Advanced Technology in Food Production

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

Citation:

Le LQ, Mahler V, Scheurer S, Foetisch K, Braun Y, Weigand D, Enrique E, Lidholm J, Paulus KE, Sonnewald S, Vieths S, Sonnewald U. Yeast profilin complements profilin deficiency in transgenic tomato fruits and allows development of hypoallergenic tomato fruits. FASEB J. 2010; 24(12): 4,939-4,947. PubMed ID: 20709910

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 complement Lyc e 1-deficient tomato plants by a non-plant profilin to examine whether the physiological function of profilin could be restored without introducing a novel allergen.

Inclusion Criteria:

Patients with tomato allergy were selected on the basis of their clinical history (oral allergy syndrome, gastrointestinal or contact urticaria on ingestion of tomato fruits) and positive response in prick-to-prick tests performed with tomato fruits or in vitro basophil histamine release assays
Sera from non-allergic individuals served as negative controls
Provided written informed consent.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment

Patients were recruited in Germany and Spain.

Design

Non-randomized crossover trial.

Blinding Used

Implied with measurements.

Intervention

Transgenic plants were generated and tested by RT-PCR and immunoblotting
Allergenicity of yeast profilin and transgenic fruits was investigated by IgE binding, basophil activation and skin-prick tests.

Data Collection Summary:

Timing of Measurements

All measurements were completed once.

Dependent Variables

Immunoblotting:
- For immunological analysis, the recombinant tomato PFN and PFYI were expressed in Escherichia coli and purified by affinity chromatography
- For IgG immunoblotting, total protein was extracted from wild-type (WT) and transgenic tomato fruits, separated on SDS-PAGE and blooted onto nitrocellulose membranes.
IgE binding: Experimental ImmunoCAP tests carrying allergens Lyc e 1 or PFYI were prepared according to Marknell DeWitt et al and assays were performed using an ImmunoCAP
Basophil activation: The basophil histamine release assay was performed with stripped and passively sensitized basophils from a nonallergic donor
Skin prick tests.

Independent Variables

Yeast profilin
Transgenic fruits.

Description of Actual Data Sample:

Initial N: N=16 tomato-allergic patients
Attrition (final N): N=16 tomato-allergic patients, sex not described
Location: Germany.

Summary of Results:

Key Findings

Among tomato-allergic patients pre-selected for sensitization to Lyc e 1 none showed significant reactivity to yeast profilin
Yeast profilin did not indece mediator release, and co-expression of yeast profilin did not enhance the allergenicity of Lyc e 1-reduced fruits
Simultanous co-expression of yeast profilin allows silencing of tomato profilin and generation of viable plants with Lyc e 1-deficient tomato fruits.

Author Conclusion:

This proof-of-concept study presented here provides a strategy to enable transgenic reduction or elimination of functionally important allergens from fruits and vegetables while maintaining biological viability by means of complementation with non-allergenic orthologous proteins.

Funding Source:

Not-for-profit

German Research Foundation grants DFG-Vi 165/6-1, DFG-Ma 1997/3-1 and DFG-So 300/13-1

Other non-profit:

Other:

Deutsche Forschungsgemeinschaft (DFG) grant PAK 132

Reviewer Comments:

Small sample size
Sample was not well described
Statistical analysis was not described.

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?	No
	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?	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?		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?	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?	N/A
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)?	N/A
	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?		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