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S T Holgate

Publications and source records attributed to S T Holgate.

At least 289 records · Page 16Linked to original sources
PubMed1995

IgE-dependent expression of mRNA for IL-4 and IL-5 in human lung mast cells.

By using the reverse transcriptase (RT)-PCR and in situ hybridization we have studied the expression of mRNA for IL-5 and IL-4 in human lung mast cells induced by cross-linkage of high affinity Fc epsilon Rs. Lung mast cells were purified using affinity magnetic selection with mAb YB5.B8 against c-kit to achieve a final mast cell purity > 93%. Purified mast cells were precultured with stem cell factor (SCF) (10 ng/ml) and myeloma IgE (3 micrograms/ml) for 16 h before challenge with anti-IgE (1 or 10 micrograms/ml). IgE-dependent activation of lung mast cells caused expression of IL-5 mRNA, which was evident by 2 h and persisted for up to 48-72 h in all of 12 experiments, whereas IL-4 mRNA expression was of a shorter duration and was demonstrable in 6 of 13 experiments. We confirmed that mast cells, and not T cells, were the source of these cytokine messages by using reverse transcriptase-PCR in cell preparations containing known numbers of mast cells and T cells, in situ hybridization in enriched mast cell preparations, and double in situ hybridization-immunocytochemical staining. IL-5 mRNA expression did not require the pretreatment of cells with SCF, whereas expression of IL-4 mRNA seemed to require both anti-IgE and SCF. The strength of IL-5 mRNA signal was related to anti-IgE concentration. Immunoreactive IL-5 was detectable 8 h after anti-IgE challenge, and 10(6) mast cells generated a mean of 731 +/- 400 pg of IL-5 into the supernatant during 48-h culture, but no IL-4 product was detectable. These findings demonstrate the capacity of human lung mast cells to transcribe IL-4 and IL-5 after IgE-dependent activation and to synthesize and release immunoreactive IL-5.

Base Sequence↗
PubMed1995

Heterogeneity of human mast cells based on cytokine content.

Human mast cells can be divided into two distinct phenotypes based on their content of neutral serine proteases, suggesting that they serve differing biologic and pathologic roles. Recently, it has been demonstrated that human mast cells are a source of several pleiotropic cytokines including IL-4, IL-5, IL-6, IL-8, and TNF-alpha, but not all mast cells contain all of these cytokines, suggesting that there is also functional heterogeneity with respect to cytokine expression. In this study, we have examined the relationship between mast cell neutral protease expression and cytokine content using immunohistochemistry. Bronchial mucosal biopsies from five normal subjects and five patients with allergic asthma, and nasal mucosal biopsies from five normal subjects and three patients with allergic rhinitis were embedded in glycol methacrylate. Sections (2 microns) were stained for IL-4, IL-5, and IL-6, adjacent to serial sections stained for tryptase and chymase. The distribution of cytokines among the tryptase+ chymase- mast cells (MCT) and tryptase+ chymase+ mast cells (MCTC) was examined by co-localization of cytokines to MCTC or MCT in serial sections using the camera-lucida. Although IL-4 was distributed among both mast cell phenotypes, it was expressed preferentially by the MCTC subset (overall 85% MCTC:15% MCT). In contrast, IL-5 and IL-6 were restricted almost exclusively to the MCT subset. Immunostaining of isolated skin mast cells (> 99% MCTC) supported these findings, with strong immunoreactivity present for IL-4 but very little for IL-5 or IL-6. These results indicate that in addition to exhibiting heterogeneity with respect to neutral protease content of the secretory granules, human mast cells are also heterogeneous with respect to cytokine content. This suggests that the biologic functions of MCTC and MCT cells differ as a result of their capacity to generate and release different cytokine profiles.

Adult↗
PubMed1995

Analysis of Ig VH region genes encoding IgE antibodies in splenic B lymphocytes of a patient with asthma.

An atopic patient with hypersensitivity against house dust mite died as a result of an asthmatic attack. A portion of the spleen was obtained and was used to analyze the spectrum of Ig heavy chain V regions involved in encoding IgE Abs. A nested PCR technique generated 14 cloned VH sequences that had distinct CDR3 regions; 5 of 14 were derived from the minor VH5 family, and the remainder derived from the larger families, VH3 (6 of 14) and VH4 (3 of 14). One of the VH3-derived sequences was present as a repeated sequence in three clones. A control PCR with the same VH primers in combination with JH primers yielded only 1 of 13 sequences from VH5, indicating preferential VH5 usage only for IgE. Analysis of VH5-C epsilon sequences revealed usage of a single gene, DP73, with extensive mutations and several "hot spots" containing common replacement amino acids. However, there was no concentration of replacement mutations in the CDRs, which conventionally would indicate a role for Ag selection. The VH3 and VH4 genes in combination with C epsilon also harbored extensive somatic mutations. From these findings in splenic B lymphocytes, and those of a previous study of blood lymphocytes, it seems that preferential usage of VH5 genes and extensive somatic hypermutation are characteristic of B cells synthesizing IgE in patients with allergic disease.

