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Aims/introduction: Autoantibodies to pancreatic islet antigens identify young children at high risk of type 1 diabetes. On a background of genetic susceptibility, islet autoimmunity is thought to be driven by environmental factors, of which enteric viruses are prime candidates. We sought evidence for enteric pathology in children genetically at-risk for type 1 diabetes followed from birth who had developed islet autoantibodies ("seroconverted"), by measuring mucosa-associated cytokines in their sera., Materials and Methods: Sera were collected 3 monthly from birth from children with a first-degree type 1 diabetes relative, in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Children who seroconverted were matched for sex, age, and sample availability with seronegative children. Luminex xMap technology was used to measure serum cytokines., Results: Of eight children who seroconverted, for whom serum samples were available at least 6 months before and after seroconversion, the serum concentrations of mucosa-associated cytokines IL-21, IL-22, IL-25, and IL-10, the Th17-related cytokines IL-17F and IL-23, as well as IL-33, IFN-γ, and IL-4, peaked from a low baseline in seven around the time of seroconversion and in one preceding seroconversion. These changes were not detected in eight sex- and age-matched seronegative controls, or in a separate cohort of 11 unmatched seronegative children., Conclusions: In a cohort of children at risk for type 1 diabetes followed from birth, a transient, systemic increase in mucosa-associated cytokines around the time of seroconversion lends support to the view that mucosal infection, e.g., by an enteric virus, may drive the development of islet autoimmunity., (© 2023 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Aims/hypothesis: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation., Methods: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme., Results: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets., Conclusions/interpretation: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes., (© 2023. The Author(s).)

CD4 + T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4 + T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4 + T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4 + T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in T reg cells. Expression of MEOX1 was comparable to FOXP3 in T reg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in T reg cells. Knockdown of MEOX1 in T reg cells revealed a profound impact on downstream gene expression programs and T reg cell suppressive capacity. These findings in the context of CD4 + T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4 + T cell functionality, which opens new avenues for future therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baßler, Schmidleithner, Shakiba, Elmzzahi, Köhne, Floess, Scholz, Ohkura, Sadlon, Klee, Neubauer, Sakaguchi, Barry, Huehn, Bonaguro, Ulas and Beyer.)

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation., (Published by Oxford University Press on behalf of Nucleic Acids Research 2023.)

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories., (© 2023. The Author(s).)

Background: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D., Results: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research., Conclusions: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs., Competing Interests: Declaration of interests A.S. is currently a consultant for Gritstone, Flow Pharma, Arcturus, Epitogenesis, Oxfordimmunotech, Caprion, and Avalia. La Jolla Institute for Immunology (A.S. and D.W.) has filed for patent protection for various aspects of T cell epitope and vaccine design work. P.G.-V., C.M.H., M.G.M., A.E.L.Y., H.B., Z.A.M., Z.A.-D., A. Abayasingam, D.A., A.O.S., A. Aggarwal, G.B., J.G., C.F., S.O., E.M.M., D.J.L., G.M., E.J.G., B.A.J.R., D.S., C.K.-L., S.G.T., M.R.B., D.W., R.A.B., S.C.B., and B.G.-B. declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Engineered T-cell therapies have proven highly efficacious for the treatment of haematological cancers, but translation of this success to solid tumours has been limited, in part, due to difficulties in maintaining high doses at specific target sites. Hydrogel delivery systems that provide a sustained release of T-cells at the target site are emerging as a promising strategy. Therefore, in this study we aimed to develop an injectable hydrogel that gels in situ via efficient Diels-Alder cycloaddition (DAC) chemistry and provides a sustained release of T-cells through gradual hydrolysis of the hydrogel matrix. Hydrogels were prepared via the DAC between fulvene and maleimide functionalised poly(ethylene glycol) (PEG) derivatives. By adjusting the concentration and molecular weight of the functionalised PEGs in the hydrogel formulation the in vitro gelation time ( T gel ), initial Young's modulus ( E ) and degradation time ( T d ) could be tailored from 15-150 min, 5-179 kPa and 7-114 h, respectively. Prior to gelation, the formulations could be readily injected through narrow gauge (26 G) needles with the working time correlating closely with the T gel . A 5 wt% hydrogel formation with conjugated cyclic RGD motif was found to be optimal for the encapsulation and release of CD3 + T-cells with a near linear release profile and >70% cell viability over the first 4 d and release continuing out to 7 d. With their tuneable T gel , T d and stiffness, the DAC hydrogels provide the opportunity to control the release period and profile of encapsulated cells.

