Total serum immunoglobulin E levels in a case-control study in asthmatic/allergic patients, their family members, and healthy subjects from India.

BACKGROUND: Total immunoglobulin E (IgE) is an important indicator of allergic disorders. However, its role in allergic patients in India has not been evaluated in relation to atopic status for a reference range as compared with healthy subjects. OBJECTIVE: The aim of the study was to establish serum IgE levels in a diseased group, study its relationship with atopy, and to compare the same with healthy volunteers in Indian subjects. METHODS: Four hundred and eighty asthmatics/allergic patients, 100 first-degree relatives of asthmatics, and 120 unrelated normal healthy volunteers from Delhi region were recruited for the study. Atopy was established by family history and skin test to common indigenous allergens and, total and specific IgE measurements. Statistical analysis was performed with the help of SPSS software program. RESULTS: The mean IgE levels were the highest in asthmatic patients and the lowest in the control healthy group. IgE was significantly high in the male than the female healthy volunteers (P<0.05), but not in the diseased group. Prosopis juliflora among pollen allergens and Alternaria alternata among fungal allergens were important sensitizers in allergic patients with 34.7% and 17.7% skin positivity, respectively. Atopic status and asthma were found to be the best predictor of IgE, which was highly significant (r(2)=0.239, P<0.00001). However, at 95% confidence interval as many as 50% of asthmatic patients had their IgE values in the normal range. CONCLUSION: The IgE levels in Indian allergic patients is significantly related to atopy, but due to wide overlap of IgE levels in patients and healthy subjects, its diagnostic significance in Indian population seems to be limited.

Characterization of recombinant respiratory syncytial viruses with the region responsible for type 2 T-cell responses and pulmonary eosinophilia deleted from the attachment (G) protein.

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpDeltaG150-222 or rA2cpDeltaG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37 degrees C, but pinpoint morphology was observed at 39 degrees C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log(10)-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpDeltaG150-222 or rA2cpDeltaG177-220. These findings are important for the development of attenuated RSV vaccines.

Risk factors for development of bronchial asthma in children in Delhi.

BACKGROUND: Information on the magnitude of the problem of childhood asthma in India and the factors influencing its occurrence is inadequate. OBJECTIVE: To measure the prevalence of asthma in schoolchildren in Delhi and study the factors determining its occurrence. METHODS: A questionnaire-based study carried out in nine randomly selected schools in Delhi. The age range was 5 to 17 years. The questionnaires were distributed to all the children (n = 21,367) for answering by either parent. The key questions relate to complaints of recurrent wheezing in the past, during the immediate last 1-year, and also wheezing exclusively induced by exercise or colds. In all, 19,456 questionnaires were received back (response rate 91%). Out of these, 18,955 were complete and analyzed. RESULTS: The prevalence of current asthma was 11.9% while past asthma was reported by 3.4% of children. Exclusive exercise-induced asthma was reported by 2.1% while that associated with colds by 2.4% of children. Boys had a significantly higher prevalence of current asthma as compared with girls (12.8% and 10.7%, respectively). Multiple logistic regression analysis showed that male sex, a positive family history of atopic disorders, and the presence of smokers in the family were significant factors influencing the development of asthma while economic class, air pollution (total suspended particulates), and type of domestic kitchen fuel were not. CONCLUSIONS: The prevalence of current asthma in children in Delhi is 11.9%. Significant risk factors for its development are male sex, a positive family history of atopic disorders, and the presence of smokers in the family.

Prevalence of bronchial asthma in schoolchildren in Delhi.

