Optimization of blood handling and peripheral blood mononuclear cell cryopreservation of low cell number samples

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 × 10⁶ cells/mL to 1.67 × 10⁶ 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 × 10⁶ 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.Christopher M. Hope, Dao Huynh, Ying Ying Wong, Helena Oakey, Griffith Boord Perkins, Trung Nguyen, Sabrina Binkowski, Minh Bui, Ace Y.L. Choo, Emily Gibson, Dexing Huang, Ki Wook Kim, Katrina Ngui, William D. Rawlinson, Timothy Sadlon, Jennifer J. Couper, Megan A.S. Penno, Simon C. Barry, and on behalf of the ENDIA Study Grou

Women with type 1 diabetes exhibit a progressive increase in gut Saccharomyces cerevisiae in pregnancy associated with evidence of gut inflammation.

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.Esther Bandala-Sanchez, Alexandra J. Roth-Schulze, Helena Oakey Megan A.S. Penno, Naiara G. Bediaga, Gaetano Naselli, Katrina M. Ngui, Alannah D. Smith, Dexing Huang, Enrique Zozaya-Valdes, Rebecca L. Thomson, James D. Brown, Peter J. Vuillermin, Simon C. Barry, Maria E. Craig, William D. Rawlinson, Elizabeth A. Davis, Mark Harris, Georgia Soldatos, Peter G. Colman, John M. Wentworth, Aveni Haynes, Grant Morahan, Richard O. Sinnott, Anthony T. Papenfuss, Jennifer J. Couper, Leonard C. Harrison, on behalf of the ENDIA Study Grou

Bunbury to Bundaberg, Darwin to Dover: establishing a successful Regional Participation Program for the ENDIA type 1 diabetes cohort study

Abstract not availableMegan Ann Sabine Penno, Rebecca Louise Thomson, Jennifer J Couper, ENDIA Study Grou

Experiences of Caregivers and At-Risk Children Enrolled in a Prospective Pregnancy-Birth Cohort Study into the Causes of Type 1 Diabetes: The ENDIA Study

Background: We sought research experiences of caregivers and their children were enrolled in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Methods: ENDIA is a pregnancy–birth cohort investigating early-life causes of type 1 diabetes (T1D). Surveys were sent to 1090 families between June 2021 and March 2022 with a median participation of >5 years. Caregivers completed a 12-item survey. Children ≥ 3 years completed a four-item survey. Results: The surveys were completed by 550/1090 families (50.5%) and 324/847 children (38.3%). The research experience was rated as either “excellent” or “good” by 95% of caregivers, and 81% of children were either “ok”, “happy” or “very happy”. The caregivers were motivated by contributing to research and monitoring their children for T1D. Relationships with the research staff influenced the experience. The children most liked virtual reality headsets, toys, and “helping”. Blood tests were least liked by the children and were the foremost reason that 23.4% of the caregivers considered withdrawing. The children valued gifts more than their caregivers. Only 5.9% of responses indicated dissatisfaction with some aspects of the protocol. The self-collection of samples in regional areas, or during the COVID-19 pandemic restrictions, were accepted. Conclusions: This evaluation identified modifiable protocol elements and was conducted to further improve satisfaction. What was important to the children was distinct from their caregivers.Kelly J. McGorm, James D. Brown, Alison G. Roberts, Susan Greenbank, Daniella Brasacchio, Alyssa C. P. Sawyer, Helena Oakey, Peter G. Colman, Maria E. Craig, Elizabeth A. Davis, Georgia Soldatos, Rebecca L. Thomson, John M. Wentworth, Jennifer J. Couper, Megan A. S. Penno, and on behalf of The ENDIA Study Grou

Associations between diet, the gut microbiome and short chain fatty acids in youth with islet autoimmunity and type 1 diabetes

First published: 20 January 2021Aim: 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.Jessica E. Harbison, Rebecca L. Thomson, John M. Wentworth, Jennie Louise, Alexandra Roth-Schulze, Rachel J. Battersby ... et al

Gut Virome Dynamics during Pregnancy in Mothers with Type 1 Diabetes

Session LB-192 - Late Breaking Poster SessionKi Wook Kim, Digby W. Allen, Thomas Briese, Chi Nam Pang, Komal Jain, Jessica L. Horton, Sonia R. Isaacs, Marc R. Wilkins, Jennifer Couper, Megan A.S. Penno, Leonard C. Harrison, Grant Morahan, Mark Harris, Andrew M. Cotterill, Claire Morbey, Simon C. Barry, Aveni Haynes, Elizabeth Davis, Peter Colman, Lynne Giles, Jodie Dodd, John Wentworth, Richard Sinnott, Tony A. Papenfuss, Peter Vuillermin, W. Ian Lipkin, William D. Rawlinson, Maria E. Crai

Mental Health During Late Pregnancy and Postpartum in Mothers With and Without Type 1 Diabetes: The ENDIA Study

This article contains supplementary material online at https://doi.org/10.2337/figshare.18551261.Objective: Pregnancy and type 1 diabetes are each associated with increased anxiety and depression, but the combined impact on well-being is unresolved. We compared the mental health of women with and without type 1 diabetes during pregnancy and postpartum and examined the relationship between mental health and glycemic control. Research Design and Methods: Participants were women enrolled from 2016 to 2020 in the Environmental Determinants of Islet Autoimmunity (ENDIA) study, a pregnancy to birth prospective cohort following children with a first-degree relative with type 1 diabetes. Edinburgh Postnatal Depression Scale (EPDS) and Perceived Stress Scale (PSS) were completed during the third trimester (T3) (median [interquartile range] 34 [32, 36] weeks) and postpartum (14 [13, 16] weeks) by 737 women (800 pregnancies) with (n = 518) and without (n = 282) type 1 diabetes. Results: EPDS and PSS scores did not differ between women with and without type 1 diabetes during T3 and postpartum. EPDS scores were marginally higher in T3: predicted mean (95% CI) 5.7 (5.4, 6.1) than postpartum: 5.3 (5.0, 5.6), independent of type 1 diabetes status (P = 0.01). HbA1c levels in type 1 diabetes were 6.3% [5.8, 6.9%] in T3 and did not correlate with EPDS or PSS scores. Reported use of psychotropic medications was similar in women with (n = 44 of 518 [8%]) and without type 1 diabetes (n = 17 of 282 [6%]), as was their amount of physical activity. Conclusions: Overall, mental health in late pregnancy and postpartum did not differ between women with and without type 1 diabetes, and mental health scores were not correlated with glycemic control.Madeleine Hall, Helena Oakey, Megan A.S. Penno, Kelly McGorm, Amanda J. Anderson, Pat Ashwood, Peter G. Colman, Maria E. Craig, Elizabeth A. Davis, Mark Harris, Leonard C. Harrison, Aveni Haynes, Claire Morbey, Richard O. Sinnott, Georgia Soldatos, Peter J. Vuillermin, John M. Wentworth, Rebecca L. Thomson, Jennifer J. Couper on behalf of the ENDIA Study Grou