John Croese

Effect of Hookworm Infection on Wheat Challenge in Celiac Disease – A Randomised Double-Blinded Placebo Controlled Trial

Background and Aims The association between hygiene and prevalence of autoimmune disease has been attributed in part to enteric helminth infection. A pilot study of experimental infection with the hookworm Necator americanus was undertaken among a group of otherwise healthy people with celiac disease to test the potential of the helminth to suppress the immunopathology induced by gluten. Methods In a 21-week, double-blinded, placebo-controlled study, we explored the effects of N. americanus infection in 20 healthy, helminth-naïve adults with celiac disease well controlled by diet. Staged cutaneous inoculations with 10 and 5 infective 3rd stage hookworm larvae or placebo were performed at week-0 and -12 respectively. At week-20, a five day oral wheat challenge equivalent to 16 grams of gluten per day was undertaken. Primary outcomes included duodenal Marsh score and quantification of the immunodominant α-gliadin peptide (QE65)-specific systemic interferon-γ-producing cells by ELISpot pre- and post-wheat challenge. Results Enteric colonisation with hookworm established in all 10 cases, resulting in transiently painful enteritis in 5. Chronic infection was asymptomatic, with no effect on hemoglobin levels. Although some duodenal eosinophilia was apparent, hookworm-infected mucosa retained a healthy appearance. In both groups, wheat challenge caused deterioration in both primary and several secondary outcomes. Conclusions Experimental N. americanus infection proved to be safe and enabled testing its effect on a range of measures of the human autoimmune response. Infection imposed no obvious benefit on pathology. Trial Registration ClinicalTrials.gov NCT00671138

Human enteric infection with Ancylostoma caninum: hookworms reappraised in the light of a “new” zoonosis

Recent studies in northeastern Australia indicate that enteric infection with Ancylostoma caninum is a leading cause of human eosinophilic enteritis. Much more frequent accompaniments of this infection are obscure abdominal pain with or without blood eosinophilia, while a large part of the population is probably infected asymptomatically. These conclusions are based on extensive serological investigations in patients and control subjects, as well as 15 cases in which single, adult hookworms were identified in situ in patients. In no case has more than one worm been identified, and none has been fully mature, so the infections have never been patent. Aphthous ulcers of the terminal ileum, caecum and colon have been seen in association with this infection and have also been observed in almost 5% of patients who are colonoscoped in north Queensland. Serodiagnosis has relied on an IgG and IgE ELISA using excretory-secretory antigens from adult A. caninum, but Western blot using these antigens to identify IgG4 antibodies to a protein of molecular weight 68 kDa (Ac68) promises to be more specific and sensitive. However, identical antigens appear to be secreted by the anthropophilic hookworms as well. The clinical, public health and biological significance of these findings are discussed in detail.

The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation

Summary Intestinal helminths are potent regulators of their host’s immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions.

Suppression of Inflammatory Immune Responses in Celiac Disease by Experimental Hookworm Infection

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)γ and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-γ and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.

