Biomedical subjects
P Fitzharris
Publications and source records attributed to P Fitzharris.
Characteristics and kinetics of proopiomelanocortin mRNA expression by human leucocytes.
Northern blot analysis of total and poly(A)+ RNA demonstrated that human leucocytes contain several proopiomelanocortin (POMC) mRNA species, including 0.8-, 1.2-, 1.5-, and 9.5-kb transcripts. Peripheral blood lymphocytes (PBL) were found to express all four species. The other cell types had either the 0.8-kb species alone or both 0.8- and 9.5-kb species. Neutrophils were the only cells to express the 9.5-kb transcript alone. In T cell clones, both interleukin (IL)-2 alone or the antigen for which the clone was specific induced POMC accumulation within 18-24 h. Cytoplasmic dot blot analysis of PBL RNA demonstrated that POMC expression could be induced by corticotrophin releasing factor, rIL-1, and phorbol ester, but not by calcium ionophore (A23187). In PBL activated in a mixed lymphocyte culture there was increased expression, particularly of the smaller species (0.8, 1.2, and 1.5 kb), within 1-3 days of activation. The intensity of the bands began to decline thereafter. The 9.5-kb POMC transcript essentially disappeared by Day 3.
Characterization of immunologically important antigens and allergens of Aspergillus fumigatus.
Using a variety of immunochemical methods, including quantitative immuno-electrophoretic techniques, combined with gel filtration and iso-electric focusing, and production of monospecific antisera for identification and affinity purification, 4 major components of Aspergillus fumigatus have now been partially characterized. Numbering of these was derived from a reference allergic bronchopulmonary aspergillosis (ABPA) self-crossed radio-immuno-electrophoresis pattern of reactivity. Two major intracellular/cytoplasmic, concanavalin A (Con A)-binding antigens, Ag 7 and Ag 13, of molecular weights 150-200 and 70 kilodaltons (kD), respectively, were confirmed to be of importance for both ABPA and aspergilloma in specific sandwich enzyme-linked immunosorbent assays. A rapidly released component, Ag 5, of molecular weight 35 kD, proved both antigenic and allergenic, with aspergilloma patients having especially high-titre IgG antibodies. The major allergenic component Ag 3, of molecular weight 24 kD by gel filtration and 18 kD by SDS-PAGE was, like Ag 5, relatively heat-labile and non-Con-A-binding. Interestingly, T cell clones have been identified which respond primarily to an 18-kD fraction.
Lymphocyte sensitization to Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.
Peripheral blood mononuclear cell (PBMC) proliferation induced by an extract of Aspergillus fumigatus (AF) was examined in patients with allergic bronchopulmonary aspergillosis (ABPA), all of whom had an immediate skin prick test reaction (SPT) and increased RAST binding to AF, and, for comparison, in individuals without immediate SPT reactivity or increased RAST binding to AF. The proliferative responses of PBMC from the ABPA patients were greater than those from the comparison donors. A substantial proportion of the comparison group, however, showed evidence of a specific immune response to AF, with AF-specific IgG measured by ELISA and specific lymphoproliferative responses. AF-responsive T cell lines and T cell clones were established from both ABPA patients and IgE-negative individuals. These clones, of helper/inducer (CD4+) phenotype, showed antigenic specificity and MHC restriction. The stimulating antigen was determined for four of six clones derived from a skin-prick-test-negative individual, and found to be of Mr 18 kD, possibly the major allergen, 'Ag 3'. ABPA patients showed a marked diminution of the proliferative response during disease exacerbation.
Leukotriene C4 generation from human eosinophils stimulated with IgG-Aspergillus fumigatus antigen immune complexes.
Sepharose beads coated with IgG stimulate eosinophils to produce leukotriene C4 (LTC4). This observation has been extended with specific immobilized IgG/antigen immune complexes to elicit mediator generation. An extract of Aspergillus fumigatus was covalently coupled to Sepharose beads and incubated with the IgG fraction of immune serum from patients with allergic bronchopulmonary aspergillosis. These beads elicited generation of 7.72 +/- 1.7 pmol of LTC4 immunoreactive material (n = 5) from 1 X 10(6) normal eosinophils of greater than 86% purity, and significantly less LTC4 (0.73 +/- 0.19 pmol per 10(6) cells; n = 3) was produced by eosinophils after incubation with beads treated with IgG from normal nonimmune serum. The maximum antibody-dependent release achieved represented approximately 20% of that induced by the calcium ionophore (A23187). LTC4 was measured by radioimmunoassay and validated by reverse-phase high-performance liquid chromatography. The amount of LTC4 generated was dependent on the concentration of A. fumigatus-specific IgG, and mediator release was completely abolished by prior adsorption of the IgG fraction onto Sepharose-protein A (Staphylococcus aureus). Grass pollen-specific IgG antibody/antigen complexes, in combination with Sepharose beads, also triggered generation of LTC4 immunoreactive material. There was no evidence to suggest that IgE/A. fumigatus immune complexes triggered LTC4 generation, although IgE myeloma protein, in association with Sepharose beads, was a weak stimulus. The efficacy of the IgG immune complex-dependent stimulation of eosinophils suggests a possible physiologic mechanism whereby these cells could participate in the inflammatory changes associated with allergic bronchopulmonary aspergillosis and similar allergic disorders.
