MR. Mannan (0.1 mg/ml) decreased internalized MBSA uptake in the HAM at 37 degrees suggesting the presence of the specific MR. In contrast, Wil-2wt cells did not bind MBSA. Mannan also reduced IgE uptake in the HAM at 37 degrees but had no effect on IgE uptake by Wil-2wt cells. Anti-CD23 monoclonal antibody (mAb) 135 also partially reduced membrane IgE uptake in HAM while completely inhibiting it by Wil-2wt cells. However, there did not appear to be competition for binding sites between IgE and MBSA in HAM. If only CD23 is involved in IgE uptake by HAM its function appears to be different to that in Wil-2wt cells. Definite involvement of the MR in IgE uptake will require further investigation as it may have an important role in allergic states.

B-Lymphocytes↗
PubMed1993

Molecular cloning of murine FLT and FLT4.

A wide range of growth and differentiation processes are regulated by the signalling of receptor tyrosine kinases (RTKs). We have developed a nested polymerase chain reaction (PCR) procedure with degenerate primers, and used it to identify RTKs expressed in murine fetal thymus. A novel RTK, called FLT4, and the murine homologue of FLT were found, and their PCR fragment sequences were used to isolate larger cDNA clones spanning the complete coding regions of these receptors. FLT4 was found to contain an extracellular region similar to the corresponding sequences of FLT and Flk-1, containing seven immunoglobulin domains.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of the cDNA encoding a receptor tyrosine kinase-related molecule with a catalytic region homologous to c-met.

Receptor tyrosine kinases mediate a range of growth and differentiation processes in multiple biological systems. In this work, we report the identification of a novel tyrosine kinase-related molecule, nyk-r, and the molecular cloning of its complete cDNA. Its extra-cellular domain bears no apparent homology with other receptor families, but its intracellular kinase-related region has considerable similarity with members of the insulin-receptor family such as c-met and trk B. Also, the nyk-r gene is expressed in a wide range of tissues and cell lines.

Amino Acid Sequence↗
PubMed1991

The influence of a family history of asthma and parental smoking on airway responsiveness in early infancy.

BACKGROUND: Airway responsiveness to inhaled nonspecific bronchoconstrictive agents has been demonstrated in normal, healthy infants. However, it is unknown whether airway responsiveness is present from birth or if it develops as a result of subsequent insults to the respiratory tract. To investigate this question, we assessed airway responsiveness in 63 normal infants at a mean age of 4 1/2 weeks. METHODS: Respiratory function was measured with use of the partial forced expiratory flow-volume technique to determine the maximal flow at functional residual capacity (VmaxFRC). The infants inhaled nebulized histamine at sequentially doubled concentrations (0.125 to 8.0 g per liter), until a concentration was reached at which the VmaxFRC fell by 40 percent from the base-line value (PC40) or until a concentration of 8.0 g per liter was reached. We also assessed maternal serum levels of IgE, cord-serum levels of IgE, the infants' skin reactivity to several allergens, and the parents' responsiveness to histamine and obtained family histories of asthma and smoking. RESULTS: Airway responsiveness was increased in infants with a family history of asthma (n = 19; median PC40, 0.78 g per liter; 95 percent confidence interval, 0.44 to 1.15; P less than 0.01), parental smoking (n = 13; median PC40, 0.52 g per liter; 95 percent confidence interval, 0.43 to 5.40; P less than 0.05), or both (n = 20; median PC40, 0.69 g per liter; 95 percent confidence interval, 0.37 to 2.10; P less than 0.05), as compared with the infants with no family history of asthma or smoking. The infants with no family history of asthma or smoking had a median PC40 of 2.75 g per liter (95 percent confidence interval, 1.48 to 4.00). No significant relations were detected between the immunologic variables and the PC40 in the infants. CONCLUSIONS: This study indicates that airway responsiveness can be present early in life and suggests that a family history of asthma or parental smoking contributes to elevated levels of airway responsiveness at an early age.

Asthma↗
PubMed1991

The characteristics of antigen define the cellular source and kinetics of histamine-releasing factor induced by human peripheral-blood mononuclear cells.

