PubMed Health⌕ Search

Biomedical subjects

B J Holt

Publications and source records attributed to B J Holt.

35 records · Page 2Linked to original sources

Genetic 'risk' for atopy is associated with delayed postnatal maturation of T-cell competence.

Recent in vitro studies suggest that IgE production in adults is co-ordinately regulated by negative signals from gamma IFN-producing CD4+ T-helper-1 (TH-1) and positive signals from IL-4 producing (TH-2) T-cells. Additionally, seroepidemiological evidence has pinpointed infancy as the period of maximum lifetime risk for T-cell sensitization to ubiquitous environmental antigens. The present study sought to elucidate the relationship between these observations, by examination of CD4+ T-cell function in normal children and those genetically at 'high risk' for atopy, spanning the age range (up to 4 years) in which IgE responses to environmental allergens is typically manifest. Immunocompetent T-cell precursor frequencies (determined by cloning at limiting dilution) were markedly reduced in 'high risk' children relative to normals (0.53 +/- 0.29 vs 0.26 +/- 0.19; P = 0.0025). Consistent with reports from other laboratories employing bulk T-cell culture techniques, the gamma IFN producing capacity of CD4+ T-cell clones from both groups of children were markedly reduced relative to adults, and was lowest in the high risk group (P < 0.02). IL-4 production by CD4+ T-cell clones from the normal children was within the adult range, but again was significantly lower in the high risk group (P < 0.00005). This indicates that initial immune responses to environmental allergens in early childhood occur against a background of maturational 'deficiency' in CD4+ T-cell function, and suggests the possibility that variations in the rate of postnatal maturation of T-cell competence may be a contributing factor in the development of differing patterns of immunological responsiveness to environmental allergens.

Adult↗

A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals.

The characteristics of the T-cell population in the healthy human lung have been investigated by analysing the properties of T-cell clones derived from bronchoalveolar lavage (BAL) samples and comparing them with T cells cloned from the blood of the same individuals. The proportions of CD4+ and CD8+ T cells in the starting populations from BAL and blood were similar although only 14% of BAL T cells were CD45RA+ compared to 70% of blood T cells. The precursor frequency of T-cell clones derived from BAL was less than from blood. The cytokine profiles [after phytohaemagglutinin (PHA) stimulation] of the clones derived from both sources were markedly different and these differences lay in the CD4+ population. BAL-derived CD4+ clones produced interferon-gamma (IFN-gamma) more frequently than did those from blood while blood-derived clones were more likely to produce interleukin-2 (IL-2) than those from BAL. IL-4 was produced by the majority of BAL- or blood-derived clones (93% and 88% respectively) either along with IFN-gamma (BAL) or IL-2 (blood). The cytokine profiles of BAL-derived T-cell clones are consistent with those derived from lung interstitium and suggest that the BAL T-cell populations reflect those in the lung wall. Whether the unique properties of lung T cells are acquired after leaving the blood or whether there is selective entry of T-cell subpopulations into the lung remains to be determined.

Adult↗

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↗

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↗

Regulation of T-cell sensitization at epithelial surfaces in the respiratory tract: suppression of IgE responses to inhaled antigens by CD3+ Tcr alpha-/beta- lymphocytes (putative gamma/delta T cells).

Repeated exposure of Brown Norway rats to an aerosol of ovalbumin (OVA) induced a state of antigen-specific immunological tolerance, particularly in the IgE isotype. Tolerance was transferable to naive syngeneic animals by inoculation of splenic T cells from tolerant rats. Sequential depletion of tolerant spleen cells by sorting techniques prior to adoptive transfer, employing T-cell subset-specific monoclonal antibodies, indicated that the cells mediating tolerance were CD3+, CD4-, CD5+ and CD8+, but lacked alpha or beta chains in the T-cell receptor (TcR), suggesting that they may be part of the gamma/delta T-cell lineage. Consistent with this suggestion, the sorted population demonstrated considerable enrichment for TcR gamma chain-specific mRNA. As few as 2 x 10(3) cells are sufficient to adoptively transfer tolerance in 200-g adult rats in this model.

Administration, Inhalation↗

Immunoregulation of asthma: control of T-lymphocyte activation in the respiratory tract.

Allergic asthma results ultimately from inappropriate responses to non-pathogenic airborn antigens by the respiratory tract T-cell system. In order to understand how sensitization to inhaled antigens develops, it is necessary to precisely define the tissue microenvironments within which T-cells occur in the respiratory tract, and to analyse the factor(s) which regulate their local activation. This review focuses upon recent data on the distribution of T-cells and antigen presenting cells within the lung and airway tissues of man and experimental animals, and examines the available information on their responses to antigenic stimulation. We argue that in the steady state, respiratory tract T-cells are tightly regulated by a series of inherent and acquired immunosuppressive control mechanisms, which normally limit local T-cell activation to situations where antigenic exposure is accompanied by an inflammatory stimulus, such as the case with incoming pathogenic microorganisms.

Allergens↗

A contiguous network of dendritic antigen-presenting cells within the respiratory epithelium.

