Controlled infection with cryopreserved human hookworm induces CTLA-4 expression on Tregs and upregulates tryptophan metabolism.

Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed to prevent or treat various intestinal and extraintestinal diseases. However, full-scale clinical trials examining hookworm therapy are limited by the inability to scale-up the production of hookworm larvae to infect sufficient numbers of patients. With the aim of overcoming this challenge, this study infected four healthy individuals with hookworm larvae that had been reanimated from cryopreserved eggs to examine their viability and immunogenicity. We demonstrate that reanimated cryopreserved hookworm larvae establish a viable hookworm infection and elicit a similar immune response to larvae cultured from fresh stool. Furthermore, a refined understanding of the therapeutic mechanisms of hookworm is imperative to determine which diseases to target with hookworm therapy. To investigate potential therapeutic mechanisms, this study assessed changes in the immune cells, microbiome, and plasma metabolome in the four healthy individuals infected with cryopreserved hookworm larvae and another nine individuals infected with larvae cultured from freshly obtained stool. We identified potential immunoregulatory mechanisms by which hookworm may provide a beneficial effect on its host, including increased expression of CTLA-4 on regulatory T cells (Tregs) and upregulation of tryptophan metabolism. Furthermore, we found that a participant's baseline microbiome predicted the severity of symptoms and intestinal inflammation experienced during a controlled hookworm infection. In summary, our findings demonstrate the feasibility of full-scale clinical trials examining hookworm therapy by minimizing the reliance on human donors and optimizing the culturing process, thereby enabling viable hookworm larvae to be mass-produced and enabling on-demand inoculation of patients. Furthermore, this study provides insights into the complex interactions between helminths and their host, which could inform the development of novel therapeutic strategies.

Helminth infection induces a distinct subset of CD101hi lung tissue-infiltrating eosinophils that are differentially regulated by type 2 cytokines.

Eosinophils play divergent roles in health and disease, contributing to both immunoregulatory and proinflammatory responses. Helminth infection is strongly associated with eosinophilia and the induction of the type 2 cytokines interleukin (IL)-5, IL-4 and IL-13. This study aimed to elucidate the heterogeneity of pulmonary eosinophils in response to helminth infection and the roles of IL-5, IL-4 and IL-13 in driving pulmonary eosinophil responses. Using the murine helminth model Nippostrongylus brasiliensis (Nb), we characterize a subtype of eosinophils, defined by high expression of CD101, that is induced in the lungs of Nb-infected mice and are phenotypically distinct from lung eosinophils that express low levels of CD101. Strikingly, we show that the two eosinophil subtypes have distinct anatomical localization within the lung: CD101low eosinophils are predominantly localized in the lung vasculature, whereas Nb-induced CD101hi eosinophils are predominantly localized in the extravascular lung niche. We show that CD101hi eosinophils are also induced across other models of pulmonary infection and inflammation, including a nonlung-migrating helminth infection, house dust mite-induced allergic inflammation and influenza infection. Furthermore, we demonstrate that the induction of CD101hi tissue eosinophils is independent of IL-5 and IL-4 signaling, but is dependent on intact IL-13 signaling. These results suggest that IL-13 produced during helminth infection and other disease states promotes a pulmonary tissue-infiltrating program in eosinophils defined by high expression of CD101.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Controlled Hookworm Infection for Medication-free Maintenance in Patients with Ulcerative Colitis: A Pilot, Double-blind, Randomized Control Trial.

BACKGROUND: Human hookworm has been proposed as a treatment for ulcerative colitis (UC). This pilot study assessed the feasibility of a full-scale randomized control trial examining hookworm to maintain clinical remission in patients with UC. METHODS: Twenty patients with UC in disease remission (Simple Clinical Colitis Activity Index [SCCAI] ≤4 and fecal calprotectin (fCal) <100 ug/g) and only on 5-aminosalicylate received 30 hookworm larvae or placebo. Participants stopped 5-aminosalicylate after 12 weeks. Participants were monitored for up to 52 weeks and exited the study if they had a UC flare (SCCAI ≥5 and fCal ≥200 µg/g). The primary outcome was difference in rates of clinical remission at week 52. Differences were assessed for quality of life (QoL) and feasibility aspects including recruitment, safety, effectiveness of blinding, and viability of the hookworm infection. RESULTS: At 52 weeks, 4 of 10 (40%) participants in the hookworm group and 5 of 10 (50%) participants in the placebo group had maintained clinical remission (odds ratio, 0.67; 95% CI, 0.11-3.92). Median time to flare in the hookworm group was 231 days (interquartile range [IQR], 98-365) and 259 days for placebo (IQR, 132-365). Blinding was quite successful in the placebo group (Bang's blinding index 0.22; 95% CI, -0.21 to 1) but less successful in the hookworm group (0.70; 95% CI, 0.37-1.0). Almost all participants in the hookworm group had detectable eggs in their faeces (90%; 95% CI, 0.60-0.98), and all participants in this group developed eosinophilia (peak eosinophilia 4.35 × 10^9/L; IQR, 2.80-6.68). Adverse events experienced were generally mild, and there was no significant difference in QoL. CONCLUSIONS: A full-scale randomized control trial examining hookworm therapy as a maintenance treatment in patients with UC appears feasible.