Aims: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes., Methods: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured., Results: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA., Conclusions: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects., Methods: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection., Results: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not., Conclusions: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals., (© 2022. The Author(s).)

Chimeric antigen receptor T (CAR-T) cell therapy is rapidly becoming a frontline cancer therapy. However, the manufacturing process is time-, labor- and cost-intensive, and it suffers from significant bottlenecks. Many CAR-T products fail to reach the viability release criteria set by regulators for commercial cell therapy products. This results in non-recoupable costs for the manufacturer and is detrimental to patients who may not receive their scheduled treatment or receive out-of-specification suboptimal formulation. It is demonstrated here that inertial microfluidics can, within minutes, efficiently deplete nonviable cells from low-viability CAR-T cell products. The percentage of viable cells increases from 40% (SD ± 0.12) to 71% (SD ± 0.09) for untransduced T cells and from 51% (SD ± 0.12) to 71% (SD ± 0.09) for CAR-T cells, which meets the clinical trials' release parameters. In addition, the processing of CAR-T cells formulated in CryStor yields a 91% reduction in the amount of the cryoprotectant dimethyl sulfoxide. Inertial microfluidic processing has no detrimental effects on the proliferation and cytotoxicity of CAR-T cells. Interestingly, ≈50% of T-regulatory and T-suppressor cells are depleted, suggesting the potential for inertial microfluidic processing to tune the phenotypical composition of T-cell products., (© 2021 Wiley-VCH GmbH.)

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4 + and CD8 + cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4 + and CD8 + T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1-2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially 'corrected' after birth.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7 ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7 ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated., Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10 6 cells/mL to 1.67 × 10 6 cells/mL., Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10 6 cells/mL., Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Background: The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D., Results: Women with and without T1D exhibited compositional and functional changes in the gut microbiome across pregnancy. Profiles in women with T1D were distinct, with an increase in bacteria that produce lipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid-binding protein (I-FABP), a marker of intestinal epithelial damage., Conclusions: Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means. Video abstract., (© 2021. The Author(s).)

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA + to CD45RO + cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Aim: We aimed to characterize associations between diet and the gut microbiome and short chain fatty acid (SCFA) products in youth with islet autoimmunity or type 1 diabetes (IA/T1D) in comparison with controls., Research Design and Methods: Eighty participants (25 diagnosed with T1D, 17 with confirmed IA, 38 sibling or unrelated controls) from the Australian T1D Gut Study cohort were studied (median [IQR] age 11.7 [8.9, 14.0] years, 43% female). A Food Frequency Questionnaire characterized daily macronutrient intake over the preceding 6 months. Plasma and fecal SCFA were measured by gas chromatography; gut microbiome composition and diversity by 16S rRNA gene sequencing., Results: A 10 g increase in daily carbohydrate intake associated with higher plasma acetate in IA/T1D (adjusted estimate +5.2 (95% CI 1.1, 9.2) μmol/L p = 0.01) and controls (adjusted estimate +4.1 [95% CI 1.7, 8.5] μmol/L p = 0.04). A 5 g increase in total fat intake associated with lower plasma acetate in IA/T1D and controls. A 5% increase in noncore (junk) food intake associated with reduced richness (adjusted estimate -4.09 [95%CI -7.83, -0.35] p = .03) and evenness (-1.25 [95% CI -2.00, -0.49] p < 0.01) of the gut microbiome in IA/T1D. Fiber intake associated with community structure of the microbiome in IA/T1D., Conclusions: Modest increments in carbohydrate and fat intake associated with plasma acetate in all youth. Increased junk food intake associated with reduced diversity of the gut microbiome in IA/T1D alone. These associations with the gut microbiome in IA/T1D support future efforts to promote SCFA by using dietary interventions., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo , either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter-enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions., (Copyright © 2020 Brown, Sadlon, Hope, Wong, Wong, Liu, Withers, Brown, Bandara, Gundsambuu, Pederson, Breen, Robertson, Forrest, Beyer and Barry.)

