Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.

Smoking, alcohol consumption, and leukocyte counts.

Blood was collected from 684 healthy volunteers and examined for total and differential white blood cell (WBC) counts. A subgroup also was tested for numbers of T cells, B cells, and CD4 and CD8 subsets. Smoking status and alcohol consumption were determined by means of questionnaire, and smoking status was verified with serum cotinine concentration. High smoking rate was associated with increases in all counts. Former smokers abstinent less than 5 years still demonstrated elevated counts, whereas those abstinent more than 5 years had WBC counts comparable to those in persons who were never smokers. Compared with levels in those who had never smoked, total WBC counts were 27% higher in current smokers and 14% higher in former smokers who were abstinent for less than 5 years. Lymphocyte counts were 9% higher in those consuming more than one alcoholic drink per day than in those consuming less alcohol, but drinking was not associated with other cell populations.

Experimental rhinovirus infection in volunteers.

Experimental viral disease studies in volunteers have clarified many aspects of the pathogenesis of human viral disease. Recently, interest has focused on rhinovirus-associated asthma exacerbations, and new volunteer studies have suggested that airway responsiveness (AR) is enhanced during a cold. For scientific, ethical and safety reasons, it is important to use validated methods for the preparation of a virus inoculum and that the particular virological characteristics and host responses should not be altered. We have prepared a new human rhinovirus (HRV) inoculum using recent guidelines and assessed whether disease characteristics (for example, severity of colds or changes in AR) were retained. Studies were conducted in 25 clinically healthy volunteers using a validated HRV inoculum in the first 17 and a new inoculum in the subsequent eight subjects. Severity of cold symptoms, nasal wash albumin levels and airway responsiveness were measured, and the new inoculum was prepared from nasal washes obtained during the cold. The new inoculum was tested using standard virological and serological techniques, as well as a polymerase chain reaction for Mycoplasma pneumoniae. No contaminating viruses or organisms were detected and the methods suggested were workable. Good clinical colds developed in 20 of the 25 subjects and median symptom scores were similar in the validated and new inoculum groups (18 and 17.5, respectively; p=0.19). All subjects shed virus, and there were no differences noted in viral culture scores, nasal wash albumin and rates of seroconversion in the two groups. Although airway responsiveness increased in both groups (p=0.02 and p=0.05), the degree of change was similar. We have performed experimental rhinovirus infection studies and demonstrated similar clinical disease in two inoculum groups. Amplified airway responsiveness was induced; continuing studies will define the mechanisms and suggest modes of treatment.

The relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis.

We have shown that viruses are associated with 80 to 85% of asthma exacerbations in school-age children in the community. We hypothesize that viral infections are also associated with severe attacks of asthma precipitating hospital admissions. To investigate this, we conducted a time-trend analysis, comparing the seasonal patterns of respiratory infections and hospital admissions for asthma in adults and children. During a 1-yr study in the Southampton area of the United Kingdom, 108 school-age children monitored upper and lower respiratory symptoms and took peak expiratory flow rate (PEFR) recordings. From children reporting a symptomatic episode or a decrease in PEFR, samples were taken for detection of viruses and atypical bacteria. A total of 232 respiratory viruses and four atypical bacteria were detected. The half-monthly rates of upper respiratory infection were compared with the half-monthly rates for hospital admissions for asthma (International Classification of Diseases [ICD] code 493) for the same time period for the hospitals serving the areas from which the cohort of schoolchildren was drawn. The relationships of upper respiratory infections and hospital admissions for asthma with school attendance were studied. Strong correlations were found between the seasonal patterns of upper respiratory infections and hospital admissions for asthma (r = 0.72; p < 0.0001). This relationship was stronger for pediatric (r = 0.68; p < 0.0001) than for adult admissions (r = 0.53; p < 0.01). Upper respiratory infections and admissions for asthma were more frequent during periods of school attendance (87% of pediatric and 84% of total admissions), than during school holiday periods (p < 0.001). These relationships remained significant when allowance was made for linear trend and seasonal variation using multiple regression analysis (p < 0.01). Not surprisingly, school attendance, because it is a major factor in respiratory virus transmission, was found to be a major confounding variable in children. This study demonstrates that upper respiratory viral infections are strongly associated in time with hospital admissions for asthma in children and adults. Rhinoviruses were the major pathogen implicated, and the majority of viral infections and asthma admissions occurred during school attendance.

Community study of role of viral infections in exacerbations of asthma in 9-11 year old children.

