Clinical Drug Experience Knowledgebase

A Phase 2a, Randomized, Double Blinded, Placebo Controlled, Study Evaluating Immunity and Gluten-sensitivity by Inoculating Celiac Disease Patients With the Human Hookworm Necator Americanus.

Background and aims: A number of lines of evidence suggest that the disappearance of helminths from human populations in developed countries may be responsible for the upsurge in autoimmune diseases, the so-called hygiene hypothesis. Using a small cohort of healthy subjects with quiescent celiac disease, our aim is to test if the ubiquitous hookworm (HW) of humans, Necator americanus, inhibits immune responsiveness to a gluten challenge (GC). As well as being of potential benefit to people with gluten intolerance, this study will provide the opportunity to undertake detailed investigation at the mucosal level of the host-parasite interaction, and the underlying immune response, and by extension, the potential of nematode infection to modulate the inflammatory response in IBD. Unlike experimental helminth infections in either animals genetically predisposed to colitis or in clinical IBD (where a range of complicating cofactors are present), this study addresses what happens in healthy humans who do not have a background of helminth exposure, and who are not currently compromised by either inflammation or immunosuppressive drugs.

Methods: Twenty healthy adults with well-documented celiac disease (DQ2 phenotype) compliant with a gluten-free (GF) diet for ≥ 6 months (based on history and a normal tissue transglutaminase [tTG]) will be recruited and randomly assigned to two groups of ten. Ten will be inoculated with hookworm (HW) larvae and 10 will serve as uninfected controls. Study subjects and Investigators will be blinded to allow comparison of disease activity and immune profiles. HW larvae will be cultured from feces supplied by a volunteer donor, previously infected for this purpose. Conventional endoscopy and biopsy will be performed twice per subject, before and after oral and rectal GCs (2x50g slices of wheat bread twice daily for 3-5 days and 6g of gluten in a 40ml slurry instilled into the rectum per endoscope, respectively). Rectal biopsy will be undertaken 4 hr and duodenal biopsy collected six days following GC; blood will be collected before and after acquiring hookworm infection and on day six following GC. Thus, four data sets will be accrued for comparison: ten GF celiac-subjects before HW infection; 10 GF celiac-s after HW infection; ten GC celiac-s before HW infection; and 10 GC celiac-s after HW infection.

Safety: A large body of observational data documenting the safety of experimental hookworm infection is available, both from our own and studies by others. Two of our researchers have maintained infections including occasional "top-up" inoculations for three years without ill effects.

Outcome Measures: These will include: subject symptom diary, full blood analysis, C-reactive protein, total and specific IgE and serum tryptase activity. The duodenal (Marsh classification) and rectal histology will be graded by a single pathologist. Goblet cells and mucosal T cells will be stained to aid quantification of responses. Peripheral blood mononuclear cells (PBMCs) and mucosal lymphocytes from intestinal biopsies will be grown ex vivo and challenged with gluten antigen immunodominant peptide (alpha-gliadin 57-73 Q65E, QLQPFPQPELPYPQPQS). Cell proliferation and cytokine profiles in response to HW and gluten antigens will be measured from PBMCs and intestinal biopsies. Varying the timing of the inoculations may provide worthwhile direction on the importance of the Th2 response only if there is a profound difference between newly established (Th2-dominant) versus mature (Th-neutral) parasite infections, as suggested by our earlier work with experimental human infections. Levels of transcription of genes of interest will be assessed using quantitative real time PCR and microarrays.

Outcomes: The null hypothesis is that Necator americanus does not change the immune process sufficiently to suppress gluten sensitivity in people with celiac disease. The measured outcomes reflect the activity of celiac disease, including the severity of mucosal inflammation, and the character and intensity of the immune processes. This study though is as much about IBD. Celiac disease is not IBD, but this model of IBD affords a previously unexplored opportunity to test quasi autoimmune responses in the duodenum and rectum to a specific antigen, one that can be introduced or excluded on demand. The immune profiling will focus on the characteristics that drive inflammation in IBD providing a clear insight as to the potential of helminths in Crohn's disease and ulcerative colitis.

Extension Study; Control patients invited to enroll in an extension of the study; each to be inoculated, challenged and investigated as per the original hookworm cohort (as above).

Low dose gluten challenge Study: This trial extension seeks to establish if hookworm infection might improve tolerance to small amounts of gluten in patients with celiac disease. The study is open. It utilises celiac patients already infected with hookworm and in whom the blood and mucosal baseline characteristics have been carefully documented. The gluten exposure will apply doses that have been demonstrated by others in a trial setting to be safe and well-tolerated. Effectiveness of hookworm infection to mitigate gluten intolerance will be measured by the quantifiable changes that occur in biopsies previously taken (pre-challenge) compared with tissue collected post-challenge.Histology will be performed as previously described. Biopsies are to be fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 μm) will be stained with haematoxylin and eosin (H&E) and immunostained with anti-CD8 (or anti-CD3 depending on availability) antibodies (Novocastra Laboratories Ltd). The intraepithelial lymphocytes (IELs) per 100 nucleated enterocytes (100NE) will be counted at 24 randomly selected sites between the villous tip and the base of the crypt (Vh/Cd) in each biopsy. Individual and collective outcomes on tissue collected after hookworm infection, but whilst on a gluten free diet, will be compared with those from tissue collected after the pasta challenge.