Alpha-Interferon Responses in Children With Respiratory Syncytial Virus Associated Bronchiolitis

Salas, Maria del Pilar (1987) Alpha-Interferon Responses in Children With Respiratory Syncytial Virus Associated Bronchiolitis. MSc(R) thesis, University of Glasgow.

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From October 1985 to March 1986 208 children (128 males, 80 females) were admitted to the Royal Hospital for Sick Children, Glasgow with suspected respiratory infection. This hospital is the largest paediatric centre in the West of Scotland with annual admissions for respiratory infections of 1903 children (79.5%) . When comparing the total number of children under study and total number of admissions during the study period, the majority of admissions (45%) were children under one year of age with the highest incidence of respiratory infections in November (30.7%) at the RHSC. In this study RSV infection was much greater in infants from Inner Glasgow predominantly Drumchapel, Castlemilk, Toryglen, High Possil (65.8%) , and Clydebank (12.6%) which have a higher proportion of low income families and households with two or more indicators of deprivation. The reverse situation was found in Bearsden/Milngavie and Eastwood where the proportion of RSV infections was 2.7% and 5.4% respectively. A full history and clinical examination was carried out in each case, and data recorded on a special form suitable for computer analysis. This included admission history, diagnosis classified into Upper Respiratory Tract Infection (URTI), pneumonia, bronchiolitis, chronic lung disease and others. Clinical parameters such as maximum respiratory rate, time to establish normal respiratory rate, physiotherapy and length of tube feeding were evaluated. Nasopharyngeal secretions (NPS) were collected on admission or first day of symptoms if already admitted, and subsequently repeated on days 3, 5, and 7 when possible. Plastic disposable collecting mucus traps with an attached feeding tube (5fg) were used to obtain the secretions. A portion of the secretions was cultured in bacteriological media and the remainder was transferred to virus transport medium (VTM) in a 1/10 dilution. After centrifugation of the specimen suspension, the supernatant fluid was used for virus isolation, embryonated hen egg inoculations and assay for alpha-interferon (IFN-alpha). The cell pellet was examined by direct immunofluorescence using a respiratory syncytial virus (RSV) specific monoclonal antibody. IFN-alpha levels were determined by a commercially available immunoradiometric assay. The test utilises an 125I labelled highly specific monoclonal antibody (Yok 5/19) to leucocyte IFN-alpha. Prior to application of the test in the clinical study it was important to determine the assay performance in terms of sensitivity and specificity parameters with special reference to the clinical specimens to be examined. The reproducibility of the test was evaluated when the mean values for each point of different standard curves were plotted together. A standard curve was used to interpolate IFN-alpha values for unknown samples of NPS. The variability found between the standard curves using different kits of reagents were similar for each point and varied from 21 to 31%. The sensitivity of IRMA for NPS was also evaluated and the test was able to detect as little as 0.2 IU/ml in a diluted clinical specimen. Experimentation on the specimens was carried out to determine heat stability and lability to trypsin. Using recombinant IFN-alpha or a pool of specimens containing endogenous IFN-alpha, treatment with heat caused partial inactivation. The protein characteristics of IFN-alpha was confirmed when standard preparations of IFN-alpha and clinical specimens containing endogenous IFN- a were treated with trypsin for trypsin sensitivity. In these experiments trypsin treatment completely inactivated both recombinant and endogenous IFN-alpha. In this study neutralization-blocking experiments were performed in order to demonstrate the specificity of the Yok 5/19 monoclonal antibody in detecting IFN-alpha. In both cases using standards and specimens, it was possible to neutralize almost 100% of the activity with very little residual IFN-alpha after blocking, demonstrating an absolute specificity in this test. It was clear for these studies that this technique is well devised, reproducible, rapid and sensitive. (Abstract shortened by ProQuest.).

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Medicine, Immunology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-77594
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 09:04
Last Modified: 14 Jan 2020 09:04

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