Adult↗
PubMed1995

Cross refractoriness between bradykinin and hypertonic saline challenges in asthma.

BACKGROUND: Repeated inhalation of bradykinin and hypertonic saline leads to refractoriness of the bronchoconstrictor response in asthma. It is not known whether cross-refractoriness exists between these stimuli. OBJECTIVE: We postulated that repeated bradykinin and hypertonic saline bronchial challenges might reduce the airway response to subsequent hypertonic saline and bradykinin challenges, respectively. METHODS: Eleven atopic asthmatic subjects underwent two concentration-response studies, separated by 1 hour, with either inhaled histamine or bradykinin. After recovery, a hypertonic saline challenge was performed. During the next phase, nine subjects underwent two concentration-response studies, separated by 1 hour, with hypertonic saline. After recovery, a bradykinin challenge was performed. RESULTS: On the histamine study day, the mean provocative volume of agonist required to produce 20% drop in forced expiratory volume in 1 second (PD20) hypertonic saline was 220.7 L (+/- 42.7 L) and this was not significantly different from that measured at baseline. On the bradykinin study day, the geometric mean provocative concentration of agonist required to produce a 20% drop in forced expiratory volume in 1 second (PC20) was 0.39 mg/ml (0.01 to 11.73 mg/ml) for the first test and significantly higher at 1.38 mg/ml (0.01 to > 16.0 mg/ml) for the second test (p = 0.006). The hypertonic saline PD20 increased significantly from a baseline of 159.2 L (+/- 27.3 L) to 377.6 (+/- 64.7 ) (p = 0.003). On the hypertonic saline study day, the mean PD20 was 152.8 L for the first test, and 337.7 L for the second test (p = 0.01). PC20 bradykinin increased significantly from a baseline of 0.57 to 2.56 mg/ml (p = 0.02). A significant correlation was found between loss of response to bradykinin and to hypertonic saline (rs, 0.63 and 0.76). CONCLUSION: Refractoriness produced by repeated exposure of the airways to bradykinin and hypertonic saline results in loss of responsiveness to hypertonic saline and bradykinin respectively, suggesting a shared mechanism for refractoriness produced by these stimuli.

Adolescent↗
PubMed1995

The reproducibility and effect on non-specific airway responsiveness of inhaled prostaglandin D2 and leukotriene D4 in asthmatic subjects.

1. Mast cell mediators PGD2 and LTD4 may play important roles in asthma pathogenesis. There is little information on the repeatability of inhalation challenge with these agonists in the laboratory. 2. We assessed the repeatability of inhalation challenges using PGD2 and LTD4 in two groups of 10 asthmatic volunteers. Non-specific bronchial responsiveness was assessed by histamine inhalation challenges. 3. Using the Bland-Altman method, we found the coefficient of repeatability to be 1.2 doubling doses for LTD4 and 2.1 for PGD2 at a 1 week interval. Repeatability for histamine inhalation challenge over the same time period was similar at 1.4 and 2.1 doubling doses respectively. 4. Non-specific bronchial responsiveness following LTD4 challenge decreased significantly, mean PD20FEV1 increasing from 169 nmol on day 1 to 278 nmol on day 3 (P = 0.001), before returning to baseline levels. 5. A progressive decrease in non-specific bronchial responsiveness occurred following PGD2 challenge. Baseline PD20FEV1 was 195 nmol, increasing to 238 nmol by day 3 (NS) and 313 nmol by day 8 (P = 0.016). 6. PGD2 inhalation challenges performed a week apart are less reproducible than LTD4 challenges, possibly as a result of significant changes in histamine bronchial responsiveness. Our findings allow accurate power calculations to be made for studies to assess new pharmacological antagonists to these mediators.

Administration, Inhalation↗
PubMed1995

A double-blind, placebo controlled study of the effect of the specific histamine H1-receptor antagonist, terfenadine, in chronic severe asthma.