This pilot study has shown a full-scale RCT examining hookworm therapy as maintenance therapy in patients with ulcerative colitis is feasible, safe, and will be well-tolerated.

Tissue-specific immunity in helminth infections.

A characteristic feature of host responses to helminth infections is the development of profound systemic and tissue-localised Type 2 immune responses that play critical roles in immunity, tissue repair and tolerance of the parasite at tissue sites. These same Type 2 responses are also seen in the tissue-associated immune-pathologies seen in asthma, atopic dermatitis and many forms of allergies. The recent identification of new subtypes of immune cells and cytokine pathways that influence both immune and non-immune cells and tissues creates the opportunity for reviewing helminth parasite-host responses in the context of tissue specific immunity. This review focuses on the new discoveries of the cells and cytokines involved in tissue specific immune responses to helminths and how these contribute to host immunity against helminth infection and allow the host to accommodate the presence of parasites when they cannot be eliminated.

Could anaesthesia be a key factor for the good outcome of bone ablation procedures? A retrospective analysis of a musculoskeletal interventional centre.

OBJECTIVE: To choose the best anaesthetic approach through the retrospective review of different bone ablation procedures. METHODS AND MATERIALS: We retrospectively evaluated 118 ablation procedures carried out in our institute over the last 30 months. Three different anaesthetic approaches were used: general anaesthesia, i.v. sedation/analgesia and loco-regional anaesthesia (brachial plexus block, spinal anaesthesia). The outcomes were evaluated based on three parameters: technical success, patient comfort (Scale 1-5) and operator comfort (Scale 1-5). RESULTS: The 118 interventional procedures were carried out on 62 benign and 56 malignant bone lesions. The overall procedural success rate was 100%. Three cases were treated under general anaesthesia: patient comfort was 5/5 in all cases; operator comfort was 5/5 in one case, and 4/5 in two cases. Twenty-one patients underwent sedation/analgesia: in three patients with benign bone lesions, patient comfort was 1/5 and operator comfort 3/5; in two patients with malignant bone lesions, patient comfort was 3/5 and operator comfort 4/5. Ninety-four patients underwent loco-regional anaesthesia: patient and operator comfort was 5/5 in all cases. CONCLUSION: Based on our experience, loco-regional anaesthesia is probably the best anaesthetic approach during bone ablation procedures. Benign bone lesions ablation are the more painful procedures. ADVANCES IN KNOWLEDGE: This is the first paper that systematically investigates about the best anaesthesiological support for IR procedures.

A Truncated Form of HpARI Stabilizes IL-33, Amplifying Responses to the Cytokine.

The murine intestinal nematode Heligmosomoides polygyrus releases the H. polygyrus Alarmin Release Inhibitor (HpARI) - a protein which binds to IL-33 and to DNA, effectively tethering the cytokine in the nucleus of necrotic cells. Previous work showed that a non-natural truncation consisting of the first 2 domains of HpARI (HpARI_CCP1/2) retains binding to both DNA and IL-33, and inhibited IL-33 release in vivo. Here, we show that the affinity of HpARI_CCP1/2 for IL-33 is significantly lower than that of the full-length protein, and that HpARI_CCP1/2 lacks the ability to prevent interaction of IL-33 with its receptor. When HpARI_CCP1/2 was applied in vivo it potently amplified IL-33-dependent immune responses to Alternaria alternata allergen, Nippostrongylus brasiliensis infection and recombinant IL-33 injection, in direct contrast to the IL-33-suppressive effects of full-length HpARI. Mechanistically, we found that HpARI_CCP1/2 is able to bind to and stabilize IL-33, preventing its degradation and maintaining the cytokine in its active form. This study highlights the importance of IL-33 inactivation, the potential for IL-33 stabilization in vivo, and describes a new tool for IL-33 research.

A helminth-derived suppressor of ST2 blocks allergic responses.

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Methylation analysis of the phosphates and tensin homologue on chromosome 10 gene (PTEN) in multiple myeloma.

BACKGROUND: Aberrant DNA methylation of promoter region CpG islands is an alternative mechanism that leads to genetic defects in the inactivation of tumor suppressor genes during myelomagenesis. The aim of this study was to examine the promoter methylation status of the phosphates and tensin homologue on chromosome 10 (PTEN) gene in a cohort of multiple myeloma patients. FINDINGS: The PTEN gene was hypermethylated in 7 out of 58 (12%) primary myeloma samples. The correlation between functional inactivation and PTEN mRNA levels was not statistically significant. The multiple myeloma subgroup with an aberrant PTEN status had a prevalence of the component IgG, Salmon Durie stage I, lower lactate dehydrogenase levels, intermediate-standard cytogenetic risk and longer overall survival with the respect to the unmethylated subgroup. CONCLUSIONS: This is the first report demonstrating the presence of PTEN promoter hypermethylation in multiple myeloma.