ATFP: Human Consumption of Plant Foods Produced Using Genetic Engineering (GE) Technologies (2015)
Mondal HA, Chakraborti D, Majumder P, Roy P, Roy A, Bhattacharya SG, Das S. Allergenicity assessment of Allium sativum leaf agglutinin, a potential candidate protein for developing sap sucking insect resistant food crops. PLoS One, 2011; 6 (11): e27,716.PubMed ID: 22110739
D - Click here for explanation of classification scheme.
To determine the allergenic and non-allergenic source of the gene by analyzing sequence homology to reported allergens (food and environmental); by serum screening for cross-reactivity with sera from patients who are allergic to materials that are broadly related to the source material of the protein; by assessing the pepsin resistance of the gene product; and by monitoring digestibility of the protein in animal models were considered to address the issue based on the guidelines of FAO/WHO.
- Diagnosed allergic patients and allergic to foods of plant origin, such as brinjal, spinach, drumstick, pumpkin and okra and each of reported that his or her regular diet contained garlic
- Non-atopic as control subjects
- Male to female ratio 7:10
- Signed consent form.
- Perennial or severe asthma, pregnancy or lactation and malignancy or other systemic diseases during sera collection
- To avoid the masking of possible symptoms, the use of corticosteroids and antihistamines was prohibited.
Description of Study Protocol:
- Recruitment: Allergic patients were selected from the outpatient clinic of the Allergy Department, Institute of Child Health, Kolkata, India, on the basis of clinical history and diagnosis
- Design: Non-controlled trial
- Blinding used: Implied with measurements
- Intervention: Not applicable
- Statistical analysis: None described.
Data Collection Summary:
Timing of Measurements
All measurements made in selected subjects.
- ASAL sequence
- Serum IgE on mammalian systems
- Pepsin resistance and thermostability of the protein.
Independent VariablesASAL proteins in allergic and control patients.
Description of Actual Data Sample:
- Initial N: 216 allergic patients (male-to-female ratio, 7:10) and 25 non atopic subjects as control (male-to-female ratio, 7:9)
- Attrition (final N): As above
- Mean age
- Allergic patients: 33.4 years
- Control subjects: 32.8±0.9 years.
- Ethnicity: Asian Indian
- Other relevant demographics: Not reported
- Anthropometrics: Not reported
- Location: Kolkata, India.
Summary of Results:
- No significant homology to the ASAL sequence was detected when compared to known allergenic proteins
- The ELISA of blood sera collected from known allergy patients also failed to show significant evidence of cross-reactivity
- In vitro and in vivo assays both indicated the digestibility of ASAL in the presence of pepsin in a minimum time period
- ASAL showed single bands of approximately 12.2kDa
- No significant matches (35% homology or at a stretch of six amino acids) were observed with any of the known allergens of either the Swiss-Prot or the WHO-International Union of Immunological Societies (IUIS) database
- Using a setting of a 29% cut-off value or above, no allergens from Swiss-Prot or WHO-IUIS were matched to the ASAL sequence. When an exact hit of six amino acids in a sequence in the databases (SwisProt and WHO-IUIS), by analysis of 107 windows [(112-6)+1=107] was searched, no significant matches were found.
- The highest percentage of identity was obtained at 22.5 with two allergens (Peptidase 1 of the American house dust mite and polygalacturonase of timothy grass) from the WHO-IUIS and Swiss-Prot database. The P/N value for ASAL did not exceed 1.25.
- ASAL was not detected by SDS-PAGE and Coomassie Brilliant Blue staining after two minutes of incubation in pepsin-amended SGF in one mcg ASAL with 45.6 units of pepsin and in one mcg ASAL with 10 units of pepsin. A western blot assay (one mcg ASAL with 10 units of pepsin) to detect the digestion profile after different time points showed no bands after two minutes of ASAL incubation with pepsin.
- In a thermal stability experiment, ASAL was stable up to 45°C, but labile at 55°C upon incubation for 30 minutes, which resulted in loss of agglutination activity. By optimizing the temperature across the range of 40° to 55°C, ASAL lost biological activity by a 30-minute incubation at 50°C.
- ASAL does not possess any apparent features of an allergen
- This is the first report regarding the monitoring of the allergenicity of any mannose-binding monocot lectin having insecticidal efficacy against hemipteran insects
- Both in vitro and in vivo experiments showed that ASAL was easily digested and thus the possibility of this lectin being allergenic is very low. This result was further confirmed by in vitro tests that showed no IgE-mediated hypersensitivity reactions.