This study utilises a simple technique to section airway epithelium in a plane parallel to the basement membrane, thus providing a unique plan view of the intra-epithelial cell populations. Immunoperoxidase staining of these tissue sections for class II major histocompatibility complex Ia antigen reveals a virtually contiguous network of Ia-positive dendritic cells (DC) within the epithelium. These DC are shown to be capable of binding inhaled antigens in vivo in a form suitable for presentation to T cells. The strategic location of these cells and the fact that they account for virtually all staining in the airway epithelium during the steady state is convincing evidence that the DC network functions as the 'first line of defence' in surveillance for inhaled antigens and further suggests a major role for the intra-epithelial DC in allergic and infectious disease(s) in the respiratory tract.

Animals↗

Ultrastructural localization of IgE in peripheral blood monocytes from atopics.

Monocyte-enriched preparations derived from peripheral blood leukocytes of atopics were probed via a cocktail comprising peroxidase-conjugated (Fab1)2 fragments of two monoclonal antibodies against human IgE. Reaction product indicative of intracellular IgE was identified by electron microscopy in both large and small vacuoles, and at high magnification exhibited a characteristic granular deposition pattern consistent with highly concentrated (perhaps insolublized) material. IgE-containing vacuoles were observed with comparable frequency to those containing IgG, despite the greater than 10,000-fold relative excess of the latter in serum suggesting highly selective uptake of IgE by the monocytes. These results are similar to those reported recently for IgA in human milk macrophages.

Antibodies, Monoclonal↗

Functional heterogeneity within the human peripheral blood B cell pool engaged in IgE synthesis.

Fractionation of human peripheral blood leukocytes (PBL) B cells by differential sedimentation on percoll gradients separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, in their response to identical populations of autologous T cells and in their response to soluble T cell factors. This B cell fractionation technique often reveals the presence of active IgE-secreting cells which are totally suppressed within unfractionated PBL B cell preparations and greatly improves the efficiency of detection of T cell-responsive, IgE-producing B cells in PBL of atopics.

Adult↗

In vitro synthesis of IgE by human peripheral blood leucocytes: V. Functional heterogeneity within the IgE-B-cell pool.

Fractionation of human peripheral blood leucocytes (PBL) B cells by differential sedimentation on a discontinuous Percoll gradient separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, often revealing the presence of active IgE secreting cells which are totally suppressed within unfractionated PBL B cell preparations. The production in vitro of IgE by separated B cell populations from the same individual may respond disparately to an identical population of autologous T cells and to pokeweed mitogen. Kinetic studies revealed major differences in both the rates of release of cell-associated IgE between these B cell populations, and their rates of de novo IgE synthesis. From a methodological viewpoint, the use of this B cell fractionation technique is demonstrated to improve greatly the efficiency of detection of T cell-responsive IgE producing B cells in peripheral blood from atopics, and from a mechanistic standpoint raises the possibility that B cell heterogeneity may modulate the functional expression of IgE-regulatory T cells signals.

Adult↗

In vitro synthesis of IgE by human peripheral blood leucocytes. IV. Longitudinal study of regulatory T-cell activity in atopics.

The assessment of IgE production in cultures of T- and B-cells from peripheral blood is proving a useful tool to probe IgE immunoregulation in human atopics. The present study contrasts secretion and synthesis as indices of IgE production, and demonstrates that these measures yield comparable data upon the magnitude and direction of regulatory T-cell effects (help vs. suppression) in severe atopics. The majority of peripheral blood B-cell samples from the atopics in this study exhibited spontaneous IgE synthesis and secretion, and in vitro T-cell help and suppression were observed with equal frequency within the sample population. Repeated testing of individual atopics indicated that the direction of T-cell effects remained stable in some (but not all) atopics over periods as long as 3 years.

Adolescent↗

In vitro synthesis of IgE by human peripheral blood leucocytes. III. Release of pre-formed antibody.

The appearance of IgE in supernatant fluids from cultures of human peripheral blood leucocytes (PBL) is employed in many laboratories as an index of antibody 'synthesis'. This study demonstrates that, unlike IgG, the majority of cell associated IgE is sequestered by PBL in a tightly bound form, which resists extraction via the freeze-thawing (F/T) techniques in current use. A method is presented for the quantitative extraction of cell associated IgE, involving brief acid treatment of whole cells, and is shown to yield up to 100% more IgE than the F/T procedure. The use of this extraction process on PBL before and after culture permits discrimination between release of pre-formed IgE and de novo synthesis, both of which are demonstrated to occur to widely differing degrees in PBL cultures from different donors.

Cells, Cultured↗

In vitro synthesis of IgE by human peripheral blood leucocytes. II modulation of IgE B cell activity by isotype-specific regulatory T cells.