There is a paucity of information on the prevalence of asthma in children in India. Some evidence suggests that asthma is less common in developing than in the developed countries. The present study was carried out to estimate its current magnitude in children in Delhi. The questionnaire-based study was carried out in two randomly selected schools in Delhi. All the children were eligible. The age range was 4-17 years. The questionnaires were distributed to all the children present (n = 2867) to be answered by either parent. The key questions were related to complaints of recurrent wheezing in the past, in the last 1 year, and also wheezing exclusively induced by exercise or colds. In all, 2609 questionnaires were completed and returned (response rate 91%). There was a slight excess of males (54%). The prevalence of current asthma was 11.6% and past asthma was reported by 4.1% of children, giving a cumulative prevalence of 15.7%. Exclusive exercise-induced asthma was 2.8% and that associated with colds, 2.3%. The current prevalence of all wheezing was thus 16.7% and cumulative prevalence was 20.8%. While there was no sex-related difference in prevalence, wheezers were the highest in the 9-13 year age group. A significant association was found between the prevalence of wheezing and a family history of asthma (odds ratio 3.65) and presence of smokers in the family (odds ratio 1.62). When both the above factors combined, the odds ratio for risk of asthma was 4.58. There was no significant association with any economic class. Only 11% of asthmatics had been labeled so by their physicians. The prevalence of bronchial asthma and wheezing in children in Delhi is quite high and comparable to that reported from several developed countries. A positive family history of asthma and presence of smokers in the family emerged as significant risk factors.

Stabilization of respiratory syncytial virus (RSV) against thermal inactivation and freeze-thaw cycles for development and control of RSV vaccines and immune globulin.

A high-titered and stable respiratory syncytial virus (RSV) is essential for the development of RSV vaccines and quality control of vaccines and RSV immune globulin. We increased the virus titer of RSV seed stock, and virus preparations made from this seed stock, 100 times by removing defective interfering particles using limiting dilution procedure. RSV preparations made from the new seed stock had infectivity titers ranging from 10(7.6) to 10(8.2) TCID50 per ml for five lots made over a period of 3 years. Unstabilized RSV lost most of its infectivity at -86 degrees C within 2-3 weeks, at 37 degrees C within 24 hr, at 56 degrees C within 3 min and after five freeze-thaw cycles. The high titered virus was stabilized at -86 degrees C for 3 years, at 37 degrees C for 3 days, at 56 degrees C for 6 min and against five freeze-thaw cycles. Most effective stabilizers included 25% sucrose, 10% trehalose and 45% fetal bovine serum (FBS) in Medium 199 whereas 3.5% dimethyl sulfoxide, > or = 45% FBS in phosphate buffered saline, 40% glycerol and 10% sorbitol also stabilized RSV to lesser and variable degrees. A mixture of 0.5% gelatine and 0.3% sodium glutamate stabilized the virus for a short period whereas 0.1 M MgCl2 and 25% FBS did not stabilize the virus. The stabilized high-titered virus is very useful for achieving reproducibility in serologic assays. A broad spectrum of stabilizers, such as those evaluated in this study, would be useful in choosing the most suitable formulation for stabilizing a live RSV vaccine.

IgG subclass antibodies to human cytomegalovirus (CMV) in normal human plasma samples and immune globulins and their neutralizing activities.

An enzyme-linked immunoabsorbent assay (ELISA) was developed for quantitation of IgG subclass antibodies to human cytomegalovirus (CMV) in human serum or plasma samples and in immune globulin (IG) preparations. The assay was based on the parallel titration of known concentrations of purified IgG subclass myeloma proteins and a specific CMV antiserum. The purified IgG subclass myeloma proteins were captured on an ELISA plate pre-coated with anti-human kappa, anti-human lambda or a mixture of anti-human kappa and lambda antibodies and the specific antiserum was titrated against CMV antigen coated on the plate. IgG subclass antibodies, captured or bound to antigen, were quantitated with IGG subclass heavy chain specific monoclonal antibodies. The method was highly reproducible, specific and sensitive. Using this method, 257 human plasma samples and 50 IG preparations were assayed for CMV specific IgG subclass and IgM antibodies. The major IgG subclass antibody to CMV was IgG1 which represented more than 96% of CMV IgG antibodies, followed by IgG3 (mean CMV IgG3 antibody content was 3% of IgG antibodies in IG preparations and 1.8% in plasma samples). A majority of the samples had low levels of IgG2 antibodies and a few samples exhibited low levels of IgG4 antibodies. IG preparations showed very low levels of CMV IgM antibodies whereas plasma samples had 14.2% of CMV antibodies (IgG and IgM) as IgM antibodies. Virus neutralizing (Nt) activity of these samples showed a significant correlation with CMV IgG1 antibodies. Nine samples of plasma and IGs were further evaluated for Nt activity of IgG1 and IgG3 antibodies by separating IgG3 from the rest of the antibodies with protein A agarose. IgG3 antibodies showed much higher Nt activity than IgG1 antibodies suggesting that enrichment of IgG3 antibodies in IG preparations may be useful in preparing CMV specific IG.

