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IntroductionKidney transplant recipients (KTRs) are at an increased risk of hospitalisation and death from COVID-19. Vaccination against SARS-CoV-2 is our primary risk mitigation strategy, yet vaccine effectiveness in KTRs is suboptimal. Strategies to enhance vaccine efficacy are therefore required. Current evidence supports the role of the gut microbiota in shaping the immune response to vaccination. Gut dysbiosis is common in KTRs and is a potential contributor to impaired COVID-19 vaccine responses. We hypothesise that dietary fibre supplementation will attenuate gut dysbiosis and promote vaccine responsiveness in KTRs.Methods and analysisRapamycin and inulin for third-dose vaccine response stimulation-inulin is a multicentre, randomised, prospective, double-blinded, placebo-controlled pilot trial examining the effect of dietary inulin supplementation prior to a third dose of COVID-19 vaccine in KTRs who have failed to develop protective immunity following a 2-dose COVID-19 vaccine schedule. Participants will be randomised 1:1 to inulin (active) or maltodextrin (placebo control), administered as 20 g/day of powdered supplement dissolved in water, for 4 weeks prior to and following vaccination. The primary outcome is the proportion of participants in each trial arm that achieve in vitro neutralisation of live SARS-CoV-2 virus at 4 weeks following a third dose of COVID-19 vaccine. Secondary outcomes include the safety and tolerability of dietary inulin, the diversity and differential abundance of gut microbiota, and vaccine-specific immune cell populations and responses.Ethics and disseminationEthics approval was obtained from the Central Adelaide Local Health Network (CALHN) Human Research Ethics Committee (HREC) (approval number: 2021/HRE00354) and the Sydney Local Health District (SHLD) HREC (approval numbers: X21-0411 and 2021/STE04280). Results of this trial will be published following peer-review and presented at scientific meetings and congresses.Trial registration numberACTRN12621001465842.

Kidney transplant recipients are at an increased risk of severe COVID-19-associated hospitalisation and death. Vaccination has been a key public health strategy to reduce disease severity and infectivity, but the effectiveness of COVID vaccines is markedly reduced in kidney transplant recipients. Urgent strategies to enhance vaccine efficacy are needed. Methods: RIVASTIM-rapamycin is a multicentre, randomised, controlled trial examining the effect of immunosuppression modification prior to a third dose of COVID-19 vaccine in kidney transplant recipients who have failed to develop protective immunity to a 2-dose COVID-19 vaccine schedule. Participants will be randomised 1:1 to either remain on standard of care immunosuppression with tacrolimus, mycophenolate, and prednisolone (control) or cease mycophenolate and commence sirolimus (intervention) for 4 weeks prior to and following vaccination. The primary outcome is the proportion of participants in each trial arm who develop protective serological neutralisation of live SARS-CoV-2 virus at 4–6 weeks following a third COVID-19 vaccination. Secondary outcomes include SARS-CoV-receptor binding domain IgG, vaccine-specific immune cell populations and responses, and the safety and tolerability of sirolimus switch. Discussion: Immunosuppression modification strategies may improve immunological vaccine response. We hypothesise that substituting the mTOR inhibitor sirolimus for mycophenolate in a triple drug regimen will enhance humoral and cell-mediated responses to COVID vaccination for kidney transplant recipients. Trial registration: Australia New Zealand Clinical Trials Registry ACTRN12621001412820. Registered on 20 October 2021; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382891&isReview=true

Introduction/Rationale: In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD. Methods: Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence. Results: Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis. Conclusion: LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

Kidney transplant recipients are at an increased risk of hospitalisation and death from SARS-CoV-2 infection, and standard two-dose vaccination schedules are typically inadequate to generate protective immunity. Gut dysbiosis, which is common among kidney transplant recipients and known to effect systemic immunity, may be a contributing factor to a lack of vaccine immunogenicity in this at-risk cohort. The gut microbiota modulates vaccine responses, with the production of immunomodulatory short-chain fatty acids by bacteria such as Bifidobacterium associated with heightened vaccine responses in both observational and experimental studies. As SCFA-producing populations in the gut microbiota are enhanced by diets rich in non-digestible fibre, dietary supplementation with prebiotic fibre emerges as a potential adjuvant strategy to correct dysbiosis and improve vaccine-induced immunity. In a randomised, double-bind, placebo-controlled trial of 72 kidney transplant recipients, we found dietary supplementation with prebiotic inulin for 4 weeks before and after a third SARS-CoV2 mRNA vaccine to be feasible, tolerable, and safe. Inulin supplementation resulted in an increase in gut Bifidobacterium, as determined by 16S RNA sequencing, but did not increase in vitro neutralisation of live SARS-CoV-2 virus at 4 weeks following a third vaccination. Dietary fibre supplementation is a feasible strategy with the potential to enhance vaccine-induced immunity and warrants further investigation.