Recent advances in the understanding of immune function and the interactions with tumour cells have led to the development of various cancer immunotherapies and strategies for specific cancer types. However, despite some stunning successes with some malignancies such as melanomas and lung cancer, most patients receive little or no benefit from immunotherapy, which has been attributed to the tumour microenvironment and immune evasion. Although the US Food and Drug Administration have approved immunotherapies for some cancers, to date, only the anti-angiogenic antibody bevacizumab is approved for the treatment of epithelial ovarian cancer. Immunotherapeutic strategies for ovarian cancer are still under development and being tested in numerous clinical trials. A detailed understanding of the interactions between cancer and the immune system is vital for optimisation of immunotherapies either alone or when combined with chemotherapy and other therapies. This article, in two main parts, provides an overview of: (1) components of the normal immune system and current knowledge regarding tumour immunology, biology and their interactions; (2) strategies, and targets, together with challenges and potential innovative approaches for cancer immunotherapy, with attention given to epithelial ovarian cancer.

Backgrounds: We aimed to monitor pancreatic exocrine function longitudinally in relation to the development of islet autoimmunity (IA) and type 1 diabetes (T1D) in at-risk children with a first-degree relative with T1D, who were followed prospectively in the Environmental Determinants of Islet Autoimmunity (ENDIA) study., Methods: Fecal elastase-1 (FE-1) concentration was measured longitudinally in 85 ENDIA children from median age 1.0 (IQR 0.7,1.3) year. Twenty-eight of 85 children (progressors) developed persistent islet autoantibodies at median age of 1.5 (IQR 1.1,2.5) years, of whom 11 went on to develop clinical diabetes. The other 57 islet autoantibody-negative children (non-progressors) followed similarly were age and gender-matched with the progressors. An adjusted linear mixed model compared FE-1 concentrations in progressors and non-progressors., Results: Baseline FE-1 did not differ between progressors and non-progressors, or by HLA DR type or proband status. FE-1 decreased over time in progressors in comparison to non-progressors (Wald statistic 5.46, P = .02); in some progressors the fall in FE-1 preceded the onset of IA., Conclusions: Pancreatic exocrine function decreases in the majority of young at-risk children who progress to IA and T1D., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The immune-regulatory microRNA miR-155 is reduced in recurrent miscarriage, suggesting that miR-155 contributes to immune tolerance in pregnancy. Here we show miR-155 is induced in the uterine mucosa and draining lymph nodes (dLN) during the female immune response to male seminal fluid alloantigens. Mice with null mutation in miR-155 (miR-155 -/- ) exhibited a reduced CD4 + T cell response after mating, with a disproportionate loss of CD25+FOXP3+ Treg cells. miR-155 deficiency impaired expansion of both peripheral and thymic Treg cells, distinguished by neuropilin-1 (NRP1), and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. Altered Treg phenotype distribution in miR-155 -/- mice was confirmed by t-distributed neighbor embedding (tSNE) analysis. Fewer dendritic cells (DCs) and macrophages trafficked to the dLN of miR-155 -/- mice, associated with lower CCR7 on DCs, and reduced uterine Ccl19 expression, implicating compromised antigen presentation in the stunted Treg cell response. miR-155 -/- mice exhibited elevated susceptibility to inflammation-induced fetal loss and fetal growth restriction compared with miR-155 +/+ controls, but outcomes were restored by transfer of wild-type Tregs. Thus miR-155 is a key regulator of immune adaptation to pregnancy and is necessary for sufficient Tregs to achieve robust pregnancy tolerance and protect against fetal loss.

Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Background: Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing., Methods: Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first-degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three-monthly in the first year of life., Results: Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking., Conclusions: We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Injectable, thermoresponsive hydrogels are promising candidates for the delivery, maintenance and controlled release of adoptive cell therapies. Therefore, there is significant interest in the development of cytocompatible and biodegradable thermoresponsive hydrogels with appropriate gelling characteristics. Towards this end, a series of thermoresponsive copolymers consisting of poly(caprolactone) (PCL), poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) segments, with various PEG:PPG ratios, were synthesised via ring-opening polymerisation (ROP) of ε-caprolactone and epoxy-functionalised PEG and PPG derivatives. The resultant PCL-PEG-PPG copolymers were characterised via proton nuclear magnetic resonance (1H NMR) spectroscopy, gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The thermoresponsive characteristics of the aqueous copolymer solutions at various concentrations was investigated using the inversion method. Whilst all of the copolymers displayed thermoresponsive properties, the copolymer with a ratio of 1:2 PEG:PPG exhibited an appropriate sol-gel transition (28 °C) at a relatively low concentration (10 wt%), and remained a gel at 37 °C. Furthermore, the copolymers were shown to be enzymatically degradable in the presence of lipases and could be used for the encapsulation of CD4+ T-cell lymphocytes. These results demonstrate that the thermoresponsive PCL-PEG-PPG hydrogels may be suitable for use as an adoptive cell therapy (ACT) delivery vehicle., Competing Interests: The authors declare no conflict of interest.