- According to the FAO/WHO decision tree, when negative results are obtained in both the pepsin digestibility assay and animal model experiments, the expressed protein is unlikely to become an allergen. Thus, ASAL can be an important component of an integrated pest management program as a safe insecticidal lectin.
|University/Hospital:||Bose Institute, Kolkata, India|
- Exposure time may influence results
- It is not a representative sample and their history of exposure and onset of allergic reaction were not reported in their medical history
- Details of all allergic reactions are not provided in the report
- They are outpatient clinic patients, not a well controlled sample
- No medical and family history of allergy reported
- Unknown whether the allergy is by birth or on set diagnosis
- Their current demographic details and prescriptions are not reported
- Appropriate statistical analysis was not reported to compare clinical chemistries between allergic patients and non atopic.
Quality Criteria Checklist: Primary Research
|1.||Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)||N/A|
|2.||Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?||Yes|
|3.||Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?||Yes|
|4.||Is the intervention or procedure feasible? (NA for some epidemiological studies)||N/A|
|1.||Was the research question clearly stated?||Yes|
|1.1.||Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?||Yes|
|1.2.||Was (were) the outcome(s) [dependent variable(s)] clearly indicated?||Yes|
|1.3.||Were the target population and setting specified?||Yes|
|2.||Was the selection of study subjects/patients free from bias?||No|
|2.1.||Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?||???|
|2.2.||Were criteria applied equally to all study groups?||Yes|
|2.3.||Were health, demographics, and other characteristics of subjects described?||No|
|2.4.||Were the subjects/patients a representative sample of the relevant population?||No|
|3.||Were study groups comparable?||Yes|
|3.1.||Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)||Yes|
|3.2.||Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?||Yes|
|3.3.||Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)||Yes|
|3.4.||If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?||N/A|
|3.5.||If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)||N/A|
|3.6.||If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?||N/A|
|4.||Was method of handling withdrawals described?||Yes|
|4.1.||Were follow-up methods described and the same for all groups?||Yes|
|4.2.||Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)||Yes|
|4.3.||Were all enrolled subjects/patients (in the original sample) accounted for?||Yes|
|4.4.||Were reasons for withdrawals similar across groups?||Yes|
|4.5.||If diagnostic test, was decision to perform reference test not dependent on results of test under study?||N/A|
|5.||Was blinding used to prevent introduction of bias?||Yes|
|5.1.||In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?||N/A|
|5.2.||Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)||Yes|
|5.3.||In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?||N/A|
|5.4.||In case control study, was case definition explicit and case ascertainment not influenced by exposure status?||N/A|
|5.5.||In diagnostic study, were test results blinded to patient history and other test results?||N/A|
|6.||Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?||N/A|
|6.1.||In RCT or other intervention trial, were protocols described for all regimens studied?||N/A|
|6.2.||In observational study, were interventions, study settings, and clinicians/provider described?||N/A|
|6.3.||Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?||N/A|
|6.4.||Was the amount of exposure and, if relevant, subject/patient compliance measured?||N/A|
|6.5.||Were co-interventions (e.g., ancillary treatments, other therapies) described?||N/A|
|6.6.||Were extra or unplanned treatments described?||N/A|
|6.7.||Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?||N/A|
|6.8.||In diagnostic study, were details of test administration and replication sufficient?||N/A|
|7.||Were outcomes clearly defined and the measurements valid and reliable?||Yes|
|7.1.||Were primary and secondary endpoints described and relevant to the question?||Yes|
|7.2.||Were nutrition measures appropriate to question and outcomes of concern?||Yes|
|7.3.||Was the period of follow-up long enough for important outcome(s) to occur?||Yes|
|7.4.||Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?||Yes|
|7.5.||Was the measurement of effect at an appropriate level of precision?||Yes|
|7.6.||Were other factors accounted for (measured) that could affect outcomes?||Yes|
|7.7.||Were the measurements conducted consistently across groups?||Yes|
|8.||Was the statistical analysis appropriate for the study design and type of outcome indicators?||No|
|8.1.||Were statistical analyses adequately described and the results reported appropriately?||No|
|8.2.||Were correct statistical tests used and assumptions of test not violated?||No|
|8.3.||Were statistics reported with levels of significance and/or confidence intervals?||No|
|8.4.||Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?||N/A|
|8.5.||Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?||No|
|8.6.||Was clinical significance as well as statistical significance reported?||No|
|8.7.||If negative findings, was a power calculation reported to address type 2 error?||No|
|9.||Are conclusions supported by results with biases and limitations taken into consideration?||No|
|9.1.||Is there a discussion of findings?||Yes|
|9.2.||Are biases and study limitations identified and discussed?||No|
|10.||Is bias due to study's funding or sponsorship unlikely?||Yes|
|10.1.||Were sources of funding and investigators' affiliations described?||Yes|
|10.2.||Was the study free from apparent conflict of interest?||Yes|