Birthweight and risk markers for type 2 diabetes and cardiovascular disease in childhood: the Child Heart and Health Study in England (CHASE).

AIMS/HYPOTHESIS: Lower birthweight (a marker of fetal undernutrition) is associated with higher risks of type 2 diabetes and cardiovascular disease (CVD) and could explain ethnic differences in these diseases. We examined associations between birthweight and risk markers for diabetes and CVD in UK-resident white European, South Asian and black African-Caribbean children. METHODS: In a cross-sectional study of risk markers for diabetes and CVD in 9- to 10-year-old children of different ethnic origins, birthweight was obtained from health records and/or parental recall. Associations between birthweight and risk markers were estimated using multilevel linear regression to account for clustering in children from the same school. RESULTS: Key data were available for 3,744 (66%) singleton study participants. In analyses adjusted for age, sex and ethnicity, birthweight was inversely associated with serum urate and positively associated with systolic BP. After additional height adjustment, lower birthweight (per 100 g) was associated with higher serum urate (0.52%; 95% CI 0.38, 0.66), fasting serum insulin (0.41%; 95% CI 0.08, 0.74), HbA1c (0.04%; 95% CI 0.00, 0.08), plasma glucose (0.06%; 95% CI 0.02, 0.10) and serum triacylglycerol (0.30%; 95% CI 0.09, 0.51) but not with BP or blood cholesterol. Birthweight was lower among children of South Asian (231 g lower; 95% CI 183, 280) and black African-Caribbean origin (81 g lower; 95% CI 30, 132). However, adjustment for birthweight had no effect on ethnic differences in risk markers. CONCLUSIONS/INTERPRETATION: Birthweight was inversely associated with urate and with insulin and glycaemia after adjustment for current height. Lower birthweight does not appear to explain emerging ethnic difference in risk markers for diabetes

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV

Abstracts from the NIHR INVOLVE Conference 2017

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31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Conjugation of peptides with polymers synthesised via living radical polymerisation

The reaction of salmon calcitonin (sCT) with various linear PEG and PolyPEGs was investigated under a variety of reaction conditions. The optimal experimental conditions for the production of high yield monoconjugated sCT were determined using RP-HPLC, SEC-HPLC, IE-FPLC and MALDI-TOF MS analysis. Semi-preparative IE-FPLC was used to purify high yield mono conjugated sCT-5 kDa linear PEG, sCT -6.5 kDa PolyPEG, sCT-20 kDa PolyPEG and sCT-40 kDa PolyPEG coupled to the N-terminal primary amine. The products were analysed using RP-HPLC, SEC-HPLC, SDS-PAGE and the site of attachment was determined by Tryptic mapping combined with LCMS, RP-HPLC and MALDI-TOF MS. Biological activity was measured invitro, sCT derivates were incubated with T47D cells and an ELISA assay was used to measure the amount of cAMP released. The purified conjugates retained a high degree of biological activity compared to the unmodified peptide. Incubation of sCT and sCTconjugates with proteolytic enzymes showed that for sCT-5 kDa linear PEG and 6.5kDa PolyPEG a high degree (60-70 % of the activity at time zero) of biological activity remained after 30 minutes compared to 0% activity for native sCT.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Conjugation of peptides with polymers synthesised via living radical polymerisation

The reaction of salmon calcitonin (sCT) with various linear PEG and PolyPEGs was investigated under a variety of reaction conditions. The optimal experimental conditions for the production of high yield monoconjugated sCT were determined using RP-HPLC, SEC-HPLC, IE-FPLC and MALDI-TOF MS analysis. Semi-preparative IE-FPLC was used to purify high yield mono conjugated sCT-5 kDa linear PEG, sCT -6.5 kDa PolyPEG, sCT-20 kDa PolyPEG and sCT-40 kDa PolyPEG coupled to the N-terminal primary amine. The products were analysed using RP-HPLC, SEC-HPLC, SDS-PAGE and the site of attachment was determined by Tryptic mapping combined with LCMS, RP-HPLC and MALDI-TOF MS. Biological activity was measured invitro, sCT derivates were incubated with T47D cells and an ELISA assay was used to measure the amount of cAMP released. The purified conjugates retained a high degree of biological activity compared to the unmodified peptide. Incubation of sCT and sCTconjugates with proteolytic enzymes showed that for sCT-5 kDa linear PEG and 6.5kDa PolyPEG a high degree (60-70 % of the activity at time zero) of biological activity remained after 30 minutes compared to 0% activity for native sCT

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel comb-shaped end-functionalised (poly(PEG) methyl ether methacrylate) (sCT-P) polymers, to yield conjugates of total molecular weights (MW) inclusive of sCT: 6.5, 9.5, 23 and 40 kDa. The conjugates were characterised by HPLC and their in vitro and in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of Emax and an EC50 of 3.7 nM. In vivo, while free sCT and the conjugates reduced serum [calcium] to a maximum of 15–30% over 240 min, the half-life (T1/2) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40 kDa). Mathematical modelling accurately predicted the MW relationships to in vitro efficacy, potency, in vivo PK and enzymatic stability. With a significant increase in T1/2 for sCT, the 40 kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation

Site-specific N-terminus conjugation of poly(mPEG(1100)) methacrylates to salmon calcitonin : synthesis and preliminary biological evaluation

Recent advances in polymerization strategies have led to the development of novel polymer (poly) peptide biohybrid materials with potential application in the field of macromolecular therapeutics. In this current work, comb-shaped alpha-aldehyde poly(monomethoxy polyethylene glycol)methacrylates (p(mPEG)MA) with molecular masses in the 6.5-109 kDa range were prepared and conjugated, via reductive amination, to the Cys1 N-terminus of salmon calcitonin (sCT), a calcitropic hormone currently administered for the treatment of a number of hypercalcaemia-related diseases. The conjugation site was determined by tryptic digestion of the sCT-p(mPEG(1100)) MA biohybrids in conjunction with LC-MS MALDI-TOF spectrometry. Preliminary in vitro biological tests show that the polymer conjugation does not interfere with the biological activity of the sCT