Advanced Technology in Food Production

ATFP: Human Consumption of Plant Foods Produced Using Genetic Engineering (GE) Technologies (2015)

Citation:

Liao EC, Chen JT, Chao ML, Yu SC, Chang CY, Chu WS, Tsai JJ. Nonadverse effects on allergenicity of isopentenyltransferase-transformed broccoli. J Investig Allergol Clin Immunol. 2013; 23(2): 112-119. PubMed ID: 23654078

Study Design:

Non-Randomized Crossover Trial

Class:

C - Click here for explanation of classification scheme.

Quality Rating:

NEUTRAL: See Quality Criteria Checklist below.

Research Purpose:

To investigate the allergenicity of isopentenyltransferase (ipt)-transformed broccoli compared with non-GM broccoli.

Inclusion Criteria:

Individuals who attended outpatient clinics belonging to the Division of Allergy, Immunology and Rheumatology at the Taichung Veterans General Hospital
The blood sera of patients with specific IgE to broccoli and the house dust mite (HDM)
Written informed consent.

Exclusion Criteria:

Not having blood sera specific IgE to broccoli and house dust mite (HDM).

Description of Study Protocol:

Recruitment

Individuals who attended outpatient clinics belonging to the Division of Allergy, Immunology and Rheumatology

Design

Non-randomized crossover trial.

Blinding Used

Implied with measurements.

Intervention

Sera from allergic individuals were used to identify the allergenicity of GM and non-GM broccoli
Immunoglobulin (Ig) binding of different lines of GM and non-GM broccoli was identified using immunoblotting, enzyme-linked immunosorbent assay, and the histamine release assay
Healthy volunteers with negative responses to broccoli and D pteronyssinus by specific IgE measurements were selected as negative controls.

Statistical Analysis

Statistical comparisons of IgE levels between non-GM broccoli and GM broccoli were performed using the unpaired T-test
Two-tailed P values of lower than 0.05 were considered statistically significant
Correlation between levels of broccoli-specific IgE and total IgE (IU per ml) in serum samples from patients with broccoli allergy (allergic group: A1–A13) was assessed using the Spearman rank correlation test.

Data Collection Summary:

Timing of Measurements

Blood samples of 5ml were collected in serum separator tubes and processed within four hours.

Dependent Variables

Allergen specific IgE was measured by ImmunoCap using the UniCAP 250 system, following the supplier's instructions
IgE antibodies to broccoli and D pteronyssinus were measured using the Phadia ImmunoCap system, the assay procedure was performed automatically and results were calibrated against the WHO standard for IgE
The protein solution of broccoli extract was mixed with 4x sodium dodecyl sulfate (SDS) sample buffer and incubated for 10 minutes (300mcg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-Page) for one hour at 70V and for one hour, 30 minutes at 120V. The separated proteins were electrophoretically transferred onto a polyvinyl difluoride membrane for two hours at 350mA.
Human IgE was detected with alkaline phosphatase-conjugated anti-human IgE secondary antibody r-nitrophenyl phosphate.

Independent Variables

Specific IgE to broccoli: Sera from allergic individuals were used to identify the allergenicity of GM and non-GM broccoli
Immunoglobulin (Ig) binding of different lines of GM and non-GM broccoli was identified using immunoblotting, enzyme-linked immunosorbent assay and the histamine release assay
Healthy volunteers with negative responses to broccoli and D pteronyssinus by specific IgE measurements were selected as negative controls.

Description of Actual Data Sample:

Initial N: N=185 individuals who attended outpatient clinics belonging to the Division of Allergy, Immunology and Rheumatology at the Taichung Veterans General Hospital were enrolled in this study and underwent further testing. Based on the specific IgE results, 15 individuals were recruited for further analysis.
Attrition (final N): N=15 subjects. A total of 13 broccoli-allergic patients (seven males, six women) and two healthy volunteers
Age: Mean age 28 years
Location: Taichung, Taiwan.

Summary of Results:

Key Findings

Positive reactions to broccoli were observed in 7.02% of individuals
Specific IgE to broccoli and total IgE from allergic individuals were well correlated
The different tests performed showed no significant differences in the allergenicity of conventionally raised and GM broccoli, indicating the absence of unexpected effects on allergenicity in ipt-transformed plants
Using Western Blot analysis, we detected heterogenous IgE-reactive allergenic components in broccoli-allergic sera, but no significant differences between GM and non-GM broccoli were observed in serum from the same patients.

Author Conclusion:

There are no differences between GM (ipt-transformed) broccoli and non-GM broccoli, as determined by specific IgE in sera from broccoli-allergic patients. This indicates that there were no unexpected effects on allergenicity in this GM broccoli.

Funding Source:

Government:

Food and Drug Administration, Grant # DOH-FS031 and FDA99-FS033

Reviewer Comments:

A small number of broccoli-allergic subjects was studied. The strength of this study is that the findings show that the most predominant allergen in the study population was HDM (positive IgE to D pteronyssinus in 68.6% of individuals).

Quality Criteria Checklist: Primary Research
Relevance Questions
	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)	Yes
	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)	Yes

Validity Questions
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?	Yes
	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?		N/A
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	N/A
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	N/A
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	N/A
	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?	N/A
	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?		Yes
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	Yes
	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?	Yes
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	Yes
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	Yes
	6.6.	Were extra or unplanned treatments described?	Yes
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	Yes
	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?		Yes
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	Yes
	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)?	Yes
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	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?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
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