Basil Hetzel Institute

Jack Jumper ant (JJA) venom immunotherapy (VIT) is highly efficacious but the lowest effective dose is unknown. Delta-inulin adjuvant (Advax) is known to enhance honeybee VIT immunogenicity. This phase 1/2 single-blind, randomised controlled trial aimed to compare the efficacy and safety of JJA VIT with different doses of venom ± Advax. Adults aged 18-65 with a history of immediate systemic reaction (SR) to JJA stings were randomised to receive JJA VIT at a maintenance dose of 25 mcg or 50 mcg ± Advax; participants were blinded to treatment allocation. Primary outcomes were the response to sting challenges after 12 months and venom-specific IgE and IgG4 responses to treatment. Forty-nine of 50 screened subjects met inclusion criteria and were randomised; 44 started treatment (25 mcg n = 12; 25 mcg + Advax n = 13; 50mcg n = 12, 50 mcg + Advax n = 12). Subsequently, two withdrew due to SRs to treatment, and two withdrew due to unrelated factors. The higher JJA venom maintenance dose was associated with reduced likelihood of SRs (OR 0.53 (95% CI, 0.28-0.98)), while Advax did not have an effect (OR 1.17 (95% CI, 0.59-2.32)). Forty proceeded to sting challenge, with six developing SRs. There was no difference between groups for sting challenge outcome (p = 0.98), and the ORs for 25 mcg vs. 50 mcg venom dose (0.89, 95% CI, 0.38-2.09) and Advax vs. no Advax (0.99, 95% CI 0.42-2.33) indicated no effect. There were no differences between groups for venom sIgE (p = 0.78), sIgG4 (p = 0.25), sIgE/IgG4 ratio (p = 0.42), intradermal (p = 0.77), and basophil activation test (BAT) (p = 0.69) responses to treatment. Subjects with high baseline BAT sensitivity, which reduced markedly in response to treatment, were less likely to have a positive sting challenge (p = 0.006). Challenge outcomes were similar for all groups, with no significant difference found between 25 and 50 mcg maintenance dose or between treatment with and without Advax. Further research of low dose JJA VIT is warranted to confirm its efficacy and tolerability. NCT03066986.

Faecal microbiota transplantation (FMT) is the standard-of-care for recurrent or refractory Clostridioides difficile infection (CDI) [1]. Numerous variables impact the effectiveness of FMT, including donor, recipient, processing and administration factors. With the expanding use of FMT, it is important to determine the optimal treatment regimen to improve outcomes for patients with CDI and beyond. Paaske et al. carried out an eloquent multi-centre quality-improvement process, aimed at increasing CDI cure rates for patients undergoing FMT [2]. They implemented a number of interventions to enhance the effectiveness of FMT, showing that dose and donor matter. In this real-world study, eight-week cure rates of CDI-associated diarrhoea (CDAD) were increased across seven centres from 50% (95% confidence interval [CI] 45%–56%) to 59% (55%–63%), after the exclusion of three low-performing donors. Two donors had previous cure rates of less than 35%, and the third was observed to have a sudden decrease in effectiveness. Whilst the concept of an FMT super-donor has gained traction in inflammatory bowel disease, its relevance in CDI remains unclear, owing to consistently high cure rates [3]. The current data highlight the need for improved FMT regimens, with real-world efficacy lower than that reported in clinical trials [2, 4]. However, donor factors are not clearly elucidated in this study; rather donor performance is measured on prior outcomes. Donor factors previously associated with reduced efficacy include antibiotics within 3–12 months of donation, and donations with a Bristol Stool Form Scale of ≤ 3 [5]. Donor sex, BMI, smoking status, H. pylori status, birth-mode and donation-weight have not been found to influence the effectiveness of FMT in CDI. Selection of a super-donor has been trialled in one small pilot study in CDI, in which a single donor was selected from nine healthy candidates based on balanced Bacteroidetes/Firmicutes ratio, high stool butyrate concentration, and high alpha-diversity and phylogenetic-diversity [6]. CDI cure at week 10 was achieved in 10/10 patients with recurrent CDI, demonstrating that donor selection is feasible and may offer increased efficacy. Paaske et al. further increased their CDAD cure rates from 59% (55%–63%) to 72% (65%–77%) by implementing an optimised dosing regimen of two-dose capsule FMT [2]. Repeated FMT has been shown to increase clinical effect at week eight compared to single-dose FMT (91% vs. 84%), although it remains unclear which patients would benefit most from repeated doses [4]. In a separate cohort, Paaske and colleagues implemented a three-dose capsule regimen in high-risk patients, including those with fulminant CDI, patients expected to receive antibiotics in the week following FMT, and those aged 65 years or older with severe or antibiotic-refractory CDI [2]. This change did not result in increased overall efficacy (70% [60%–78%] vs. 67% [57%–76%]). FMT is highly effective in the treatment of CDI, but real-world cure rates lag behind those of well-designed clinical trials. Ongoing development of optimal donor, recipient and administration protocols is needed. Repeated dosing increases the efficacy of FMT in CDI, and emerging evidence supports donor selection in this population. To advance the field, we must now define microbial factors associated with efficacy. Perhaps the future is a rationally designed microbial therapeutic, with a defined and reproducible mechanism of action. D. Bogatic: conceptualization, writing – original draft, writing – review and editing. S. P. Costello: conceptualization, writing – original draft, writing – review and editing. R. V. Bryant: conceptualization, writing – original draft, writing – review and editing. The authors have nothing to report. D. Bogatic has received research support from The Gutsy Group and BiomeBank. S.P. Costello is a shareholder of BiomeBank. R.V. Bryant has received grant/research support/speaker honoraria/advisory board fees from AbbVie, Ferring, Janssen, Shire, Takeda, GlaxoSmithKline, Bristol Myers Squibb, and Emerge Health; and is a shareholder in BiomeBank. This article is linked to Paaske et al paper. To view this article, visit https://doi.org/10.1111/apt.70395. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