Influence of atopy on the clinical manifestations of coronavirus infection in adult volunteers.
In an attempt to understand the relationship between viral upper respiratory tract infection and the underlying virological and immunological mechanisms, thirty-four volunteers were inoculated intranasally with coronavirus 229E; subsequent virus shedding and/or antibody rises, indicating active infection, were observed in twenty-nine. There was a greater increase in independently measured scores of clinical severity, e.g. cold symptoms, in those with detectable IgE in nasal secretions (P less than 0.01). A similar association was found between clinical scores and serum IgE concentrations greater than or equal to 150 IU/ml, but the relationship with systemic atopy, as assessed by skin-prick tests to common allergens, was less marked. A more detailed study of twelve of the infected volunteers failed to explain these findings on the basis of mast cell mediator release, as concentrations of leukotriene B4, the sulphidopeptide leukotriene C4, and histamine, were not appreciably elevated in the nasal secretions following virus inoculation. Similarly, there was no evidence that circulating coronavirus specific IgE was produced. Thus, this study suggest that atopy may be related to the severity of cold symptoms produced by coronavirus 229E, although the exact connection has yet to be determined.
A T-lymphocyte-derived factor that enhances IgG-dependent release of leukotriene B4 (LTB4) from human neutrophils.
We describe a human blood mononuclear cell (MNC)-derived leukotriene release enhancing factor (LREF) which significantly increased IgG-dependent leukotriene B4 (LTB4) generation by neutrophils. MNCs incubated with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody and PHA-stimulated ER+ lymphocytes produced a 150-350% increase in LTB4 generation from IgG-stimulated neutrophils. With PHA, maximal activity was observed 48 hr after culture in serum-free medium. LREF was relatively stable when exposed to low pH (pH 2) or heat (56 degrees, 60 min). Following progressive purification by gel filtration (FPLC, Superose 12) and chromatofocusing (FPLC, Mono P) LREF was associated with proteins of molecular weight of 35-40 kD and a pI of 5.1-5.5. Partially purified LREF did not contain detectable amounts of interleukin 2 (IL-2) or interferon-gamma (IFN-gamma). Although high concentrations of recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) (greater than 2 ng/ml) and recombinant tumour necrosis factor (rTNF) (greater than 100 U/ml) gave a slight (60%) enhancement of LTB4 generation, this was considerably less than that of partially purified LREF (350%). Our results suggest that human lymphocyte-derived LREF may play a role in the amplification of inflammatory reactions involving sensitized lymphocytes, neutrophils and lipid mediators.
Leukotriene B4 generation by human neutrophils following IgG-dependent stimulation.
It has previously been shown that human neutrophils generate substantial quantities of LTB4 when stimulated with the calcium ionophore, A23187, or with unopsonized zymosan. We now report that normal human neutrophils produced substantial quantities of LTB4 (measured by radioimmunoassay and validated by RP-HPLC) when incubated with large non-phagocytosable IgG-coated beads (Sepharose 4B). LTB4 was identified in both the extra and intracellular compartments. The production of LTB4 was dependent upon the number of IgG-coated particles and the concentration of IgG bound to the beads. Release was maximal after a 15-30 min incubation time and was enhanced by prior activation of the neutrophils with the synthetic bacterial product f-met-leu-phe. Comparable LTB4 production was also observed when neutrophils were incubated with antigen (Aspergillus fumigatus)-coated beads sensitized with purified IgG obtained from the sera of patients with allergic bronchopulmonary aspergillosis. These results suggest a further mechanism by which neutrophils may be activated to produce inflammatory mediators in the tissues.
Morphological and secretory properties of bronchoalveolar lavage mast cells in respiratory diseases.