An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies.

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.

Helium/oxygen flow volume curves in normal Indian males > 50 year, and the effect of bronchodilator aerosols on airflow variables in this subject group.

Fourteen healthy males (mean age 54.7 yr) described maximal flow volume curves after Air breathing (AB) and after inspiring 10 deep breaths of a mixture of 80% Helium/20% Oxygen (He/O2). Vmax 50% FVC as obtained from the AB curves was 3.11 +/- 1.27 lps, and this increased by 38.1 +/- 17%, with He/O2. The Vmax 50% He-O2/Air ratio was 1.32 +/- 0.17, while the V iso V was 1.1 +/- 0.35 liters (30.9% of the FVC). Inhalation of 80 micrograms (4 puffs) of Ipratropium bromide, a vagolytic aerosol, or salbutamol (200 micrograms; 2 puffs), a beta 2 adrenergic stimulant did not alter the He/O2 curves significantly. As another aspect of the study, airflow variables viz. Vmax 50% of the FVC, Vmax 25%-75%, and Vmax 25% were measured from the AB curves, and the effect of bronchodilator aerosols was investigated in order to evaluate the degree of control of the bronchomotor tone by either the adrenergic receptors, or the vagus. Only the Vmax 25%-75% increased significantly with Salbutamol administration. While the degree of increase brought about by salbutomol aerosol for all of the variables under investigation was more than that seen with ipratropium br., the difference in this increase between the two bronchodilators used was not significant. The result may indicate that almost an equivocal influence is exerted by the two arms of the autonomic nervous system on bronchomotor tone in middle aged normal males, and that a more sensitive test (viz He-O2 breathing) does not elucidate this any further.

Cardiovascular responses to phenylephrine during acute experimental anaemia in anaesthetized cats.

Experiments were performed on anaesthetized artificially ventilated cats to study the effects of phenylephrine (PE) on cardiovascular responsiveness, before and after induction of experimental anaemia. Acute anaemia was induced by replacement of blood by dextran in three steps of 20% each of total estimated blood volume. The effect of PE (20 micrograms/kg) was investigated at four stages: control and after 1st, 2nd and 3rd exchanges of blood. Induction of anaemia produced a significant increase in heart rate (HR) and cardiac output (CO) and a decrease in right atrial pressure (RAP). No significant change in mean arterial pressure (MAP), LV dP/dt max and blood gas tension was observed. Administration of bolus dose of PE produced a rapid rise in MAP, LVdP/dt max, and a decrease in HR without a change in the RAP. The pattern of response to PE was similar after induction of acute anaemia, however the magnitude of the response was significantly reduced. The attenuation in the response to PE was related to the fall in the haematocrit (HCT) level. This shows that induction of experimental anaemia, produced an increase in CO due to an increase in HR and SV and the effect of PE on cardiovascular responsiveness was significantly attenuated. The reduced sensitivity to PE during acute anaemia could be due to many factors such as inadequate O2 supply, effect of local vasodilating agents or some other cardiotonic agents which are known to contribute to vascular responsiveness.

Suitability of the Vero cell method for titration of diphtheria antitoxin in the United States potency test for diphtheria toxoid.

The in vitro Vero cell method for titration of diphtheria antitoxin in immunized guinea pig sera was standardized to obtain comparable results to the in vivo toxin neutralization (TN) test in guinea pigs used to test potency of adsorbed diphtheria toxoid according to the United States Minimum Requirements. In the Vero cell method, the antitoxin titers of the guinea pig sera, obtained 4 weeks after immunization, were markedly dependent on the toxin dose level used in the assay. A toxin dose level termed the Lcd/1 dose (limit of cytopathic dose at 1 IU/ml) for Vero cell method gave comparable estimates of antitoxin activity to the in vivo TN method performed at L+/1 dose of toxin. The Lcd/1 toxin dose level for the Vero cell method is defined as the minimum concentration of diphtheria toxin required to produce a cytopathic effect on Vero cells in 4 days when the solution of diphtheria toxin is mixed with an equal volume of standard diphtheria antitoxin whose concentration is 1 IU/ml. When lower dose levels of toxin were used in the Vero cell assay (Lcd/10 to Lcd/1000), the concentrations of antitoxin in 4 weeks guinea pig sera were 2 to 11.7 times lower than with the Lcd/1 dose level and similarly lower than those measured by the in vivo TN test at L+/1 level. In contrast, the concentration of antitoxin measured in sera from guinea pigs who have been boosted with diphtheria toxoid increased approximately two-fold with the Lcd/1000 dose level.(ABSTRACT TRUNCATED AT 250 WORDS)

On the contribution of height to predict lung volumes, capacities and diffusion in healthy school children of 10-17 years.