CD4 + T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16 + Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims/hypothesis: To investigate the longitudinal relationship between the gut microbiome, circulating short chain fatty acids (SCFAs) and intestinal permeability in children with islet autoimmunity or type 1 diabetes and controls., Methods: We analyzed the gut bacterial microbiome, plasma SCFAs, small intestinal permeability and dietary intake in 47 children with islet autoimmunity or recent-onset type 1 diabetes and in 41 unrelated or sibling controls over a median (range) of 13 (2-34) months follow-up., Results: Children with multiple islet autoantibodies (≥2 IA) or type 1 diabetes had gut microbiome dysbiosis. Anti-inflammatory Prevotella and Butyricimonas genera were less abundant and these changes were not explained by differences in diet. Small intestinal permeability measured by blood lactulose:rhamnose ratio was higher in type 1 diabetes. Children with ≥2 IA who progressed to type 1 diabetes (progressors), compared to those who did not progress, had higher intestinal permeability (mean [SE] difference +5.14 [2.0], 95% confidence interval [CI] 1.21, 9.07, P = .006), lower within-sample (alpha) microbial diversity (31.3 [11.2], 95% CI 9.3, 53.3, P = .005), and lower abundance of SCFA-producing bacteria. Alpha diversity (observed richness) correlated with plasma acetate levels in all groups combined (regression coefficient [SE] 0.57 [0.21], 95% CI 0.15, 0.99 P = .008)., Conclusions/interpretation: Children with ≥2 IA who progress to diabetes, like those with recent-onset diabetes, have gut microbiome dysbiosis associated with increased intestinal permeability. Interventions that expand gut microbial diversity, in particular SCFA-producing bacteria, may have a role to decrease progression to diabetes in children at-risk., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3 + CD4 + Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1 + tTregs and 30% neuropilin 1 - pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t -stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4 + T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women., (Copyright © 2019 by The American Association of Immunologists, Inc.)

T cells from neonates (cord blood) with a tendency to develop allergic diseases express low PKCζ levels. More extensive investigations into PKC isozyme levels in T cell subsets and changes during neonatal T cell maturation are hampered by limitations of Western blot analyses. We have undertaken to validating the specificity of commercially available antibodies marketed for flow cytometry to measure PKCα, βI, βII, δ, ε, η, θ, ζ, ι/λ and μ. Western blot analyses of human peripheral blood mononuclear cell (PBMC) lysates demonstrated that some antibodies were unsuitable for flow cytometry assays. A panel of antibodies with the desirable specificity and reliability in the flow cytometry assay were identified using both PBMC and whole blood assays. The results showed that all PKC isozymes were expressed in CD4 + and CD8 + T cells, monocytes and neutrophils. Murine lymphocytes showed similar patterns of expression. A major finding was that 35.2% and 38.5% of cord blood samples have low PKCζ (≤the 5 th percentile of adult levels) in the CD4 + and CD8 + subsets, respectively, consistent with the incidence of allergy development in the population. Furthermore, these low PKCζ levels 'normalised' within 24 h after initiation of maturation of these cells in culture, providing a 'window of opportunity' for altering PKCζ levels.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E 2 (PGE 2 ) into the metabolite 15-keto PGE 2 , was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE 2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inflammation is a central feature and is implicated as a causal factor in preeclampsia and other hypertensive disorders of pregnancy. Inflammatory mediators and leukocytes, which are elevated in peripheral blood and gestational tissues, contribute to the uterine vascular anomalies and compromised placental function that characterize particularly the severe, early onset form of disease. Regulatory T (Treg) cells are central mediators of pregnancy tolerance and direct other immune cells to counteract inflammation and promote robust placentation. Treg cells are commonly perturbed in preeclampsia, and there is evidence Treg cell insufficiency predates onset of symptoms. A causal role is implied by mouse studies showing sufficient numbers of functionally competent Treg cells must be present in the uterus from conception, to support maternal vascular adaptation and prevent later placental inflammatory pathology. Treg cells may therefore provide a tractable target for both preventative strategies and treatment interventions in preeclampsia. Steps to boost Treg cell activity require investigation and could be incorporated into pregnancy planning and preconception care. Pharmacological interventions developed to target Treg cells in autoimmune conditions warrant consideration for evaluation, utilizing rigorous clinical trial methodology, and ensuring safety is paramount. Emerging cell therapy tools involving in vitro Treg cell generation and/or expansion may in time become relevant. The success of preventative and therapeutic approaches will depend on resolving several challenges including developing informative diagnostic tests for Treg cell activity applicable before conception or during early pregnancy, selection of relevant patient subgroups, and identification of appropriate windows of gestation for intervention.