1. The characteristic changes seen in asthma are widely regarded as being caused by local mediator release in the airways, with histamine the first putative mediator in asthma to be identified. 2. We performed a double-blind, randomised, placebo-controlled crossover trial of the effect of 4 weeks treatment with terfenadine 120 mg twice daily in chronic severe asthma. 3. Forty-two subjects (20 male and 22 female) completed the 10 week study. 4. Terfenadine had no significant treatment effect on the primary efficacy variables measured. Mean (95% CI) measurements for terfenadine vs placebo treatment periods were 1.5 vs 1.5 (-0.3, 0.3) l for FEV1, 259 vs 260 (-42, 40) l min-1 for morning PEF and 0.8 vs 0.8 (-0.3, 0.3) for global symptom scores. 5. Bronchodilator use and sleep disturbance, the secondary efficacy variables studied, showed an improvement during terfenadine treatment but this only reached statistical significance for the number of times subjects awoke from sleep (P = 0.04). 6. There was a similar frequency of minor adverse effects reported during placebo (13.6%) and terfenadine (16.7%) treatments. 7. Addition of the potent and specific histamine H1-receptor antagonist terfenadine to maintenance asthma treatment had no significant therapeutic benefit in this group of chronic severe asthmatics.

Adult↗
PubMed1995

Mast cell distribution and neutral protease expression in acute and chronic allergic conjunctivitis.

Allergic eye disease has a variety of clinical manifestations including seasonal atopic conjunctivitis (SAC), perennial atopic conjunctivitis (PAC), atopic keratoconjunctivitis (AKC), and atopic blepharoconjunctivitis (ABC). We have investigated the number, distribution and protease expression of mast cells in normal and diseased conjunctiva with the use of immunohistochemistry in water-miscible resin sections. The median mast cell densities in normal subjects were 17 mm-2 in the bulbar substantia propria and 9 mm-2 in tarsal substantia propria. Mast cells were absent from the normal conjunctival epithelium at both sites. Mast cell densities were increased in the bulbar substantia propria in SAC, AKC and ABC. Tarsal substantia propria showed a significant increase in mast cells in ABC and AKC disease states. Mast cells express a range of proteases which varies according to their anatomic site. Mast cells in connective tissue are described to contain tryptase, chymase, cathepsin-G and carboxypeptidase-A, whereas mucosal mast cells contain only tryptase. In the diseased conjunctiva there was a marked reduction in proteases other than tryptase in the intraepithelial mast cells. There were also significant reductions in protease expression other than tryptase in the bulbar substantia propria in AKC and ABC. There appear to be specific alterations in the distribution of mast cells in the sub-categories of allergic eye disease. The distinction between mucosal and connective tissue mast cell phenotypes is not clear-cut and may depend on the functional state of the mast cells in relation to the microenvironment.

Acute Disease↗
PubMed1995

TNF alpha is localized to nasal mucosal mast cells and is released in acute allergic rhinitis.

Allergic mucosal inflammation is characterized by tissue infiltration with eosinophils, and associated activation of mast cells and T lymphocytes. Tumour necrosis factor (TNF) alpha/cachectin is a candidate cytokine relevant to the pathogenesis of these events through its capacity to upregulate the expression of endothelial cell adhesion molecules, mediate granulocyte chemoattraction, and activate eosinophils, mast cells and T cells. To investigate the presence and localization of TNF alpha in the nasal mucosa in allergic rhinitis, nasal biopsies from perennial rhinitic (n = 13) and non-rhinitic volunteers (n = 11) were embedded in glycol methacrylate and immunostained with a monoclonal antibody directed against TNF alpha, and adjacent 2 microns sections stained for tryptase, CD3 and eosinophil cationic protein. This identified positive immunostaining for TNF alpha in the submucosa of both the rhinitic and normal subjects (median cell counts 13 and 23 cells/mm2 respectively, P = 0.24) with cellular localization to mast cells but not to T-lymphocytes or eosinophils. In a subsequent study of seven atopic subjects, nasal allergen challenge produced increases in lavage levels of histamine and albumin, which was associated with significant release of TNF alpha as early as 2 min post-allergen when compared with the saline control day (P = 0.05). This difference was also apparent when studying the area under the curve both at 30 and 60 min post-challenge t-test (P = 0.015 and 0.02 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Exclusion from proximal 11q of a common gene with megaphenic effect on atopy.

We have typed three markers on proximal 11q in 131 random families with three or more children studied for atopy. A summary map that includes the FCER1B candidate was constructed. Using a 2-locus disease model, we performed combined segregation and linkage analysis of three models, none of which suggested linkage. Nine marker loci on other chromosomes were also negative. In the regions swept by these 12 markers we cannot rule out a rare gene, perhaps of large effect, nor a common gene of small effect. However, a common gene of large effect is excluded. These results and alternative strategies are discussed in the perspective of inconsistent evidence for a major atopy gene.

Alleles↗