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2022 ◽  
Vol 12 ◽  
Author(s):  
Brian Akhaphong ◽  
Brigid Gregg ◽  
Doga Kumusoglu ◽  
Seokwon Jo ◽  
Kanakadurga Singer ◽  
...  

The risk of obesity in adulthood is subject to programming in the womb. Maternal obesity contributes to programming of obesity and metabolic disease risk in the adult offspring. With the increasing prevalence of obesity in women of reproductive age there is a need to understand the ramifications of maternal high-fat diet (HFD) during pregnancy on offspring’s metabolic heath trajectory. In the present study, we determined the long-term metabolic outcomes on adult male and female offspring of dams fed with HFD during pregnancy. C57BL/6J dams were fed either Ctrl or 60% Kcal HFD for 4 weeks before and throughout pregnancy, and we tested glucose homeostasis in the adult offspring. Both Ctrl and HFD-dams displayed increased weight during pregnancy, but HFD-dams gained more weight than Ctrl-dams. Litter size and offspring birthweight were not different between HFD-dams or Ctrl-dams. A significant reduction in random blood glucose was evident in newborns from HFD-dams compared to Ctrl-dams. Islet morphology and alpha-cell fraction were normal but a reduction in beta-cell fraction was observed in newborns from HFD-dams compared to Ctrl-dams. During adulthood, male offspring of HFD-dams displayed comparable glucose tolerance under normal chow. Male offspring re-challenged with HFD displayed glucose intolerance transiently. Adult female offspring of HFD-dams demonstrated normal glucose tolerance but displayed increased insulin resistance relative to controls under normal chow diet. Moreover, adult female offspring of HFD-dams displayed increased insulin secretion in response to high-glucose treatment, but beta-cell mass were comparable between groups. Together, these data show that maternal HFD at pre-conception and during gestation predisposes the female offspring to insulin resistance in adulthood.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3711-3711
Author(s):  
Daniel Friedman ◽  
Antony Long ◽  
Piers EM Patten ◽  
Robbert Hoogeboom

Abstract Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant CD5+ B cells in the peripheral blood (PB), secondary lymphoid tissues and bone marrow. Currently considered an incurable disease, B cell receptor (BCR) signalling plays a key role in the disease aetiology as evidenced by the therapeutic success of BCR signalling inhibitors such as ibrutinib. Previous studies using incorporation of 2H-labelling of DNA in vivo demonstrated sub-clonal heterogeneity in PB CLL cell fractions sorted based on reciprocal densities of chemokine C-X-C motif receptor 4 (CXCR4) and CD5. The CXCR4 loCD5 hi fraction was shown to be enriched in recently born proliferating cells while the CXCR4 hiCD5 lo fraction consists of resting, quiescent cells thought to reflect their migratory and BCR signalling histories in tissue. Whilst these proliferating/resting fractions have since been more closely examined, the remaining bulk PB CLL population has been left relatively unexplored leaving other therapeutically relevant cell fractions undetected. Here, we have comprehensively analysed the phenotype of subpopulations of PB cells from 11 CLL patients using flow cytometry to identify activated and proliferating cell fractions. CD19 +CD5 +cells were divided into 9 fractions based on CXCR4/CD5 densities and to permit comparisons between fractions, each cell fraction was defined as containing 1-2% of the total clonal CD19 +CD5 + population. Surprisingly, we detected enrichment for Ki67+ proliferating cells and high expression of AID in the cell fraction with highest expression levels of both CXCR4 and CD5 (CXCR4 hiCD5 hi), demonstrating that CXCR4 loCD5 hi cells are not the only proliferating fraction in the blood. Moreover, we could detect mitotic cells in the CXCR4 hiCD5 hi fraction using imaging flow cytometry of a nuclear stain. This CXCR4 hiCD5 hi fraction showed the highest surface expression levels of IgM, CD86, CCR7, CXCR3 and CXCR5 of all the fractions assessed (p<0.05), indicating they are highly activated and primed for migration to lymph nodes (LNs) for further activation and proliferation. Proliferation of CLL cells is highest in secondary lymphoid tissues, however the phenotype of proliferating cells in tissue is unknown. To examine the phenotype of proliferating CLL cells in LNs, we analysed a fine-needle aspirate obtained from an enlarged cervical node using flow cytometry and compared this to a matched PB sample. Flow cytometric gates set on the PB sample were used to define and quantify LN cell fractions. Expression levels of both Ki67 and surface IgM were highest in the CXCR4 hiCD5 hi fraction which was expanded to 20% of the CD19 +CD5 + population in the LN whilst CXCR4 loCD5 hi cells (accounting for 2% of the bulk LN population) expressed very low surface IgM and Ki67 levels, suggesting CXCR4 hiCD5 hi cells may be the most proliferative cells in CLL. The CXCR4 loCD5 hi cell fraction has been shown to be a key target of ibrutinib, however the impact of ibrutinib on the CXCR4 hiCD5 hi fraction is unknown. Administration of ibrutinib to PB CLL cells for 48hr in vitro resulted in selective targeted depletion of the CXCR4 loCD5 hi fraction, as evidenced by induction of apoptotic markers in this compartment; conversely, persistent cells after 48hr ibrutinib administration in vitro were exclusively of the CXCR4 hi phenotype. In conclusion, we have identified a potentially dangerous fraction of proliferating cells in the PB of CLL patients with high expression of CXCR4, CD5, IgM, CCR7, CXCR3 and CXCR5 open for both migration to tissue and reception of BCR signals. Furthermore, CXCR4 hiCD5 hi cells in the periphery may closely mirror tissue-resident activated cell phenotypes and may represent critical targets for therapeutic intervention, particularly in high-risk CLL patients refractory to BCR inhibitor therapies. Disclosures Patten: ROCHE: Research Funding; GILEAD SCIENCES: Honoraria, Research Funding; NOVARTIS: Honoraria; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi207-vi208
Author(s):  
Rebeca Nunez ◽  
Miguel Mayol-Del Valle ◽  
Luis Almodovar ◽  
Lilia Kucheryavykh