Lung volumes, capacities, diffusion and alveolar volumes with physical characteristics (age, height and weight) were recorded for 186 healthy school children (96 boys and 90 girls) of 10-17 years age group. The objective was to study the relative importance of physical characteristics as regressor variables in regression models to estimate lung functions. We observed that height is best correlated with all the lung functions. Inclusion of all physical characteristics in the models have little gain compared to the ones having just height as regressor variable. We also find that exponential models were not only statistically valid but fared better compared to the linear ones. We conclude that lung functions covary with height and other physical characteristics but do not depend upon them. The rate of increase in the functions depend upon initial lung functions. Further, we propose models and provide ready reckoners to give estimates of lung functions with 95 per cent confidence limits based on heights from 125 to 170 cm for the age group of 10 to 17 years.

Adjuvants--a balance between toxicity and adjuvanticity.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Biological standardization of pollen allergens from India.

Standardization of allergens are achieved by in vitro and in vivo methods. Some of the allergens from Western countries are standardized using biological potency of the extracts but no attempt has been made till now to standardize any of the pollen extracts from India based on biological units. Therefore, we have attempted to standardize two important pollen allergens Ricinus communis and Holoptelea integrifolia by biological methods. Broadly the methods adopted by Dreborg and Grimmer (1983) was followed. Skin prick tests were carried out with the extracts of R. communis and H. integrifolia on 15 allergic patients in five three fold log dilutions starting with 1:10, in 50% glycerinated buffer. Glycerinated buffer (50%) and histamine dihydrochloride (1 mg/ml) were used as negative and positive controls, respectively. The mean wheal diameter obtained with different concentrations showed a gradual systematic fall with increase in dilution. The mean relative diameter (% of histamine reaction) varied from 124.1 +/- 8.9 to 33.7 +/- 6.1 and 78.9 +/- 5.5 to 21.4 +/- 3.8 with the highest and lowest concentrations of R. communis and H. integrifolia pollen antigens, respectively. The histamine equivalent concentration of antigen 1,000 Biological Units (BU) obtained for crude pollen extracts of R. communis and H. integrifolia was 1:17 and 1:22 respectively.

Reaction in leprosy: acute phase reactant response during and after remission.

Sera from 25 patients with type 1 (Lepra), upgrading and downgrading, and type 2 (erythema nodosum leprosum [ENL]) reactions were assayed, during the reaction and after its clinical remission, for changes in levels of alpha-1-antitrypsin (A1A) and C-reactive protein (CRP). The results were compared with those from normal healthy adults and patients of leprosy without history and/or clinical evidence of reaction. The A1A levels correlated better with changes in status of type 1 reaction; whereas CRP levels correlated well with alterations in type 2 reactions and were definitely superior to A1A in this situation for monitoring the course of these episodes.

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.

Lymphocyte adenosine deaminase activity (L-ADA) in leprosy, during and after treatment of reactions.

Twenty-five patients with type 1 (lepra) and type 2 (E.N.L.) leprosy reactions were studied for lymphocyte adenosine deaminase activity (L-ADA), during and after treatment of the reactions, using a standard technique, in order to establish its pattern and if possible, its value in assessing the course of reactions. The results were compared with those from 30 control subjects, comprising 10 normal healthy adults, 10 patients with borderline tuberculoid (BT) leprosy, four patients with borderline lepromatous (BL) leprosy and six patients with lepromatous (LL) leprosy. The level of L-ADA in the leprosy controls was higher than that of normal healthy subjects. The L-ADA values in patients with different types of reactions were about 10-fold higher than those obtained from leprosy controls, emphasizing a possible role in assessing reactions in leprosy. However, there was no significant variation in L-ADA levels, either between the various leprosy controls or reaction groups, before and after treatment.