Celiac Disease
Conditions
Keywords
celiac disease, hookworm, probiotic
Brief summary
We have established that the hookworm Necator americanus (Na) dramatically alters the local and systemic immune landscape of the infected human host. Consistent with the principle of desensitisation, diet managed celiac disease subjects previously infected by us with Na will be invited to receive small incremental doses of gluten as pasta (3-25 mm straw of spaghetti) over 16 weeks. Each participant will then be carefully re-assessed to determine if it is appropriate to undertake a 12-week gluten challenge.
Detailed description
Hypothesis The adaptive Th2/regulatory profile imposed by Na will promote gluten tolerance following a micro-dose desensitising programme. Primary Aim: To determine the safety and efficacy of Na as a tolerising agent in celiac subjects Specific Aim 1. Undertake a therapeutic pilot study comparing mucosal histopathology before and after a gluten challenge, to be preceded by a programmed desensitising micro-challenge using Na as a tolerising agent. Specific Aim 2. Assess systemic and mucosal immune responses to gluten micro-challenge, Na infection, and gluten re-challenge throughout the pilot study, to be referenced against hookworm-naive people with treated and untreated celiac disease. Specific Aim 3. Utilising blood and tissue from hookworm-naive celiac disease volunteers, undertake in vitro studies focusing on the effects of Na-derived excretory/secretory (ES) products on gluten-stimulated gut mucosal cell apoptosis, cytokine and gene profiles.
Interventions
BIOLOGICALNecator americanus
Previously inoculated subjects will be further inoculated as previously undertaken with 20 3rd stage infective Na larvae (10 + 10 over 4 weeks). Four weeks after the 2nd inoculation, each participant will receive a micro-dose of gluten (10 mg daily) as pasta for 8 weeks, to be followed by a low-dose of gluten (50 mg daily) for 8 weeks. After this, a detailed assessment involving upper endoscopy and duodenal biopsy will be performed before deciding on an individual case basis that it is safe for the participant to proceed to challenge. A gluten challenge of 1 G (15-20 G of pasta or a ½ slice of standard white bread) twice weekly for 12 weeks will commence.
Sponsors
National Health and Medical Research Council, Australia
The Prince Charles Hospital
Study design
Allocation
NA
Intervention model
SINGLE_GROUP
Primary purpose
PREVENTION
Masking
SINGLE (Outcomes Assessor)
Eligibility
Sex/Gender
ALL
Age
18 Years to No maximum
Healthy volunteers
No
Inclusion criteria
* Previously enrolled adults who received an experimental hookworm infection with diet treated celiac disease.
Exclusion criteria
* Immune suppressive therapies
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Duodenal Villus Height:Crypt Depth | Week -24 to -36 | Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with H&E. Slides from both time-points were de-identified, shuffled and graded by Dr John Croese after which results from poorly orientated slides were verified by Dr Andrew Clouston. The Vh:Cd ratios were measured on 5 randomly selected well-orientated sites. The null hypothesis is that hookworm infection will not protect against mucosal damage following 12-week exposure to gluten in celiac disease. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Intraepithelial Lymphocyte Count | Week-24 and -36 | Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with anti-CD3. All slides were de-identified and graded by Dr John Croese. The IEL percentages were measured on 2 or more randomly selected well-orientated villi. The null hypothesis is that hookworm infection will not protect against mucosal IEL influx following 12-week exposure to gluten in celiac disease. |
| Number of Participants With 2 Points Increase in Marsh Score Post GC-1g | Longitudinal change between week-24 and week-36 | The Marsh score is a defined but qualitative assessment assigned a value to allow for comparison. The scores were evaluated by consensus between the primary (chief) investigator and the study pathologist. The Marsh score was graded 0, 1, 2, 3A (assigned-4), 3B (-5) and 3C (-6); rage 1-6 with normal=0 and severe inflammation=6. Because the scoring is vulnerable to artefact, only a 2-point shift was regarded as a significant intra-individual change. The scores were graded after week-36 on biopsies de-identified shuffled. An upward shift was interpreted to reflect a significant worsening of gluten-associated inflammation. The comparison reported evaluated changes from baseline (week-24) to post-low-dose gluten challenge (week-24; GC-1g). The objective for using the Marsh score was to identify individuals who might have experienced a severe worsening in pathology due to GC-1g that might not be reflected in the Vh:Cd group analysis. |
| Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) | Anti-tTG IU/mL levels pre-trial, mid-trial and after 3 gram/day gluten challenge | The trial was extended with pre-trial and mid-trial anti-tTG antibody levels used to compare with the post-trial levels. Anti-tTG is a serological measure of tissue transglutaminase-2 antibodies. In active celiac disease, levels are increased. In treated disease, levels are low (normal cut-off was \<15 IU/mL). A significant increase compared to baseline in tTG can be expected 2 weeks after consuming 3g of gluten daily for 2 weeks in people with celiac disease who have been maintaining a gluten-free diet, but who are not taking other treatment. |
Countries
Australia
Participant flow
Recruitment details
Of 20 potential candidates, 2 could not be contacted (address changed), 1 was not interested, and 5 were unavailable (travel-2, pregnancy-2 or study-1 commitments). Twelve enrolled in September 2012. 8 committed to 3 endoscopic procedures and 2 committed to two endoscopies.