IgE and IgG synthesis was demonstrated in cultures of isolated B cells from normal subjects, and those manifesting mild to severe atopy. Titration of autochthonous T cells into B cell cultures revealed both IgE isotype-specific T-help and T-suppression at low and high T:B ratios respectively. Increasing levels of help for IgG synthesis usually accompanied the stepwise addition of the same T cells to autochthonous or allogeneic B cells cultures. Experiments comparing the activity of putative IgE suppressor T cells in autochthonous versus allogeneic B cell cultures indicated that allogeneic interactions per se selectively inhibit IgE B cells. This finding seriously questions earlier conclusions of a causal relationship between suppressor cell deficiency and the expression of the allergic phenotype.

B-Lymphocytes↗

In vitro synthesis of IgE by human peripheral blood leucocytes: relationship to serum titre of leucocyte donor.

A microculture system was developed for in vitro IgE synthesis employing human peripheral blood leucocytes (PBL), comprising 1.0 x 10(6) cells in 1.0 ml serum-supplemented medium, in round-bottomed tubes. Examination of the kinetics of IgE synthesis indicated that production was relatively linear during culture for up to 12 days, and was not susceptible to stimulation with polyclonal B cell mitogen. Employing a standardized 7-day culture period, PBL IgE synthesis was compared within a group of 50 volunteers, comprising normal non-atopic individuals exhibiting serum IgE titres less than 50 units/ml, and moderate atopics with serum titres up to approximately 400 units/ml. PBL IgE synthesis was found to reflect closely the serum IgE status of the cell donor, a feature thus far unique to this culture system, and indicative of the preservation of normal in vivo regulatory mechanisms in the in vitro cultures.

Cells, Cultured↗

An immunoepidemiological approach to asthma: identification of in-vitro T-cell response patterns associated with different wheezing phenotypes in children.

BACKGROUND: Increasing evidence suggests that patterns of T-cell immunity to inhalant allergens in genetically diverse human populations are more heterogeneous than previously assumed, and that covert differences in expression patterns might underlie variations in airway disease phenotypes. We tested this proposition in a community sample of children. METHODS: We analysed data from 172 individuals who had been recruited antenatally to a longitudinal birth cohort study. Of the 194 birth cohort participants, data from the 147 probands (age range 8.6-13.5 years) who consented to blood collection were included along with data from 25 consenting siblings (mean age 11 years [range 7.4-17.4]). We ascertained clinical phenotypes related to asthma and allergy. We measured T-cell responses to allergens and mitogens, together with blood eosinophils and IgE/IgG antibodies, and assessed associations between these indices and clinical phenotypes. FINDINGS: Atopy was associated with allergen-specific T-helper (Th)2 responses dominated by interleukin 4, interleukin 5, interleukin 9, interleukin 13, whereas interleukin 10, tumour necrosis factor alpha, and interferon gamma responses were common to both atopics and non-atopics. The wheal size from skin prick with allergen was positively associated with in-vitro interleukin 5 and interferon gamma responses, and negatively associated with interleukin 10. Asthma, especially in atopics, was strongly associated with eosinophilia/interleukin 5, and bronchial hyper-responsiveness (BHR) was associated with eosinophilia plus polyclonal interferon gamma production. BHR in non-atopics was associated with elevated allergen-specific and polyclonal interleukin 10 production. INTERPRETATION: Parallel immunological and clinical profiling of children identified distinctive immune response patterns related to asthma and wheeze compared with BHR, in atopics non-atopics. Immunological hyper-responsiveness, including within the Th1 cytokine compartment, is identified as a hallmark of BHR. RELEVANCE TO PRACTICE: These findings highlight the heterogeneity of immune response patterns in asthmatic children, including those with seemingly homogeneous Th2-driven atopic asthma. Further elucidation of the covert relationships between wheezing phenotypes and underlying immunophenotypes in this age group will potentially lead to more effective treatments for what is an unexpectedly heterogeneous collection of disease subtypes.

Adolescent↗

Developing patterns of T cell memory to environmental allergens in the first two years of life.

Several recent studies have demonstrated cord blood mononuclear cell (CBMC) proliferation in response to food and inhalant allergens, suggesting that initial T-cell-priming may occur in utero. The findings below from an ongoing prospective study on 60 subjects provide initial information on the nature of accompanying T cell cytokine responses. We demonstrate CBMC proliferation following culture with house dust mite and ovalbumin (OVA) in 47 and 42% of subjects, respectively, compared to an overall rate of 3% for tetanus toxoid; the frequencies of these responses were comparable in neonates with and without atopic family history (FH). With the exception of IL-10, analysis of cytokine responses in allergen-stimulated cultures of CBMCs required the use of semiquantitative RT-PCR, which revealed low-level IL-4 and/or IL-5 mRNA production, in particular a 50% IL-5 response rate to OVA in FH-positive neonates. IFN-gamma responses were less frequent and required higher PCR cycle numbers for detection. Preliminary analysis of culture supernatants from a subgroup of CBMCs indicate high-level allergen-specific IL-10 responses in both FH-negative and -positive subjects, detectable by ELISA. Parallel PCR studies on MCs from 27 children (mean age 18 months) indicated a clear segregation at this age on the basis of FH, with Th0-like or mixed Th1/Th2 responses (IL-5 plus IFN-gamma) which were mainly restricted to the FH-positive group.

Allergens↗