Peripheral-blood leucocytes from Dermatophagoides pteronyssinus- and/or Lolium perenne-allergic patients produce in vitro histamine-releasing factor (HRF) in an allergen-specific and concentration-dependent relationship. Maximum production of HRF occurred in cultures containing as little as 10-100 pg/ml crude allergen extract, although a second peak occurred at higher concentrations (10-100 micrograms/ml). While HRF was detectable in 1-hour cultures, maximal production required 24 h in culture. In contrast, HRF induced by streptokinase/streptodornase (SK/SD) was generally maximal after 1 h. Allergen-induced HRF production was almost exclusively associated with monocytes and B cells. In contrast, peripheral-blood T cells were the major source of induced HRF production in cultures containing SK/SD. Histamine-releasing cytokines are apparently produced by different cell populations, the activation of which may be dependent upon the characteristics of the stimulating antigen.

Allergens↗
PubMed1991

Comparison of IgE expression at the mRNA and protein levels in vitro.

The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.

Cell Line↗
PubMed1990

Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay.

A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.

Allergens↗
PubMed1989

In vivo arming of cutaneous mast cell receptors by IgE released from macrophages.

Intracutaneous injection of purified peritoneal macrophages harvested from ovalbumin (OVA)-hypersensitive high-IgE-responder BN rats into naive animals sensitised the injection sites for subsequent OVA-specific passive cutaneous anaphylaxis (PCA) reactions. The underlying mechanism(s) were investigated using a macrophage cell line (WEHI 265.1), which exhibited comparable sensitising activity in rat or mouse skin, after initial pulsing in vitro with antiserum rich in OVA-specific IgE. Transfer of OVA-hypersensitivity by the cell line (1) was IgE-dependent and did not occur when the cells were pre-exposed to antiserum containing OVA-specific IgG alone, (2) was blockable by saturation of cell surface receptors in the recipient with myeloma IgE (but not myeloma IgG), and (3) did not occur in mast cell-deficient mice carrying the W/Wv mutation, in contrast to their normal heterozygous littermates which developed marked OVA-hypersensitivity at the injection site. These results are consistent with arming of IgE-receptors on cutaneous mast cells by IgE antibody released from macrophages, and hint at a possible role for phagocytes in amplifying IgE-mediated reactions in tissues.

Animals↗
PubMed1989

Identification through chemical cross-linking of distinct granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors on myeloid leukemic cells, KG-1.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high-affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM-CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.

Binding, Competitive↗
PubMed1989

Epidemiology of the allergic response.

Asthma and related common allergic diseases are significant worldwide health problems which cause considerable morbidity and appreciable mortality and make severe demands on the time and costs of medical and hospital care. The more readily available epidemiological data relate to asthma, which has a wide prevalence, ranging from a general figure of 4-8% for developed countries to less than 1% in some European, African, Indian and Melanesian countries. Earlier reports that asthma is uncommon in the tropics, particularly in rural areas, appear from later studies to be an over-generalization; the highest prevalence of asthma in the world has been reported in the Western Caroline Islands, the Maldives and Tristan da Cunha. Atopic diseases are associated with excess IgE production, which is genetically controlled, and it is likely that additional genes control allergic shock organs. However, the development of allergic disease is balanced between genetic and environmental factors, principally exposure to aggressive allergens, which is particularly relevant during the first six months of life, amplified by maternal smoking both pre- and postnatally, and IgA deficiency in breast milk. Other factors such as viral infections and atmospheric pollution play important but lesser roles. A unique feature of asthma in village communities in developing countries, in contrast to developed, is that it is much less frequent in children than in adults. Although this may be relevant to understanding the regulation of allergic disease in general the mechanism(s) remain to be determined.

Adolescent↗
PubMed1988

Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases.

A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.

Allergens↗
PubMed1988

Specific binding, internalization, and degradation of human recombinant interleukin-3 by cells of the acute myelogenous, leukemia line, KG-1.

