Patent Application: US-91329701-A

Abstract:
the present invention is concerned with methods for determining predisposition to infection in a subject exposed to stressors . in particular the present invention is concerned with methods of assessing the risk of susceptibility to infection in a subject by monitoring the levels of iga and iga1 .

Description:
the concentration of iga1 in a sample taken from a subject to be tested is determined by radial immunodiffusion ( rid ) using commercially prepared low - level rid plates and calibrators . elisa or other technologies applicable to rapid near - subject testing . the present results show that depressed concentrations of iga1 are particularly associated with a higher risk of infection . whole unstimulated saliva was collected from 25 elite swimmers ( 16 males , 9 females ) during the early ( april to june ) and late ( august to october ) phases of a 7 - month training program ( 15 ). the mean time between sample collections for individual athletes was 4 months . the saliva samples were collected prior to the first exercise session on the day of collection and at least 18 hours after the previous exercise session the swimmers aged 16 – 24 years were undertaking 20 – 25 hours of pool training and 5 hours of dry - land training per week . every episode of respiratory infection was recorded during the study and physician - verified ( 16 ). the study was conducted with the informed consent of the australian institute of sport ( ais ) swimming team and had ethics clearance from the australian sports commission . the concentration of total iga in whole unstimulated saliva was measured by electroimmuno diffusion ( 21 ). the concentrations of iga1 and iga2 were determined by radial immunodiffusion ( rid ) using one batch number of commercially prepared low level rid plates and calibrators . ( the binding site , birmingham , uk ). the detection limit of the assays was 4 . 0 mg / l for total iga , 8 . 3 mg / l for iga1 and 7 . 5 mg / l for iga2 . the between run cvs were 3 . 5 % for total iga , 4 . 1 % for iga1 and 3 . 0 % iga2 . for the purposes of this study the athletes were classified as either having no infections (‘ healthy ’) or at least one infection episode during the study period . the wilcoxon signed rank test for paired data was used to compare ratio values between ‘ healthy ’ athletes and athletes who had at least one infection during the study period . spearman &# 39 ; s correlation coefficients were used to determine the association between the number of infections and the concentrations of total iga , iga1 , iga2 , and the ratio of iga1 : iga2 in the early and late training phase saliva samples . the mann - witney rank sum test was used to compare differences between genders for the percentage of nil - detected values of iga2 . a p - value of & lt ; 0 . 05 was considered significant . the median concentrations of total iga , iga1 , iga2 or the ratio of iga1 : iga2 were not statistically significantly different between saliva samples collected in the early and late phases of the training program ( table 1 ). on average iga1 represented 80 % of the total iga saliva collected in both the early and late phases of the season ( table 1 , fig1 ). there were 11 samples with iga2 concentrations below the detection level of the rid assay ; 7 of the early phase samples and 4 of the late phase sample collections . there were no significant differences between gender for the median concentrations of iga2 or the proportions with nil detected values for iga2 in either the early or late season samples ( table 2 ). seven swimmers ( 5 males , 2 females ) has no episodes of respiratory infection during the 7 month study . the number of infection episodes in the other 18 swimmers ranged from one to seven ( fig2 ). the correlations between the number of infections and the concentrations of total iga , iga1 and iga2 ( table 3 ) showed a statistically significant association between the early season salivary iga1 concentration and the number of infection episodes in each athlete ( fig2 ). the correlation between the early season salivary iga1 and the number of infections remained significant ( p = 0 . 