Background: The importance of gut bacteria in human physiology, immune regulation, and disease pathogenesis is well established. In contrast, the composition and dynamics of the gut virome are largely unknown; particularly lacking are studies in pregnancy. We used comprehensive virome capture sequencing to characterize the gut virome of pregnant women with and without type 1 diabetes (T1D), longitudinally followed in the Environmental Determinants of Islet Autoimmunity study., Methods: In total, 61 pregnant women (35 with T1D and 26 without) from Australia were examined. Nucleic acid was extracted from serial fecal specimens obtained at prenatal visits, and viral genomes were sequenced by virome capture enrichment. The frequency, richness, and abundance of viruses were compared between women with and without T1D., Results: Two viruses were more prevalent in pregnant women with T1D: picobirnaviruses (odds ratio [OR], 4.2; 95% confidence interval [CI], 1.0-17.1; P = .046) and tobamoviruses (OR, 3.2; 95% CI, 1.1-9.3; P = .037). The abundance of 77 viruses significantly differed between the 2 maternal groups (≥2-fold difference; P < .02), including 8 Enterovirus B types present at a higher abundance in women with T1D., Conclusions: These findings provide novel insight into the composition of the gut virome during pregnancy and demonstrate a distinct profile of viruses in women with T1D.

Control of oncogenes, including ZEB1 and ZEB2, is a major checkpoint for preventing cancer, and loss of this control contributes to many cancers, including breast cancer. Thus tumour suppressors, such as FOXP3, which is mutated or lost in many cancer tissues, play an important role in maintaining normal tissue homeostasis. Here we show for the first time that ZEB2 is selectively down regulated by FOXP3 and also by the FOXP3 induced microRNA, miR-155. Interestingly, neither FOXP3 nor miR-155 directly altered the expression of ZEB1. In breast cancer cells repression of ZEB2, independently of ZEB1, resulted in reduced expression of a mesenchymal marker, Vimentin and reduced invasion. However, there was no de-repression of E-cadherin and migration was enhanced. Small interfering RNAs targeting ZEB2 suggest that this was a direct effect of ZEB2 and not FOXP3/miR-155. In normal human mammary epithelial cells, depletion of endogenous FOXP3 resulted in de-repression of ZEB2, accompanied by upregulated expression of vimentin, increased E-cadherin expression and cell morphological changes. We suggest that FOXP3 may help maintain normal breast epithelial characteristics through regulation of ZEB2, and loss of FOXP3 in breast cancer cells results in deregulation of ZEB2., Competing Interests: CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest.

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at -80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

Regulatory T cells (Treg) are critical for preventing autoimmunity and curtailing responses of conventional effector T cells (Tconv). The reprogramming of T-cell fate and function to generate Treg requires switching on and off of key gene regulatory networks, which may be initiated by a subtle shift in expression levels of specific genes. This can be achieved by intermediary regulatory processes that include microRNA and long noncoding RNA-based regulation of gene expression. There are well-documented microRNA profiles in Treg and Tconv, and these can operate to either reinforce or reduce expression of a specific set of target genes, including FOXP3 itself. This type of feedforward/feedback regulatory loop is normally stable in the steady state, but can alter in response to local cues or genetic risk. This may go some way to explaining T-cell plasticity. In addition, in chronic inflammation or autoimmunity, altered Treg/Tconv function may be influenced by changes in enhancer-promoter interactions, which are highly cell type-specific. These interactions are impacted by genetic risk based on genome-wide association studies and may cause subtle alterations to the gene regulatory networks controlled by or controlling FOXP3 and its target genes. Recent insights into the 3D organisation of chromatin and the mapping of noncoding regulatory regions to the genes they control are shedding new light on the direct impact of genetic risk on T-cell function and susceptibility to inflammatory and autoimmune conditions.