3D printing has emerged as an attractive alternative for the fabrication of microfluidic devices offering the ability for rapid prototyping and a high degree of customization, in contrast to conventional microfabrication techniques. Despite its potential, the size of internal features (such as microfluidic channels) has been limited to larger cross-sectional areas and shorter lengths. Advances in 3D printing technology and the development of custom resins have allowed for smaller features to be obtained, moving the field closer to genuine microfluidic devices. However, not all researchers can readily perform the customization or resin development required. In this work, we obtained channels with smaller cross-sections (∼100-200μm) and longer channel lengths (up to 96mm) using a commercial printer and resin, by optimizing several printing parameters as well as aspects of the microfluidic design. A chip array and custom 3D printed manifold was used to develop a workflow for high throughput post-processing, enabling facile removal of uncured resin from enclosed channels. Using a rectangular channel geometry, a layer height of 50μm, and a single layer cover, channels with specified dimensions of 150×150μm and 6mm total length were obtained with a high success rate (>98%). Channels up to 96mm could be printed using dimensions of 200×200μm. The potential for these microchips for the pre-concentration and separation of proteins was also demonstrated, by fabricating polyacrylamide separation gels within the channels. This work presents an alternative workflow for fabrication of enclosed channels that does not rely on the use of custom printers or resins. Implementation of this workflow with pre-existing 3D printing technology is expected to allow future research progress facilitating smaller channel dimensions of longer lengths more readily. If coupled with emerging 3D printing technology, this could open the door for the development of truly microfluidic devices for bioanalysis and point-of-care devices in healthcare settings.

ABSTRACTDietary therapy has an established role in managing gastrointestinal disorders, as a short‐term induction therapy (e.g., exclusive enteral nutrition for Crohn's disease), a long‐term monotherapy (e.g., gluten‐free diet for coeliac disease and a personalized FODMAP diet) or adjunct therapy (e.g., Mediterranean diet). As use of dietary therapies rises, it is becoming increasingly common for gastroenterologists and dietitians to encounter patients who are simultaneously following two or more dietary therapies to achieve optimal symptom control, to manage multiple concurrent medical issues, or for social or religious reasons. This practice is termed ‘diet stacking’. The aim of this review is to clarify the principles behind the safe and effective combination of dietary interventions with specific attention to potential risks, mitigation of risk and practical application. For gastroenterologists, awareness of current dietary practices of their patients prior to advising dietary therapy is warranted and, in those who are diet stacking, risk assessment is essential. Validated screening tools to evaluate risks, particularly of disordered eating, are limited. The management of those who currently or are at risk of diet stacking is best directed toward gastrointestinal dietitians who offer time, appropriate skills for assessment, and the delivery of pragmatic patient education to safely implement appropriate dietary interventions, and assess for the need for engaging psychological primary management or co‐management. In conclusion, diet stacking is common, and requires awareness and often multi‐disciplinary management to ensure it is implemented safely.