04 ) even after removing the athlete with the high number of infections ( n = 7 ) from the calculation . there were no statistically significant correlations for other variables ( table 3 ). although the ratio of iga1 : iga2 tended to be lower in swimmers who had no infection episodes during the 7 - month training season ( table 4 ) there were no statistically significant differences in the early or late phase samples collected from athletes who had no infections compared to those who had at least one infection episode . the results of this study of elite swimmers indicate that low concentrations of iga1 below 25 mg / l ( fig2 ) in saliva collected early in a 7 - month training season were selectively associated with a higher risk of respiratory tract infection episodes during the season . a previous study of respiratory infections in elite swimmers revealed that pre - season concentrations of total iga below 35 mg / l were associated with high numbers of infection episodes ( 16 ). in the current study the saliva samples were collected early in the training season but not prior to the commencement of the training season . iga1 represented approximately 80 % of the total iga concentration in this cohort of elite swimmers . in studies of normal non - exercising adults the proportion of iga1 in saliva is approximately 60 % ( 4 , 7 – 8 ). iga1 antibodies are primarily produced in response to protein antigens while iga2 antibodies are induced by carbohydrates and lipid - containing antigens ( 1 ). the fact that iga1 proteases produced by most mucosal pathogens can degrade iga1 antibodies , while iga2 antibodies are protected ( 1 ) is on importance when considering resistance to infections at mucosal surfaces . as iga1 is more susceptible to the proteases produced by mucosal pathogens ( 1 ), the higher proportion of iga1 and the occasional non - detectable levels of iga2 in this cohort of elite swimmers may combine to increase their risk of mucosal infections . despite a small portion of samples having iga2 concentrations below the assay detection limit there were no associations between iga2 concentration or the ratios of the subclasses with infection rates . as many of the elite swimmers in this study had been competing and training for several years , it is possible that the levels of iga1 in the early training phase saliva collections represent a cumulative effect over the years of training of exercise - induced iga1 suppression . the pan pacs championships ( swimming ) were held in sydney , australia between 22 aug . 1999 and 29 aug . 1999 . a further competition was held in canberra , australia the following week between 1 sep . 1999 and 5 sep . 1999 . the swimmers &# 39 ; iga levels and infection data were collected during the training phase leading up to and during these competitions . infection was defined as yes / no between defined time points of data collection ( table 5 ) after each saliva collection ( fig3 ). the number of subjects in the study varied at each time point ( see above ) due to unavailability of subjects on the day of saliva collection or exclusion of samples for various reason post analysis or infection logs not available . there were no associations between salivary igg , igm or albumin and infection at any of the time points studied . there was a trend for swimmers who had an infection to have lower salivary iga concentrations than those without infections ( tables 7 to 12 ) and at two time points the concentrations of pre - exercise salivary iga were statistically significantly lower in swimmers who had an infection ( table 13 ). the results suggest that the concentration of salivary iga in the early season training camp is the best predictor of later infection ( s ) during training leading up to a major competition . the median concentration for swimmers reporting no urtis after each time point decreased over the four month period ( 63 . 5 to 44 . 1 mg / l ) while the concentration for swimmers with infections showed little variation ( 32 . 7 to 37 . 3 mg / l ) ( fig4 ). this suggests swimmers who are susceptible to infections may already be below a critical threshold level of salivary iga at the beginning of the season and have little room for further reduction with training . the increase in salivary iga in samples collected on 9 aug . 1999 may be due to the fact that post this saliva collection there was an outbreak of gastrointestinal illness in the training camp , which may reflect mucosal stimulation in the elevated salivary iga , even prior to the symptoms of gastrointestinal infection . this case study presents data on the application of salivary immunoglobulin monitoring in managing an individual elite kayaker who was prone to recurrent debilitating respiratory illness that interfered with training and preparations for competitions . the male elite athlete , aged 25 years , had been competing at an international level for 10 years . five years prior to this study he contracted epstein - barr virus ( ebv ) followed by two episodes of upper respiratory tract illness ( urti ). over the proceeding years the average number of urti had increased to 5 – 6 episodes per year and were associated with fatigue . a medical examination excluded all recognised clinical causes of recurrent illness and associated fatigue . non - fasting , non - stimulated whole mixed saliva samples were collected by a standardised method ( 27 ), by the athlete before and again immediately after every exercise session over a 14 - day