Participants by arm
| Arm | Count |
|---|---|
| Gluten Micro-challenge Single arm, vertical. All twelve healthy adults enrolled subjects were successfully inoculated with hookworm and there was no serious adverse response. Individual hemoglobin levels were all normal and the group mean had significantly increased unexpectedly at completion of the study. Histology was not graded until after low-dose challenge but retrospectively confirmed enrollment Marsh scores of M0-8, M1-1, M2-2 and M3a-1. All participants were complying with a gluten-free diet, were symptomatically well and had a normal anti-tTG. | 12 |
| Total | 12 |
Withdrawals & dropouts
| Period | Reason | FG000 |
|---|---|---|
| High-dose Gluten Challenge | Withdrawal by Subject | 2 |
| Low-dose Gluten Challenge | Physician Decision | 2 |
Baseline characteristics
| Characteristic | Gluten Micro-challenge |
|---|---|
| Age, Categorical <=18 years | 0 Participants |
| Age, Categorical >=65 years | 1 Participants |
| Age, Categorical Between 18 and 65 years | 11 Participants |
| Age, Continuous | 53 years |
| Sex: Female, Male Female | 9 Participants |
| Sex: Female, Male Male | 3 Participants |
Adverse events
| Event type | EG000 affected / at risk |
|---|---|
| deaths Total, all-cause mortality | — / — |
| other Total, other adverse events | 2 / 12 |
| serious Total, serious adverse events | 0 / 12 |
Outcome results
Primary
Duodenal Villus Height:Crypt Depth
Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with H&E. Slides from both time-points were de-identified, shuffled and graded by Dr John Croese after which results from poorly orientated slides were verified by Dr Andrew Clouston. The Vh:Cd ratios were measured on 5 randomly selected well-orientated sites. The null hypothesis is that hookworm infection will not protect against mucosal damage following 12-week exposure to gluten in celiac disease.
Time frame: Week -24 to -36
Population: All 10 of 12 participants who completed micro-challenge successfully progressed and completed low-dose challenge.
| Arm | Measure | Group | Value (MEAN) |
|---|---|---|---|
| Necator Americanus, Gluten Challenge | Duodenal Villus Height:Crypt Depth | Baseline | 2.73 Ratio |
| Necator Americanus, Gluten Challenge | Duodenal Villus Height:Crypt Depth | Low-dose gluten | 2.73 Ratio |
p-value: 0.693t-test, 2 sided
Secondary
Intraepithelial Lymphocyte Count
Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with anti-CD3. All slides were de-identified and graded by Dr John Croese. The IEL percentages were measured on 2 or more randomly selected well-orientated villi. The null hypothesis is that hookworm infection will not protect against mucosal IEL influx following 12-week exposure to gluten in celiac disease.
Time frame: Week-24 and -36
| Arm | Measure | Group | Value (MEAN) |
|---|---|---|---|
| Necator Americanus, Gluten Challenge | Intraepithelial Lymphocyte Count | Baseline | 33.40 Percentage of epithelial cells |
| Necator Americanus, Gluten Challenge | Intraepithelial Lymphocyte Count | Low-dose gluten | 35.00 Percentage of epithelial cells |
p-value: =0.837t-test, 2 sided
Secondary
Number of Participants With 2 Points Increase in Marsh Score Post GC-1g
The Marsh score is a defined but qualitative assessment assigned a value to allow for comparison. The scores were evaluated by consensus between the primary (chief) investigator and the study pathologist. The Marsh score was graded 0, 1, 2, 3A (assigned-4), 3B (-5) and 3C (-6); rage 1-6 with normal=0 and severe inflammation=6. Because the scoring is vulnerable to artefact, only a 2-point shift was regarded as a significant intra-individual change. The scores were graded after week-36 on biopsies de-identified shuffled. An upward shift was interpreted to reflect a significant worsening of gluten-associated inflammation. The comparison reported evaluated changes from baseline (week-24) to post-low-dose gluten challenge (week-24; GC-1g). The objective for using the Marsh score was to identify individuals who might have experienced a severe worsening in pathology due to GC-1g that might not be reflected in the Vh:Cd group analysis.
Time frame: Longitudinal change between week-24 and week-36
Population: The outcome score was the number with a 2-point increase in Marsh 3 score post GC-1g.
| Arm | Measure | Value (NUMBER) |
|---|---|---|
| Necator Americanus, Gluten Challenge | Number of Participants With 2 Points Increase in Marsh Score Post GC-1g | 1 participants |
Comparison: The null hypothesis was that irrespective of hookworm infection, GC-1g would result in a 2-point or more deterioration in the Marsh score. A binomial (yes=deterioration or no=no deterioration) distribution was applied to pre- and post-GC-1g paired biopsies.p-value: =0.99Binomial distribution
Secondary
Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL)
The trial was extended with pre-trial and mid-trial anti-tTG antibody levels used to compare with the post-trial levels. Anti-tTG is a serological measure of tissue transglutaminase-2 antibodies. In active celiac disease, levels are increased. In treated disease, levels are low (normal cut-off was \<15 IU/mL). A significant increase compared to baseline in tTG can be expected 2 weeks after consuming 3g of gluten daily for 2 weeks in people with celiac disease who have been maintaining a gluten-free diet, but who are not taking other treatment.
Time frame: Anti-tTG IU/mL levels pre-trial, mid-trial and after 3 gram/day gluten challenge
Population: 2 of 12 participants did not progress past baseline micro-challenge, 2 of 10 participants did not progress past low-dose challenge.
| Arm | Measure | Group | Value (MEAN) |
|---|---|---|---|
| Necator Americanus, Gluten Challenge | Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) | Baseline | 4.12 International Units/mL |
| Necator Americanus, Gluten Challenge | Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) | Low-dose gluten | 2.99 International Units/mL |
| Necator Americanus, Gluten Challenge | Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) | After 3 G gluten daily | 2.11 International Units/mL |
p-value: =0.005Mixed effects model.