We have studied the interaction of 35S-labeled recombinant IL-3 with the acute myelogenous leukemia cell line, KG-1. 35S-IL-3 bound to these cells in a time dependent, saturable, and specific manner at 4 degrees C. Scatchard transformation of binding isotherms demonstrated the existence of a small number (200) of binding sites, with an apparent dissociation constant of 70-105 pM. After a temperature shift from 4 degrees C to 37 degrees C, surface-bound 35S-IL-3 was rapidly internalized and processed into a trichloroacetic acid soluble form that was released into the medium. Experiments to address the specificity of the IL-3 binding site revealed that neither human IL-2, M-CSF, erythropoietin, transferrin, bovine insulin, nor murine nerve growth factor compete with IL-3 for binding to KG-1 cells. Both human and gibbon recombinant IL-3 and, surprisingly, human recombinant GM-CSF effectively competed the binding of the labeled IL-3 to these cells at 4 degrees C. The competition by GM-CSF was found to be concentration dependent, but much higher concentrations were required to achieve the levels obtained with IL-3. These results suggest that GM-CSF may also interact with the high-affinity IL-3 binding site on KG-1 cells or, alternatively, that GM-CSF binding to its own receptor may decrease the affinity of the IL-3 receptor for its ligand.

Binding Sites↗
PubMed1988

Seroprevalence of Toxocara canis infection in tropical Venezuela.

An enzyme-linked immunosorbent assay (ELISA) was used to determine the seroprevalence of Toxocara canis infection in different socio-economic groups of the tropical population of Venezuela. The lack of definitive independent diagnostic criteria for toxocariasis required the establishment of operational upper limits of normality for Toxocara ELISA values, based upon their log-normalized distribution in a presumptive "non-toxocariasis" sub-population. Only 1.8% of urban subjects of medium-high socio-economic level were considered to be clearly positive in Toxocara ELISA, compared to 20.0% of urban slum dwellers, 25.6% rural subsistence farmers and 34.9% Amazon Indians. As the test was performed using excretory-secretory antigen, and under conditions of competitive inhibition by soluble extracts of non-homologous parasites, co-infection by common intestinal helminths, protozoa or other organisms did not give rise to false positive results. However, strong cross-reactivity with Onchocerca volvulus may have influenced the prevalence figure obtained for the Amazon Indians. These results indicate that T. canis is yet another parasite that is widely distributed in economically underprivileged tropical populations.

Adolescent↗
PubMed1988

In vitro synthesis of IgE by human peripheral blood leucocytes: VI. Seasonal variations in synthesis of rye pollen (Lolium perenne) specific IgE.

Fractionation by Percoll density centrifugation of peripheral blood leucocyte cells, from atopic subjects with seasonal hay fever, unmasked IgE-B cell populations whose individual capacities to synthesize IgE in vitro were obscured in cultures of unfractionated B cells. B cell cultures from all six subjects in the study released rye pollen-specific IgE during the 6 days of culture, but actual synthesis was significant only in October, the pollen season. Synthesis in October occurred most frequently in cultures of mature, low density B cells, which generally responded to the addition of autologous T cells with enhanced synthesis (T-help). T-help was also found for high density B cells in the mid-winter (July) cultures. Total IgE synthesis in vitro demonstrated a less seasonal relationship, although it tended to be maximal for low density B cell cultures in October and for high density B cells in May. All B cell cultures contained preformed total and rye-specific IgE antibody which persisted throughout the pre- and post-pollen seasons, particularly in the low density B cell fractions, even in the absence of de novo synthesis. Moreover, the intracellular levels of rye pollen-specific IgE antibody were often higher in the winter than in the peak of the pollen season. The relevance of this preformed IgE remains to be established.

B-Lymphocytes↗
PubMed1988

Isotype specific immunoglobulin responses to the house dust mite Dermatophagoides pteronyssinus and the purified allergen Der p 1 in asthmatic and control subjects from the Eastern Highlands of Papua New Guinea.

The IgG, IgE and IgA antibody responses to the whole mite extract and a purified major mite allergen Der p 1, in sera from asthmatic and age- and sex-matched control subjects from the South Fore region of the Eastern Highlands of Papua New Guinea, have been studied. Radio-allergosorbent studies showed that the majority of the asthmatics, in contrast to control subjects, produced IgE to whole mite extract, and that Der p 1 was a major allergen in this population with 88% of mite allergic asthmatics responding. Enzyme-linked immunosorbent assay studies on these sera showed that the geometric mean levels of whole mite, but not Der p 1, specific IgG and IgA were significantly higher in the asthmatic group than in the control group. Significant correlations between whole mite specific IgG, IgE and IgA responses were obtained. These data indicate that Papua New Guinean asthmatics are similar to Caucasian asthmatic population with regard to serological responses to mite allergens, despite differences in disease presentation, particularly the late age of onset and severity of symptoms.

Adult↗