OBJECTIVE: To study the association between upper and lower respiratory viral infections and acute exacerbations of asthma in schoolchildren in the community. DESIGN: Community based 13 month longitudinal study using diary card respiratory symptom and peak expiratory flow monitoring to allow early sampling for viruses. SUBJECTS: 108 Children aged 9-11 years who had reported wheeze or cough, or both, in a questionnaire. SETTING: Southampton and surrounding community. MAIN OUTCOME MEASURES: Upper and lower respiratory viral infections detected by polymerase chain reaction or conventional methods, reported exacerbations of asthma, computer identified episodes of respiratory tract symptoms or peak flow reductions. RESULTS: Viruses were detected in 80% of reported episodes of reduced peak expiratory flow, 80% of reported episodes of wheeze, and in 85% of reported episodes of upper respiratory symptoms, cough, wheeze, and a fall in peak expiratory flow. The median duration of reported falls in peak expiratory flow was 14 days, and the median maximum fall in peak expiratory flow was 81 l/min. The most commonly identified virus type was rhinovirus. CONCLUSIONS: This study supports the hypothesis that upper respiratory viral infections are associated with 80-85% of asthma exacerbations in school age children.

Low immunogenicity of therapeutic rat CD45 antibodies when used for pre-treatment of donor organs for transplantation.

Immunoassays for the measurement of human anti-immunoglobulin responses against the CD45 specific rat monoclonals, YTH 24.5 and YTH 54.12, have been developed. The assays are based on a 'double antigen' ELISA system and are reproducible with coefficients of variation of less than 15%. The assays were used to measure anti-immunoglobulin responses in sera from 40 patients who had received kidneys pre-treated with the pair of anti-CD45 monoclonals YTH 24.5 and YTH 54.12. Only two patients elicited a weak human antimurine antibody (HAMA) response.

Smoking, alcohol consumption, and susceptibility to the common cold.

OBJECTIVES: This study was conducted to test the supposition that both smoking and consuming alcohol suppress host resistance to viral infections. METHODS: The relations between smoking, alcohol consumption, and the incidence of documented clinical colds were prospectively studied among 391 subjects intentionally exposed to one of five respiratory viruses and 26 subjects given saline. Clinical colds were defined as clinical symptoms verified by the isolation of virus or by an increase in virus-specific antibody titer. Analyses included control variables for demographics; body weight; virus; and environmental, immunological and psychological factors. RESULTS: Smokers were at greater risk for developing colds than nonsmokers because smokers were more likely both to develop infections and to develop illness following infection. Greater numbers of alcoholic drinks (up to three or four per day) were associated with decreased risk for developing colds because drinking was associated with decreased illness following infection. However, the benefits of drinking occurred only among nonsmokers. CONCLUSIONS: Susceptibility to colds was increased by smoking. Although alcohol consumption did not influence risk of clinical illness for smokers, moderate alcohol consumption was associated with decreased risk for nonsmokers.

Signs and symptoms in common colds.

The patterns of disease caused by five common viruses which infect the respiratory tract are described. The viruses were strains of rhinovirus types 2, 9, and 14, a strain of coronavirus type 229E and of respiratory syncytial virus. Volunteers were given nasal drops containing a low infectious dose of one of the viruses, quarantined from 2 days before to 5 days after inoculation, and examined daily by a clinician using a standard checklist of respiratory signs and symptoms. Only subjects who developed clinical illness accompanied by viral shedding and/or specific antibody production were analysed [n = 116]. The results confirm indication from earlier studies that the main difference between colds induced by different viruses is in duration of the incubation period. Patterns of symptom development were not substantially different with different viruses. Analyses of signs and symptoms in different categories, e.g. nasal symptoms v. coughing, justify treatment with different drugs either successively or simultaneously.

Immunogenicity and pathogenicity of a triple temperature-sensitive modified respiratory syncytial virus in adult volunteers.

The immunogenicity and pathogenicity of a strain of respiratory syncytial (RS) virus modified by sequential induction of three temperature-sensitive (ts) mutations have been evaluated by intranasal administration to 22 adult volunteers. This modified virus, a triple ts mutant designated ts1C, was derived from a double mutant ts1B evaluated in a previous trial. The original isolate (strain RSS-2) and all its derivatives were propagated throughout in human diploid cells in a specially assigned laboratory. The triple mutant ts1C is unable to multiply in MRC-5 cells at 37 degrees C and above. Following nasal administration of ts1C, immune responses were observed in volunteers with low pre-existing neutralizing antibody titres. The ability of mutant ts1C to induce upper respiratory tract disease in adults was greatly diminished in comparison with the non-ts wild-type virus, but not markedly more so than a previously tested double ts mutant (ts1B) which replicates at 37 degrees C. Mutant ts1C, however, may have greater potential as a live vaccine in view of its inherently greater genetic stability.