Introduction: Laboratory-based ex vivo cell culture methods are largely manual in their manufacturing processes. This makes it extremely difficult to meet regulatory requirements for process validation, quality control and reproducibility. Cell culture concepts with a translational focus need to embrace a more automated approach where cell yields are able to meet the quantitative production demands, the correct cell lineage and phenotype is readily confirmed and reagent usage has been optimized. Areas covered: This article discusses the obstacles inherent in classical laboratory-based methods, their concomitant impact on cost-of-goods and that a technology step change is required to facilitate translation from bed-to-bedside. Expert opinion: While traditional bioreactors have demonstrated limited success where adherent cells are used in combination with microcarriers, further process optimization will be required to find solutions for commercial-scale therapies. New cell culture technologies based on 3D-printed cell culture lattices with favourable surface to volume ratios have the potential to change the paradigm in industry. An integrated Quality-by-Design /System engineering approach will be essential to facilitate the scaled-up translation from proof-of-principle to clinical validation.

One of the most significant hurdles to the affordable, accessible delivery of cell therapy is the cost and difficulty of expanding cells to clinically relevant numbers. Immunotherapy to prevent autoimmune disease, tolerate organ transplants or target cancer critically relies on the expansion of specialized T cell populations. We have designed 3D-printed cell culture lattices with highly organized micron-scale architectures, functionalized via plasma polymerization to bind monoclonal antibodies that trigger cell proliferation. This 3D technology platform facilitate the expansion of therapeutic human T cell subsets, including regulatory, effector, and cytotoxic T cells while maintaining the correct phenotype. Lentiviral gene delivery to T cells is enhanced in the presence of the lattices. Incorporation of the lattice format into existing cell culture vessels such as the G-Rex system is feasible. This cell expansion platform is user-friendly and expedites cell recovery and scale-up, making it ideal for translating T cell therapies from bench to bedside., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The reproductive tract environment at conception programs the developmental trajectory of the embryo, sets the course of pregnancy, and impacts offspring phenotype and health. Despite the fundamental importance of this stage of reproduction, the rate-limiting regulatory mechanisms operating locally to control fertility and fecundity are incompletely understood. Emerging studies highlight roles for microRNAs (miRNAs) in regulating reproductive and developmental processes and in modulating the quality and strength of the female immune response. Since endometrial receptivity and robust placentation require specific adaptation of the immune response, we hypothesize that miRNAs participate in establishing pregnancy through effects on key gene networks in immune cells. Our recent studies investigated miRNAs that are induced in the peri-conception environment, focusing on miRNAs that have immune-regulatory roles-particularly miR-223, miR-155, and miR-146a. Genetic mouse models deficient in individual miRNAs are proving informative in defining roles for these miRNAs in the generation and stabilization of regulatory T cells (Treg cells) that confer adaptive immune tolerance. Overlapping and redundant functions between miRNAs that target multiple genes, combined with multiple miRNAs targeting individual genes, indicate complex and sensitive regulatory networks. Although to date most data on miRNA regulation of reproductive events are from mice, conserved functions of miRNAs across species imply similar biological pathways operate in all mammals. Understanding the regulation and roles of miRNAs in the peri-conception immune response will advance our knowledge of how environmental determinants act at conception, and could have practical applications for animal breeding as well as human fertility., (© 2017 Wiley Periodicals, Inc.)

Quantification of cell-associated nanoparticles (NPs) is a paramount question in both nanomedicine and nanotoxicology. Inductively coupled plasma mass spectrometry is a well-established method to resolve cell-associated (metal) NPs in bulk cell populations, however, such analysis at single cell level remains a challenge. Here we used mass cytometry, a technique that combines single cell analysis and time-of-flight mass spectrometry, to quantitatively analyze extra- and intracellular silver (Ag) in individual Ag NP exposed human T-lymphocytes. The results revealed significant population heterogeneity: for example, in lymphocytes exposed to 3 μg of 30 nm branched polyethylene imine coated Ag NPs/mL the extracellularly bound Ag varied from 79 to 560 fg and cellular uptake from 17 to 121 fg. Similar amplitude of heterogeneity was observed in cells exposed to various doses of Ag NPs with other sizes and surface coatings, demonstrating the importance of single cell analysis when studying NP-cell interactions. Although mass cytometry has some shortcomings such as inability to analyze potential transformation or dissolution of NPs in cells, we consider this method as the most promising for quantitative assessment of cell-NP interaction at single cell level.