Poor mesh tissue integration is thought to be responsible for hernia mesh repair failures years after surgery. The Mesh Integration (MINT) Index was proposed as a solution to unify methods of evaluating in vivo mesh behaviour. The aim was to validate the Index over a 3-month period using a porcine model. Twelve brands of mesh were implanted into twelve Landrace-White pigs. Appropriate mesh was randomised to retrorectus and intraperitoneal space. Post-mortems were performed at 2, 3, 4, 8, and 12weeks. All mesh tissue samples were subjected to standardised testing as predefined in the Index. Mesh was successfully implanted into all pigs. One pig died from congenital cardiomyopathy, and another was euthanised earlier than intended due to complications of midline wound infection. There were no difficulties at post-mortem. Multilevel linear regression analysis with maximum likelihood was performed. A strong fit of models were achieved for all four domains of the Index, with minimal residues. Integration and fibrosis were predicted by time ( and , respectively). Although statistically not significant when cluster robust version 2 (CR2) standard errors were used, retrorectus mesh had a greater numerical integration over time than intraperitoneal, and numerical differences were present between mesh products. Degradation was predicted by intraperitoneal mesh ( ). A reduction in adhesion score was observed after week 4. The MINT Index is a valid framework to objectively assess in vivo mesh behaviour. Substantial research is required before full clinical utility of the Index can be achieved.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options and inconsistent response to immune checkpoint inhibitors (ICIs). Emerging evidence indicates that tumor-associated bacteria may shape immune signaling and alter immunotherapy outcomes. Here, we investigated whether Staphylococcus aureus invades TNBC cells, persists intracellularly, and modulates PD-L1 expression. Methods: Using eFluor450-labeled S. aureus for flow cytometry, gentamicin protection assays, CFU quantification, and transmission electron microscopy, we assessed bacterial uptake and persistence in six TNBC cell lines and a non-tumorigenic control. PD-L1, TLR2, and STAT1 activation were evaluated after infection or TLR2 ligand treatment ± IFN-γ. Results: At multiplicity of infection (MOI) of 10, S. aureus internalized into 67% of MDA-MB-468 and 54% of MDA-MB-231, with intermediate uptake in Hs578T (27%) and BT-549 (24%) and only 0.5-9% in low-uptake lines (MDA-MB-453, CAL-51, MCF-12A). High-uptake lines exhibited marked cytotoxicity and reduced proliferation, with MDA-MB-468 showing an 82% drop in viability at 2 h and a 74% decrease after 5 d, whereas low-uptake lines showed minimal impact. Persistence lasted >7 d in MDA-MB-231 but only 3-5 days in others. IFN-γ plus S. aureus significantly amplified PD-L1, with up to a 2.9-fold increase in MDA-MB-468 and 1.5-fold in MDA-MB-231, but no effect in low-uptake lines. TLR2 agonists modestly increased PD-L1 in high-TLR2-expressing lines and synergized with IFN-γ. These effects were accompanied by STAT1 phosphorylation, supporting a TLR2/STAT1 axis linking bacterial sensing to immune checkpoint regulation. Conclusions: Together, these findings identify S. aureus as a modulator of immune signaling in TNBC and highlight the potential for microbial factors to influence ICI responsiveness. Targeting tumor-bacteria interactions may represent a novel strategy to enhance immunotherapy efficacy in breast cancer.

We appreciate the insightful comments by Vos and colleagues on our expert position statement on the role of intestinal ultrasound (IUS) in luminal contents assessment [1]. IUS is a rapidly evolving area emerging as an alternative modality for the assessment of disorders of gut-brain interaction (DGBI) [2]. Patients with DGBI are exposed to more ionising radiation than healthy controls; IUS offers safety, cost effectiveness, and accuracy over conventional assessment methods [3]. Until recently, however, IUS for functional assessment has remained in the realm of paediatrics, with a paucity of adult studies and a lack of description or validation of sonographic parameters [4]. Our position statement [2] was intended to serve as initial guidance and a summation of current evidence. A narrative review has further summarised available evidence on luminal contents assessment [5]. A study performed in a DGBI cohort did not find a correlation between sonographic findings and symptoms, but did suggest that IUS was useful in identifying those patients who would benefit from therapy for constipation rather than treatment of diarrhoea due to identification of faecal loading [6]. Together, these set the scene for IUS in the assessment of luminal contents in clinical practice. However, they clearly illustrate knowledge gaps and the need for further research. Small studies have shown that IUS is as accurate as computed tomography [7, 8]. However, prospective, blinded, and centrally read studies are required to measure the accuracy and validity of IUS in measuring luminal contents, to improve reliability and uptake in both clinical trials and practice [9]. The Sonographic Examination and Assessment of Ulcerative Colitis Associated Constipation (SEE UCAC trial, ACTRN12622001207707) is the first prospective, blinded, and centrally read study to directly compare the accuracy of luminal contents assessment against CT scan with ultrasound. Interim results of 63 patients suggest close correlation between IUS and CT in assessing luminal contents as well as excellent inter-rater agreement between sonographic parameters [10]. The body of evidence supporting the use of IUS in clinical practice has remained in the assessment of inflammation, particularly in adults. It is now with great optimism that we see the increasing interest and growing potential of IUS in the assessment of DGBI and luminal contents emerging from the shadows into the spotlight. Ryan M. Mathias: conceptualization; writing – original draft. Thomas M. Goodsall: writing – review and editing. Claire E. Parker: writing – review and editing. Alice S. Day: writing – review and editing. Yoon K. An: writing – review and editing. Brandon Baraty: writing – review and editing. Chamara Basnayake: writing – review and editing. Jakob Begun: writing – review and editing. Ray K. Boyapati: writing – review and editing. Rebecca Burgell: writing – review and editing. Michael T. Dolinger: writing – review and editing. Giovanni Maconi: writing – review and editing. Kerri L. Novak: writing – review and editing. Shintaro Sagami: writing – review and editing. Rebecca L. Smith: writing – review and editing. Ashish R. Srinivasan: writing – review and editing. Stuart A. Taylor: writing – review and editing. Vipul Jairath: writing – review and editing. Christopher Ma: writing – review and editing. Robert V. Bryant: writing – review and editing; Supervision. The authors declare no conflicts of interest. This article is linked to Mathias et al. papers. To view these articles, visit https://doi.org/10.1111/apt.70250 and https://doi.org/10.1111/apt.70300. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.