Negative life events, perceived stress, negative affect, and susceptibility to the common cold.

After completing questionnaires assessing stressful life events, perceived stress, and negative affect, 394 healthy Ss were intentionally exposed to a common cold virus, quarantined, and monitored for the development of biologically verified++ clinical illness. Consistent with the hypothesis that psychological stress increases susceptibility to infectious agents, higher scores on each of the 3 stress scales were associated with greater risk of developing a cold. However, the relation between stressful life events and illness was mediated by a different biologic process than were relations between perceived stress and illness and negative affect and illness. That these scales have independently relations with illness and that these relations are mediated by different processes challenges the assumption that perceptions of stress and negative affect are necessary for stressful life events to influence disease risk.

Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms.

Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.

A view from the Common Cold Unit.

I have been asked to stand back and describe in broad terms the view I have had of common colds--probably the most frequent of acute human diseases and a long-lasting scientific problem--and in particular our recent work on antivirals. I should be able to do this for two reasons. Like everyone else I have suffered from colds, but in addition I have been studying the problem from the virological and clinical point of view for over 35 years--for the last 31 at the Common Cold Unit, Salisbury. As a result I may have problems with perspective--it is not possible to give a personal view and at the same time to describe something from every possible angle, and quite impossible to be comprehensive, but I have done my best and readers will make their own judgements and corrections.

The common cold, pattern sensitivity and contrast sensitivity.

Results from two studies involving challenge with respiratory syncytial viruses showed that volunteers who developed colds were more sensitive to a visually distracting pattern presented prior to virus challenge than were volunteers who did not get a cold. Volunteers with sub-clinical infections reported more illusions after virus challenge than they had done before, whereas uninfected volunteers and those with colds tended to report fewer illusions on the second test. These effects did not occur when volunteers were challenged with either a coronavirus or rhinovirus. Overall, the results confirm that behavioural measures may be related to susceptibility to subsequent illness, and that viral infections may influence visual perception. They also show that the effects vary according to the nature of the infecting agent, which agrees with results from studies looking at other aspects of behaviour.

The specificity of antibodies induced by infection with rhinovirus type 2.

Mouse monoclonal antibodies (MAbs) against three distinct antigenic sites on rhinovirus type 2 have been obtained and the sites identified. We describe how these MAbs were used in a blocking test to detect antibodies in human sera directed against the same three defined sites. Sera from twelve volunteers were studied. All had been exposed to rhinovirus type 2 by intranasal inoculation, four had been uninfected, eight were infected of whom four developed a cold while four did not. Blocking antibodies were high and did not increase in the resistant volunteers, and were lower and increased in the infected volunteers. The antibodies were almost as sensitive as other antibody assays for detecting infection. The responses to all three sites were similar. Correlations between the results of all tests were calculated and the results are summarised. Tests were also devised to measure the Ig subclass of antibodies against the whole virus particle. The A1, G1, and G4 classes showed most frequent rises in response to infection. Correlations between these results and other antibody assays were found and are presented.

Bronchial reactivity to histamine and bradykinin is unchanged after rhinovirus infection in normal subjects.

We investigated the effects of rhinovirus (RV) infection on airways reactivity. Twenty seven normal volunteers (11 atopic) were inoculated with RV 2 or RV EL. The provocative concentrations of histamine and bradykinin required to produce a 15% fall in the forced expiratory volume in one second (FEV1) (PC15FEV1) were measured before, 7 and 21 days after inoculation. Infection was determined by a fourfold rise in anti-viral antibody titre and by viral culture from nasal washings. Peak expiratory flow rate (PEF) was recorded three days before and for 21 days after inoculation. All subjects underwent the first two bronchial challenges, and 22 the third challenge. For the whole group and for atopic subjects, there were significant correlations between the PC15 values for bradykinin and histamine (r = 0.82 and r = 0.85, respectively). Twenty subjects were infected; six had clinical colds. For the 16 infected subjects who had all three challenges, the median (range) PC15FEV1 for the histamine challenges was 36 (0.89-64), 62 (1.5-64) and 34 (0.94-64) mg.ml-1, respectively, and 32 mg.ml-1 for each bradykinin challenge (range: 0.015-32, 0.088-32 and 0.033-32). There were no significant differences between study days for PC15FEV1 histamine or bradykinin for the whole group, the infected subjects, those with clinical colds or for those infected with either RV subtype. There was no significant change in mean daily PEF after viral infection. We conclude that airways reactivity to histamine and bradykinin is unchanged after experimental RV infection in normal volunteers.