Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03449537 |
| Other study ID # |
284/17 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
April 1, 2018 |
| Est. completion date |
December 31, 2020 |
Study information
| Verified date |
February 2024 |
| Source |
Federico II University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Cow's milk allergy (CMA) affects up to 3% of European children. In the absence of an
alternative to cow's milk, the management of CMA is based on the use of safe, affordable and
nutritionally adequate formulas. In Scientific Societies Guidelines, extensively hydrolyzed
casein formula (EHCF) is considered as safe first line approach for the treatment of children
with CMA, whereas amino acid-based formula (AAF) is considered as second line strategy in
children reacting to EHCF or as first line approach in children with CMA-induced anaphylaxis.
Few and not recent studies, involving a poorly characterized study population, suggested that
up to 10% of CMA children could react to the extensively hydrolysed formulas.
It has been demonstrated that EHCF supplemented with L.rhamnosus GG (LGG) maintains
hypoallergenic status and that is able to accelerate oral tolerance acquisition in children
with CMA comparing with other formulas.
The purpose of this study is to investigate the feasibility of a "step-down" approach in
children affected by Immunoglobulin E (IgE)-mediated CMA with the aim to evaluate the effects
of EHCF + LGG on oral tolerance acquisition and on immune response and gut microbiota
shaping.
Description:
60 Immunoglobulin E-mediated CMA children, consecutively observed at tertiary Centers for
Food Allergy, who will meet the inclusion criteria will be invited to participate to the
study. Anamnestic, demographic, anthropometric and clinical data, as well as information on
socio-demographic factors, family and living conditions, parental history of allergic
diseases, number of siblings, and pet ownership will be obtained from the parents of each
infant and recorded in a clinical database.
Then in all subjects an oral food challenge with EHCF + LGG will be performed. Only subjects
with negative oral food challenge will be randomly allocated to one of the two groups of
dietary interventions for a 12 months follow up period: group 1 received AAF , and group 2
received EHCF + LGG.
Effective use of the formula will be evaluated during the study by dieticians counselling
parents about issues that could arise during the elimination diet. Parents or caregivers will
be asked to keep a daily record of formula use. The amount prepared (millimetres of water and
number of formula spoons) and amount left after each consumption will be recorded in a diary
to assess the amount consumed by the child.
At enrolment, after 6 and 12 months body growth will be assessed by body weight, body length
and head circumference measured at enrolment, after 6 and 12 months of follow-up with
reference to growth charts. Unscheduled visits will be made if necessary.
In addition at enrolment, after 6 and 12 months, the investigators will perform:
1. All Oral food challenge procedures will be performed in double blind fashion in 2
consecutive days. Full emergency equipment and drugs (epinephrine, antihistamines,
steroids) will be at hand. The challenge will be stopped upon the appearance of clinical
symptoms or when the highest dose will be reached. The child will be observed for 2 h,
and then discharged.
2. Skin prick test (whole milk, casein, α-lactalbumin, β-lactoglobulin): allergens and
fresh milk will be applied to the patients volar forearm: cow's milk (CM) containing
3.5% fat. Skin prick tests were performed using a 1-mm single peak lancet (ALK,
Copenhagen, Denmark), with histamine dihydrochloride (10 mg/ml) and isotonic saline
solution (NaCl 0.9%) as positive and negative control, respectively. Reactions will be
recorded on the basis of the largest diameter (in millimetres) of the wheal and flare at
15 min. The SPT result will be considered "positive" if the wheal was 3 mm or larger,
without reaction of the negative control.
3. Total IgE and specific IgE and Immunoglobulin G 4 against proteins and epitopes of cow's
milk: we will perform a venous blood sample; serum of the patients will be collected
using tube serum separator tubes and was obtained by centrifugation for 10-15 minutes.
Serum will be flash frozen and stored at -80 °C until further analysis. From serum,
total IgE and specific IgE and IgG4 against proteins and epitopes of cow's milk will be
analyzed with enzymatic immunoassay.
4. Gut microbiota composition: a stool sample will be collect and immediately frozen to
-80°C and stored until further analysis. Total genomic DNA (gDNA) will be isolated from
fecal material using a specific DNA. Isolation kit and gut microbiota composition will
be analysed using an approach for bacteria and an internal transcribed spacer region
sequencing approach (High-throughput sequencing).
5. Short chain fatty acids (SCFAs) fecal and serum production: a stool sample and serum
will be collect. One gram of fecal samples will be weighed, diluted 1:2 in sterile
phosphate-buffered saline solution, and homogenized. Supernatants will be then obtained
by centrifugation (10 000g, 30 minutes, 4°C), filtered through 0.2-μm filters and stored
at -80°C until analysis. Serum of the patients will be collected using tube serum
separator tubes and was obtained by centrifugation for 10-15 minutes. Serum will be
flash frozen and stored at -80 °C until further analysis. Analysis of SCFAs will be
performed using gas chromatography-mass spectrometry (MS) to measure the concentrations
of acetic, propionic, and butyric acid in fecal samples.
6. Serum level of interleukin (IL)-4, IL-5, IL-13, IL-10, interferon (IFN)-γ: we will
perform a venous blood sample; serum of the patients will be collected using tube serum
separator tubes and was obtained by centrifugation for 10-15 minutes. Serum will be
flash frozen and stored at -80 °C. From serum, IL-4, IL-5, IL-13, IL-10, IFN-γ will be
determined by ELISA (specific kit for each cytokine).
7. Methylation status of the promoter region of genes involved in IgE-mediated allergy,
IL-4, IL-5, IL-13, IL-10 and IFN-γ and of FoxP3+: Venous blood will be obtained from the
patients and DNA will be extracted from leukocytes using DNA Extraction Kit. Extracted
DNA will be modified with sodium bisulfite using the Methylation Gold Kit (ZYMO Research
Co.) according to the manufacturer's instructions. The converted DNA will be stored at
-70°C until used. Methylation analyses will be performed using High resolution melting
Real Time (LightCycler® 480, Roche Applied Science). The results will be confirmed by
direct sequencing (Sanger method modified: ddNTPs labeled with four different
fluorophores).