We have studied various functional and morphological characteristics of mast cells obtained in bronchoalveolar lavage from fifty-two patients with several lung diseases. The percentage of mast cells ranged from 0.04 to 0.6% (bronchial carcinoma), 0.05-0.3% (sarcoidosis), 0.06-0.25% (asthma), 0.04-1.8% (miscellaneous) and 0.02-0.04% (normals). There were no significant differences in the mast cell counts between the disease groups. Lung mast cells exhibited heterogeneity of size, shape and intensity of staining. Cells from thirty-seven subjects were further studied for total histamine content and histamine release using various secretagogues. There was a significant correlation (P less than 0.001) between the histamine content of the total lavage cell population and mast cell counts. The calculated mean histamine content per mast cell was 6.35 pg. Histamine was released in a dose-dependent fashion after stimulation with anti-IgE, calcium ionophore and phorbol myristate acetate with a time course of histamine release characteristic of the mast cell. Unlike peripheral blood basophils, no release was observed following incubation with f-met-leu-phe (10(-6)-10(-8) M) and neither cell type released histamine following incubation with 48/80 (10 micrograms/ml). Inhibition of anti-IgE-induced histamine release was obtained following pre-incubation with salbutamol (10(-4)-10(-6) M). These studies indicate that bronchoalveolar lavage is a suitable model for the study of human lung mast cells.
The effects of eosinophil activating factor on IgG-dependent sulphidopeptide leukotriene generation by human eosinophils.
Eosinophil activating factor (EAF) is a 40 kD protein released from cultured, unstimulated human monocytes which enhances the IgG-dependent eosinophil-mediated cytotoxicity of helminthic larvae. We have recently shown that eosinophils elaborate substantial quantities of leukotriene C4 (LTC4) during incubation with IgG-coated particles and now report that EAF, partially purified by sequential chromatography on Sephacryl S-200 and DEAE-cellulose, enhanced this IgG-dependent LTC4 production by human eosinophils in a dose- and time-dependent fashion. LTC4 production by normal density eosinophils, separated on discontinuous metrizamide gradients, was significantly increased after incubation with several dilutions of EAF (P less than 0.05), although an increase was not seen with low density cells. The enhancement was similar in degree to that seen when normal density eosinophils were activated with the bacterial analogue, f-met-leu-phe (fMLP). EAF produced a time-dependent increase in LTC4 which was significantly greater (P less than 0.01) than the control. Sulphidopeptide leukotriene (LT) generation was validated by reverse phase high pressure liquid chromatography (RP-HPLC). These results indicate that there is a firm association between monocytes, eosinophils and LTC4; an observation which may be of relevance to mechanisms in chronic asthma and related disorders, and in immune reactions against migrating helminthic larvae.
The effects in volunteers of BW12C, a compound designed to left-shift the blood-oxygen saturation curve.
BW12C, a potent left-shifting anti-sickling compound in vitro, was administered to normal healthy male Caucasian volunteers. Doses of 2-20 mg kg-1 given by intravenous infusion over 1 h caused a dose-dependent left-shift of the blood-oxygen saturation curve and at the highest dose some 16% of the haemoglobin existed in a high affinity form. Peak left-shift was observed at the end of infusion and decayed thereafter with a mean half-life of approximately 3 h. There were no adverse systemic effects, either clinical, biochemical or haematological, but there was some local irritation at the intravenous infusion site if the infusion was too concentrated. Pharmacokinetic measurements indicated uptake into erythrocytes, low levels in plasma and a volume of distribution not appreciably greater than the blood volume. A pilot radiolabel study indicated extensive metabolism with elimination into the urine.
Allergen-induced release of sulphidopeptide leukotrienes (SRS-A) and LTB4 in allergic rhinitis.
Leukotrienes are a recently discovered group of arachidonic acid-derived lipid mediators. Using radioimmunoassay and high pressure liquid chromatography (HPLC), we have identified the SRS-A sulphidopeptide leukotrienes (LTC4, LTD4 and LTE4) in nasal washings from patients with allergic rhinitis who underwent nasal challenge with specific allergen. Smaller, but significant, amounts of LTB4 were also detected. The concentrations of nasal leukotrienes were directly related to the dose of allergen, and were recovered in washings in a time-dependent fashion after challenge. When the patients were subjected to methacholine nasal challenge on a control day, we found only negligible amounts of either the sulphidopeptide leukotrienes or LTB4. These findings support the view that LTC4, LTD4 and LTE4 might contribute to the pathogenesis of allergic rhinitis as a result of their recognized effects on mucous hypersecretion and vasopermeability, and that the potent chemoattractant LTB4 might be involved in the subsequent infiltration of inflammatory cells.
Null cell immunoregulation in SLE.
Fresh normal T cells do not lyse MDA-157 target cells. Normal null cells, cultured for 4 days with MDA-157 stimulators, and then mixed overnight with fresh normal T cells, induce cytotoxicity on MDA-157 targets and suppressor activity in the T-cell acceptor population. With the same normal acceptor T cells, MDA-157 activated null cells from 13/18 patients with Systemic Lupus Erythematosus (SLE), unlike activated cells from a disease control population, induce little or no T-cell cytotoxicity or suppression. These results provide further evidence for abnormal null cell function in SLE.