period . an in - house elisa measured the concentrations of iga , igg and igm and albumin concentrations were measured by rate nephelometry using a beckman array analyser ( beckman , brae , calif .). the athlete using the peats program ( 24 ) an adaptation of the method described by sharp ( 27 ), to calculate the intensity of each training session . the wilcoxon signed rank test for paired data was used to compare pre - and post - exercise session protein values . the mann - whitney u test was used to compare values between two selected time periods . spearmans correlation coefficients were used to assess the association between protein values and day of training and time of day of session . the salivary protein concentrations were all on average higher in the pre - exercise session samples compared with the post - exercise session samples ( fig5 ). the median differences were statistically significant ( table 14 ). the salivary iga concentrations fell significantly over the two week study ( correlation coefficient ( rs )=− 0 . 55 . p = 0 . 0002 ) for both the pre - exercise ( rs =− 0 . 61 , p = 0 . 003 ) and post - exercise ( rs =− 0 . 52 , p = 0 . 02 ) concentrations . there were no significant changes over time for the other salivary proteins . the magnitude of the session change ( pre minus post ) for salivary iga concentrations also decreased significantly over time ( rs =− 0 . 56 , p = 0 . 01 ), but this was not significant for the other salivary proteins . the magnitude of the session change for salivary iga concentrations , but not the other proteins , was positively correlated with the intensity of the exercise session ( rs = 0 . 53 , p = 0 . 02 ). the intensity of each exercise session decreased over the two - week study period ( rs =− 0 . 45 , p = 0 . 03 ) and was significantly higher ( p = 0 . 05 ) in the first four days ( median score ≈ 165 , range = 80 – 217 ) compared to the subsequent ten days of training ( median score = 110 , range = 56 – 185 ). the concentrations of iga in both the pre - exercise and post - exercise session samples were significantly higher ( p = 0 . 009 ) for the first four days ( median = 164 mg / l , range = 28 – 228 mg / l ) compared to the last ten days ( median = 51 mg / l , range = 30 – 72 mg / l ). the magnitude of the session change in concentrations for salivary iga were significantly greater ( p = 0 . 02 ) in the first four days ( median = 115 mg / l , range =− 44 – 137 ) compared to the next ten days ( median = 20 mg / l , range =− 30 – 47 mg / l ), being almost six times greater . this was primarily due to the drop in pre - exercise session salivary iga concentration during the fourth day of training and lack of subsequent recovery ( fig5 ). multiple exercise sessions were undertaken during some of the training days . the average percent recovery of the initial iga value for the day during the first three days was 74 % ( fig6 ). on day four the recovery for the second exercise session was only 20 %. once the initial iga concentrations fell below 50 mg / l ( days 8 – 13 ) the iga concentration showed little variation with additional training during the day ( fig5 & amp ; 6 ). an adequate level of immune protection at mucosal surfaces is important for resistance against infection ( 23 ). low levels of salivary iga have been associated with a risk of urti in cohorts of elite athletes ( 16 , 26 , 18 , 28 ). monitoring every episode of training in this case study allowed the identification of the exact exercise session that resulted in a significant suppression of the salivary iga concentration without subsequent recovery ( day 4 ). an important finding was the association between the early high intensity exercise with the magnitude of subsequent mucosal immune suppression , reflected by lower levels of salivary iga after the intense exercise period . this study highlights the benefit to the athlete and the coach if results of the salivary iga concentration assays are available rapidly . without the benefit of the knowledge of the mucosal immune suppression the athlete continued intensive training until restricted by a further episode of respiratory illness . in individual athletes who are prone to infection , or whose performance has declined , extensive monitoring of the athlete &# 39 ; s training program may assist with identifying training factors that contribute to the mucosal immune suppression and potential risk of infection . although the invention has been described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms . 1 . brandtzaeg p . humoral immune response patterns of human mucosae : induction and relation to bacterial respiratory tract infections . j . inf . dis . 1992 ; 165 ( suppl ): s167 – 76 . 2 . mazanec m b , medrud j g , kaetzel c h s and lamm m l . a three - 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