INTRODUCTION:Eosinophilic esophagitis (EoE) is associated with atopy; however, recent studies have identified an association with food-specific immunoglobulin G4 (FS-IgG4) rather than immunoglobulin E antibodies. This study aimed to evaluate the role of serum FS-IgG4 in guiding an elimination diet and its outcomes.METHODS:Patients with and without EoE were enrolled in a prospective, controlled, single tertiary center trial. Serum FS-IgG4 titers, esophageal eosinophil counts, and dysphagia symptom questionnaire scores were assessed, and participants with elevated FS-IgG4 (ImmunoCAP, cutoff of 10 mgA/L) commenced 6-week targeted elimination diet. Repeat serum FS-IgG4 and endoscopic and histologic examination were performed at 6-week follow-up.RESULTS:Twenty-two patients with active EoE and 13 controls were recruited. Serum FS-IgG4 to milk, wheat, soy, eggs, and nuts was significantly higher in EoE (P = 0.0002, P = 0.002, P = 0.003, P = 0.012, and P < 0.001, respectively). Elevated serum FS-IgG4 to 1 or more food groups (median 2) was identified in 21/22 (95.4%) patients with EoE; 20/21 underwent 6-week dietary elimination. Median reductions in dysphagia symptom questionnaire score and EoE endoscopic reference score after elimination were 8 (P = 0.0007) and 1 (P = 0.002), respectively. Nine (45%) patients had histological remission (<15 eosinophils per high-power field). Fall in median esophageal eosinophil count was not statistically significant (50 vs 23; P = 0.068). Serum FS-IgG4 did not decline by 6-week follow-up.DISCUSSION:Serum FS-IgG4 to milk, wheat, soy, egg, and nuts was present at higher levels in EoE, with targeted elimination resulting in 45% histologic remission rate. Serum FS-IgG4 has potential as a noninvasive biomarker in EoE. When successful, FS-IgG4-led elimination diet can negate need for medications and be viewed more favorably by patients because of its smaller endoscopic burden compared with empirical elimination diets.

Objectives Regulatory T cells (Tregs) are a vital sub‐population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF‐β, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α‐CD3/CD28 bead ratios on human iTreg stability. Methods We assess iTregs generated with various concentrations of rapamycin and differing ratios of α‐CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg‐specific demethylation region (TSDR) status. Results iTregs generated in the presence of TGF‐β, ATRA, rapamycin and a higher ratio of α‐CD3/CD28 beads were highly suppressive and stable upon in vitro re‐stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation. Conclusion This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re‐stimulation. Further validation in pre‐clinical models will be needed to ensure its suitability for applications in adoptive transfer. Induced regulatory T cells are an attractive alternative for adoptive transfer in an allogeneic transplant setting. We report that rapamycin and abundant TCR stimulation are required to generate stable human induced regulatory T cells.

Background A common feature of COPD is a defective lung macrophage phagocytic capacity that can contribute to chronic lung inflammation and infection. The precise mechanisms remain incompletely understood, although cigarette smoke is a known contributor. We previously showed deficiency of the LC3-associated phagocytosis (LAP) regulator, Rubicon, in macrophages from COPD subjects and in response to cigarette smoke. The current study investigated the molecular basis by which cigarette smoke extract (CSE) reduces Rubicon in THP-1, alveolar and blood monocyte-derived macrophages, and the relationship between Rubicon deficiency and CSE-impaired phagocytosis. Methodology Phagocytic capacity of CSE-treated macrophages was measured by flow cytometry, Rubicon expression by Western blot and real time polymerase chain reaction, and autophagic-flux by LC3 and p62 levels. The effect of CSE on Rubicon degradation was determined using cycloheximide inhibition and Rubicon protein synthesis and half-life assessment. Results Phagocytosis was significantly impaired in CSE-exposed macrophages and strongly correlated with Rubicon expression. CSE-impaired autophagy, accelerated Rubicon degradation, and reduced its half-life. Lysosomal protease inhibitors, but not proteasome inhibitors, attenuated this effect. Autophagy induction did not significantly affect Rubicon expression. Conclusions CSE decreases Rubicon through the lysosomal degradation pathway. Rubicon degradation and/or LAP impairment may contribute to dysregulated phagocytosis perpetuated by CSE.

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.

Eosinophilic esophagitis (EoE) is associated with atopy; however, recent studies have identified an association with food-specific immunoglobulin G 4 (FS-IgG 4 ) rather than immunoglobulin E antibodies. This study aimed to evaluate the role of serum FS-IgG 4 in guiding an elimination diet and its outcomes. Patients with and without EoE were enrolled in a prospective, controlled, single tertiary center trial. Serum FS-IgG 4 titers, esophageal eosinophil counts, and dysphagia symptom questionnaire scores were assessed, and participants with elevated FS-IgG 4 (ImmunoCAP, cutoff of 10 mgA/L) commenced 6-week targeted elimination diet. Repeat serum FS-IgG 4 and endoscopic and histologic examination were performed at 6-week follow-up. Twenty-two patients with active EoE and 13 controls were recruited. Serum FS-IgG 4 to milk, wheat, soy, eggs, and nuts was significantly higher in EoE ( P = 0.0002, P = 0.002, P = 0.003, P = 0.012, and P < 0.001, respectively). Elevated serum FS-IgG 4 to 1 or more food groups (median 2) was identified in 21/22 (95.4%) patients with EoE; 20/21 underwent 6-week dietary elimination. Median reductions in dysphagia symptom questionnaire score and EoE endoscopic reference score after elimination were 8 ( P = 0.0007) and 1 ( P = 0.002), respectively. Nine (45%) patients had histological remission (<15 eosinophils per high-power field). Fall in median esophageal eosinophil count was not statistically significant (50 vs 23; P = 0.068). Serum FS-IgG 4 did not decline by 6-week follow-up. Serum FS-IgG 4 to milk, wheat, soy, egg, and nuts was present at higher levels in EoE, with targeted elimination resulting in 45% histologic remission rate. Serum FS-IgG 4 has potential as a noninvasive biomarker in EoE. When successful, FS-IgG 4 -led elimination diet can negate need for medications and be viewed more favorably by patients because of its smaller endoscopic burden compared with empirical elimination diets.