The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/β-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141-2150, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene (IQSEC2) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippocampal cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2, which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in the morphology of developing neurons.

The objective of this study was to trial point of truth calibration (POTCal) as a novel method for disease prioritisation. To illustrate the application of this method, we used a previously described case-study of prioritisation of exotic diseases for the pig industry in Australia. Disease scenarios were constructed from criteria which described potential impact and pig-producers were asked to score the importance of each scenario. POTCal was used to model participants' estimates of disease importance as a function of the criteria, to derive a predictive model to prioritise a range of exotic diseases. The best validation of producers' estimates was achieved using a model derived from all responses. The highest weighted criteria were attack rate, case fatality rate and market loss, and the highest priority diseases were the vesicular diseases followed by swine fevers and zoonotic encephalitides. Comparison of results with a previous study in which probabilistic inversion was used to prioritise diseases for the same group of producers highlighted differences between disease prioritisation methods. Overall, this study demonstrated that POTCal can be used for disease prioritisation. An advantage of POTCal is that valid models can be developed that reflect decision-makers' heuristics. Specifically, this evaluation of the use of POTCal in animal health illustrates how the judgements of participants can be incorporated into a decision-making process. Further research is needed to investigate the influence of scenarios presented to participants during POTCal evaluations, and the robustness of this approach applied to different disease issues (e.g. exotic versus endemic) and production types (e.g. intensive versus extensive). To our knowledge, this is the first report of the use of POTCal for disease prioritisation., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Wildlife collision data are ubiquitous, though challenging for making ecological inference due to typically irreducible uncertainty relating to the sampling process. We illustrate a new approach that is useful for generating inference from predator data arising from wildlife collisions. By simply conditioning on a second prey species sampled via the same collision process, and by using a biologically realistic numerical response functions, we can produce a coherent numerical response relationship between predator and prey. This relationship can then be used to make inference on the population size of the predator species, including the probability of extinction. The statistical conditioning enables us to account for unmeasured variation in factors influencing the runway strike incidence for individual airports and to enable valid comparisons. A practical application of the approach for testing hypotheses about the distribution and abundance of a predator species is illustrated using the hypothesized red fox incursion into Tasmania, Australia. We estimate that conditional on the numerical response between fox and lagomorph runway strikes on mainland Australia, the predictive probability of observing no runway strikes of foxes in Tasmania after observing 15 lagomorph strikes is 0.001. We conclude there is enough evidence to safely reject the null hypothesis that there is a widespread red fox population in Tasmania at a population density consistent with prey availability. The method is novel and has potential wider application., Competing Interests: The authors declare they have no competing interests. All authors are employees of Data61, Commonwealth Scientific and Industrial Research Organisation.

Fluctuations in circulating estrogen and progesterone across the menstrual cycle lead to increased breast cancer susceptibility in women; however, the biological basis for this increased risk is not well understood. Estrogen and progesterone have important roles in normal mammary gland development, where they direct dynamic interactions among the hormonally regulated mammary epithelial, stromal, and immune cell compartments. The continuous fluctuations of estrogen and progesterone over a woman's reproductive lifetime affect the turnover of mammary epithelium, stem cells, and the extracellular matrix, as well as regulate the phenotype and function of mammary stromal and immune cells, including macrophages and regulatory T cells. Collectively, these events may result in genome instability, increase the chance of random genetic mutations, dampen immune surveillance, and promote tolerance in the mammary gland, and thereby increase the risk of breast cancer initiation. This article reviews the current status of our understanding of the molecular and the cellular changes that occur in the mammary gland across the menstrual cycle and how continuous menstrual cycling may increase breast cancer susceptibility in women.