Human Enteric Infection with Canine Hookworms

Since the 1920s, the common dog hookworm, Ancylostoma caninum, has been known to cause cutaneous disease in persons who have been exposed to infective larvae [1]. Sporadic accounts of its occurrence as an adult worm in the human intestine have been reported from the Philippines [2], South America [3-5], and Israel [6]; the occasional adult A. caninum was found among numerous human hookworm specimens (usually Necator americanus) recovered either after anthelminthic treatment or at autopsy. In no case was the parasite implicated in clinical disease or shown to be fully developed and producing eggs. In 1988, a published series of 33 Australian patients with abdominal pain and blood eosinophilia included a patient with ileal obstruction caused by an eosinophilic phlegmon to which a hookworm was attached [7]. Histologic sectioning precluded specific identification of the parasite. Because human hookworm infection, a persistent Third World problem, is not endemic in urban Australia and has virtually disappeared from the aboriginal communities of Queensland [8, 9], it was speculated that the cases had a common cause, possibly a dog hookworm. Shortly afterwards, two other cases of infection with a single adult A. caninum were reported, both detected at colonoscopy and one associated with eosinophilic ileitis [10, 11]. We developed an enzyme-linked immunosorbent assay (ELISA) and Western blot [12, 13] that are helpful in the diagnosis of abdominal pain with blood eosinophilia and indicate that the parasite is a common cause of this syndrome in eastern Australia. We describe six additional cases of human enteric infection with A. caninum and summarize pertinent features, including serologic findings, of all nine Australian cases so far identified. Methods From 1985 to 1992, five patients were diagnosed by us and four others were referred with single hookworm infections. They lived either in Townsville (latitude, 19 degrees S) or Brisbane (latitude, 27 degrees S). In each case, the medical records and biopsy tissue were provided by the attending clinician and pathologist. At least one serum sample collected within 4 weeks of diagnosis and the formalin- or alcohol-fixed worms were also available from all except one patient (patient 1). Measurements Clinical and demographic data were sought from the records and by interview. A complete blood examination, total serum IgE assay, and fecal microscopy were done in commercial or teaching hospital laboratories. Blood eosinophilia was ascribed to a count higher than 0.50 109/L, and the upper normal value for IgE was 288 g/L. Tissue biopsy specimens were examined and reported by histopathologists not associated with the study. Hookworms were examined microscopically by experienced parasitologists and were drawn and measured using standard techniques. Species identification was based on accepted criteria, which included overall length; esophageal length; body width; number of teeth in the buccal cavity; and, in male worms, length of spicules and structural details of the copulatory bursa [14, 15]. Sera were stored at 15C and tested, among numerous control sera, for IgG and IgE antibodies by one of the investigators who was unaware of the patient histories. The ELISAs incorporated excretory-secretory antigens from adult A. caninum [12]. In the Western blots, these antigens were separated electrophoretically on polyacrylamide gels and transferred to nitrocellulose paper, which was then incubated with patient sera diluted 1:10 [13]. Controls were sera from blood donors in the state of Tasmania, where A. caninum does not exist. The cut-off absorbance levels for positivity in IgG and IgE ELISAs were 0.147 and 0.155 optical density units, respectively. Both the IgG and IgE Western blots were reported as positive when a protein band (Ac68) with a molecular weight of 68 kd was recognized. Results All patients were white; six were men and three were women, with a mean age of 48 years (range, 30 to 61 years). Patients 1 to 3 were diagnosed from 1985 to 1990; the others were diagnosed from 1991 to 1993. Each patient lived in a freestanding house with lawns and garden and kept one or more dogs as pets. By gardening or walking outside barefooted, all patients had been potentially exposed to soil contaminated with canine feces. Except for patient 7, none had recently travelled to an area endemic for human hookworm. None had had atopic or immunologic diseases, and none reported cutaneous or respiratory symptoms that may occur with human or zoonotic ancylostomiasis [1]. After removal of the worm, all patients recovered and have remained well (Table 1). Table 1. A Summary of Demographic, Laboratory, and Parasitologic Findings in Nine Patients Infected by Solitary Hookworms* Patients 1 to 3 The clinical features of these patients have been published previously and are summarized in Table 1 [7, 10, 11]. A 61-year-old woman (patient 1) with a 4-week history of recurrent bowel obstructions had a laparotomy with resection of an inflamed ileal segment. A single hookworm was attached to the mucosa and was sectioned for histologic study, which precluded a species identification. Her case was reported to one of us several months after her recovery; serum had not been stored. A 50-year-old tradesman (patient 2) recurrent abdominal pain and a positive fecal occult blood test was diagnosed at colonoscopy. A feeding hookworm, species A. caninum, was removed from an inflamed and ulcerated terminal ileum. A 53-year-old male teacher (patient 3), having colonoscopy for vague abdominal pain and minor rectal bleeding thought to be caused by hemorrhoids, had a single feeding worm, species A. caninum, removed from his rectum. The colon and rectum appeared healthy. Patient 4 A 41-year-old woman from Townsville described three attacks during the preceding 5 weeks of mid-abdominal pain, which aroused her from sleep, lasted a few hours, and did not require urgent care. Her stools were loose and more frequent after each episode but she felt well between attacks. Results of a physical examination, abdominal ultrasonography, and upper gastrointestinal endoscopic examinations were normal. Colonoscopy, completed 20 cm into the ileum, showed two ulcers, 2 mm in diameter, in the cecum. A feeding nematode, 5 cm proximal to the ileocecal valve, was removed with biopsy forceps (Figure 1). Damage to the buccal cavity prevented specific identification of this 8-mm long, male hookworm, with spicules 750 and 785mlong. Tissue obtained from near the worm and the cecal ulcers was excessively infiltrated with eosinophils and plasma cells. Figure 1. Feeding hookworm in ileum (patient 4). Patient 5 A 45-year-old Brisbane man, with mild chronic dyspepsia that had not been investigated, was hospitalized after 24 hours of increasingly severe colic. He was afebrile and his abdomen was distended and tender; radiologic findings showed dilated loops of small bowel with fluid levels. A complete blood examination showed initial neutrophilia (10.25 109/L), with a normal eosinophil count of 0.37 109/L. After early improvement, the signs worsened; a laparotomy was done 36 hours later and showed increased serous peritoneal fluid and a 24-mm, inflamed ileal stricture, which was resected. An 8-mm long, female hookworm, without eggs, was attached to the lumen of the opened segment, but damage to the buccal capsule precluded its specific identification. Histologic findings showed intense eosinophilic ileitis. Mild blood eosinophilia (0.68 109/L and 0.76 109/L) was recorded 4 and 14 days after admission and resolved (0.15 109/L) by 4 weeks. Patient 6 A 30-year-old Townsville woman complained of anorexia, abdominal pains, diarrhea, and a 6-kg weight loss during a period of 4 weeks. She described the pain as a persistent, dull ache with severe exacerbations lasting 5 minutes, often provoked by eating. Her normal bowel habit, three formed stools daily, had increased to 10 liquid movements, often at night; passing stool gave temporary pain relief. A clinical examination was normal, but the blood eosinophil count was 2.96 109/L. The results of a fecal culture and microscopic examination were normal. The colonoscope was passed approximately 20 cm into the ileum. The only abnormal finding was a hookworm feeding in the cecum; it was removed with biopsy forceps and identified as a 10-mm long, adult male A. caninum, with spicules 785 and 800mlong (Figure 2). Random ileal and cecal biopsy samples showed intense eosinophilic inflammation. Three weeks later, her blood eosinophil count (0.26 109/L) was normal. Figure 2. Scanning electronmicrograph (patient 6). A. caninum Patient 7 A 51-year-old Brisbane man had a laparotomy for suspected acute appendicitis. He had presented 3 days after developing generalized abdominal pain, which improved initially but became severe again just before assessment. Six weeks earlier, he had returned from a 3-week vacation in Vanuatu, in the South Pacific, where he had walked barefooted and had observed many freely roaming dogs and extensive environmental fouling with their feces. He bred and trained greyhound dogs professionally, treating his animals regularly with anthelminthic agents. A clinical examination showed maximum tenderness over the right iliac fossa, with absence of bowel sounds. The operative findings were peritonitis, with slightly turbid fluid (culture sterile; cells not identified) and an intensely inflamed ileal segment 60 cm proximal to the ileocecal junction, surrounding a 2-cm thickened area that at first appeared to be a tumor. The ileum was opened to show a small ulcer that, histologically, was intensely inflamed and infiltrated with eosinophils. An 8-mm long, female A. caninum, without eggs, was attached to the center of the ulcer. Patient 8 A 50-year-old Townsville man with atherosclerosis was hospitalized with a 6-hour history of severe abdominal pain coming in waves lasting several seconds. He had noticed a mild inc

Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.

Impact of Experimental Hookworm Infection on the Human Gut Microbiota

The interactions between gastrointestinal parasitic helminths and commensal bacteria are likely to play a pivotal role in the establishment of host-parasite cross-talk, ultimately shaping the development of the intestinal immune system. However, little information is available on the impact of infections by gastrointestinal helminths on the bacterial communities inhabiting the human gut. We used 16S rRNA gene amplification and pyrosequencing to characterize, for the first time to our knowledge, the differences in composition and relative abundance of fecal microbial communities in human subjects prior to and following experimental infection with the blood-feeding intestinal hookworm, Necator americanus. Our data show that, although hookworm infection leads to a minor increase in microbial species richness, no detectable effect is observed on community structure, diversity or relative abundance of individual bacterial species.

Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma

A secreted hookworm protein in recombinant form acts on dendritic cells to drive the expansion and mucosal homing of regulatory T cells that protect against airway inflammation in mice, and also dampens human dendritic cell and T cell activation. Airway allergy alleviated by hookworm protein One reason for allergy prevalence in the developed world may be a lack of exposure to parasites, which can influence immune development and function. Because administering live parasites to people might pose safety issues, Navarro et al. tested the ability of the hookworm protein AIP-2 to treat airway allergic sensitization. Administration of AIP-2 could prevent or treat asthma symptoms in a mouse model, in a mechanism that was dependent on dendritic cells and regulatory T cells. Encouragingly, AIP-2 also reduced activation of human dendritic cells and T cells, indicating that these findings may readily translate to the clinic. In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103+ DCs and suppression of airway inflammation was dependent on both DCs and Foxp3+ regulatory T cells (Tregs) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2–treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted Treg induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases.

Guaiac versus immunochemical tests: faecal occult blood test screening for colorectal cancer in a rural community

Objective: To describe patient participation and clinical performance in a colorectal cancer (CRC) screening program utilising faecal occult blood test (FOBT).

Experimental hookworm infection and escalating gluten challenges are associated with increased microbial richness in celiac subjects.

The intestinal microbiota plays a critical role in the development of the immune system. Recent investigations have highlighted the potential of helminth therapy for treating a range of inflammatory disorders, including celiac disease (CeD); however, the mechanisms by which helminths modulate the immune response of the human host and ameliorate CeD pathology are unknown. In this study, we investigated the potential role of alterations in the human gut microbiota in helminth-mediated suppression of an inflammatory disease. We assessed the qualitative and quantitative changes in the microbiota of human volunteers with CeD prior to and following infection with human hookworms, and following challenge with escalating doses of dietary gluten. Experimental hookworm infection of the trial subjects resulted in maintenance of the composition of the intestinal flora, even after a moderate gluten challenge. Notably, we observed a significant increase in microbial species richness over the course of the trial, which could represent a potential mechanism by which hookworms can regulate gluten-induced inflammation and maintain intestinal immune homeostasis.