Objective: To compare the incidence of complications with a primary gastrostomy versus gastrostomy with concurrent fundoplication and evaluating the impact of the method of gastrostomy tube placement. Neurologically impaired children were compared with neurologically normal children. Two low profile devices were compared for longevity., Methods: Ninety-eight patients (58 boys, mean age 4.66 years) with 107 gastrostomies inserted between April 2004 and May 2008 were included in this retrospective, single institution audit. Minimum follow-up period was 1 year. Specific complications reviewed were tube and site related. Logistic regression analysis examined the relationship between complications, type of procedure, method of placement, and neurological status. Survival analysis with log-rank test was used to compare the duration of the low-profile devices., Results: There were 63 primary gastrostomies and 44 with concurrent fundoplication, 71 children were neurologically impaired. Mean (±SD) follow-up time was 35.6 ± 1.4 months. There was a significant association between concurrent gastrostomy insertion with fundoplication and incidence of infection (odds ratio = 2.4, 95% confidence interval (CI) 1.02-5.56, P = 0.02) and excoriation (odds ratio = 2.5, 95% CI 1.09-5.71, P = 0.015). There were no associations between the complications with gastrostomy placement and neurological status. Failure rate of the balloon device was significantly greater than the fixed bolster device with a Hazard Ratio for survival of 3.2 (95% CI 2.2-4.6)., Conclusions: Gastrostomy site-related problems were more common than generally reported. There was a higher incidence of site infection and skin excoriation for gastrostomy placement with concurrent fundoplication. There was no significant difference in complications between the method of gastrostomy placement or neurological status. Balloon devices were changed 3 times more often than bolster retention devices.

The cost of an uncontrolled incursion of invasive alien species (IAS) arising from undetected entry through ports can be substantial, and knowledge of port-specific risks is needed to help allocate limited surveillance resources. Quantifying the establishment likelihood of such an incursion requires quantifying the ability of a species to enter, establish, and spread. Estimation of the approach rate of IAS into ports provides a measure of likelihood of entry. Data on the approach rate of IAS are typically sparse, and the combinations of risk factors relating to country of origin and port of arrival diverse. This presents challenges to making formal statistical inference on establishment likelihood. Here we demonstrate how these challenges can be overcome with judicious use of mixed-effects models when estimating the incursion likelihood into Australia of the European (Apis mellifera) and Asian (A. cerana) honeybees, along with the invasive parasites of biosecurity concern they host (e.g., Varroa destructor). Our results demonstrate how skewed the establishment likelihood is, with one-tenth of the ports accounting for 80% or more of the likelihood for both species. These results have been utilized by biosecurity agencies in the allocation of resources to the surveillance of maritime ports., (© 2015 Society for Risk Analysis.)

Maternal interleukin (IL) 10 deficiency elevates susceptibility to fetal loss induced by the model Toll-like receptor agonist lipopolysaccharide, but the mechanisms are not well elucidated. Here, we show that Il10 null mutant (Il10(-/-)) mice exhibit altered local T cell responses in pregnancy, exhibiting pronounced hyperplasia in para-aortic lymph nodes draining the uterus with >6-fold increased CD4(+) and CD8(+) T cells compared with wild-type controls. Among these CD4(+) cells, Foxp3(+) T regulatory (Treg) cells were substantially enriched, with 11-fold higher numbers at Day 9.5 postcoitum. Lymph node hypertrophy in Il10(-/-) mice was associated with more activated phenotypes in dendritic cells and macrophages, with elevated expression of MHCII, scavenger receptor, and CD80. Affymetrix microarray revealed an altered transcriptional profile in Treg cells from pregnant Il10(-/-) mice, with elevated expression of Ctse (cathepsin E), Il1r1, Il12rb2, and Ifng. In vitro, Il10(-/-) Treg cells showed reduced steady-state Foxp3 expression, and polyclonal stimulation caused greater loss of Foxp3 and reduced capacity to suppress IL17 in CD4(+)Foxp3(-) T cells. We conclude that despite a substantially expanded Treg cell pool, the diminished stability of Treg cells, increased numbers of effector T cells, and altered phenotypes in dendritic cells and macrophages in pregnancy all potentially confer vulnerability to inflammation-induced fetal loss in Il10(-/-) mice. These findings suggest that IL10 has a pivotal role in facilitating robust immune protection of the fetus from inflammatory challenge and that IL10 deficiency could contribute to human gestational disorders in which altered T cell responses are implicated., (© 2015 by the Society for the Study of Reproduction, Inc.)