Abstract Glioblastoma (GBM) is the most aggressive and highly invasive primary brain tumor in adults. Evidence suggests that microglia create a microenvironment favoring glioma invasion and proliferation. Indeed, previous reports indicate the involvement of focal adhesion kinase (FAK) signaling cascades in glioma cell proliferation. Besides, studies from our laboratory support a critical role of Pyk2, a relative of FAK, in glioma invasion by tumor-infiltrating microglia. However, the microglial-released factors modulating Pyk2 and FAK signaling pathways are unknown. In this study, 20 human GBM specimens were evaluated to identify the cytokine expression patterns in purified microglia and FAK and Pyk2 phosphorylation in glioma cell fraction by RT-PCR and western blot. A Pierson correlation test demonstrated a high correlation (0.8-1.0) of gene expression for PDGFα, PDGFβ, SDF-1α, IL-6, IL-8, and EGF in percoll-purified microglia, and pPyk2(Y579/580) and pFAK(Y925) levels in glioma cell fraction. The role of cytokines in cell invasion and proliferation by Pyk2/FAK activation was further investigated in primary cell lines from three patients. Thirty percent up-regulation of pPyk2 and pFAK was detected in glioma cells treated (2 hrs.) with microglia conditioned media (MCM) compared to control cells. siPyk2 or siFAK knockdown identified IL-6 (100 μM) and EGF (1 μM) as key factors of Pyk2- and FAK-dependent activation in all glioma cell lines. Similar results with siPyk2 or siFAK were observed for matrix degradation, invadopodia formation, cell viability, and mitosis. Indeed, Tocilizumab (IL-6R blocker, 100 ng/mL) and Gefitinib (EGFR blocker, 1 μM) reversed the effect of MCM on glioma cell proliferation and invasion in all cell lines evaluated. These findings support a pivotal role of Pyk2 and FAK in enhancing proliferation and invasion of glioma tumors through IL-6 and EGF-dependent pathways. The latter could be of clinical relevance for new therapeutic developments in GBM patients.


Nature ◽  
2021 ◽  
Author(s):  
Robert Bentham ◽  
Kevin Litchfield ◽  
Thomas B. K. Watkins ◽  
Emilia L. Lim ◽  
Rachel Rosenthal ◽  
...  

Cell Systems ◽  
2021 ◽  
Author(s):  
Gryte Satas ◽  
Simone Zaccaria ◽  
Mohammed El-Kebir ◽  
Benjamin J. Raphael

Biorheology ◽  
2021 ◽  
pp. 1-10
Author(s):  
Alper Turgut ◽  
Özlem Yalçin

BACKGROUND: Measurement of abnormal Red Blood Cell (RBC) deformability is a main indicator of Sickle Cell Anemia (SCA) and requires standardized quantification methods. Ektacytometry is commonly used to estimate the fraction of Sickled Cells (SCs) by measuring the deformability of RBCs from laser diffraction patterns under varying shear stress. In addition to estimations from model comparisons, use of maximum Elongation Index differences (ΔEImax) at different laser intensity levels was recently proposed for the estimation of SC fractions. OBJECTIVE: Implement a convolutional neural network to accurately estimate rigid-cell fraction and RBC concentration from laser diffraction patterns without using a theoretical model and eliminating the ektacytometer dependency for deformability measurements. METHODS: RBCs were collected from control patients. Rigid-cell fraction experiments were performed using varying concentrations of glutaraldehyde. Serial dilutions were used for varying the concentration of RBC. A convolutional neural network was constructed using Python and TensorFlow. RESULTS: Our measurements and model predictions show that a linear relationship between ΔEImax and rigid-cell fraction exists only for rigid-cell fractions less than 0.2. Our proposed neural network architecture can be used successfully for both RBC concentration and rigid-cell fraction estimations without a need for a theoretical model.


2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3563-3563
Author(s):  
Emil Lou ◽  
Yasmine Baca ◽  
Joanne Xiu ◽  
Andrew Nelson ◽  
Subbaya Subramanian ◽  
...  

3563 Background: The tumor microenvironment (TME) of colorectal cancers (CRC) is modulated by oncogenic drivers such as KRAS. The TME comprises a broad landscape of immune infiltration. How tumor genomics associates with the immune cell landscape is less known. We aim to characterize immune cell types in RAS wild-type (WT) and mutant (MT) CRC, and to examine the prevalence of immuno-oncologic (IO) biomarkers (e.g. tumor mutation burden (TMB), PD-L1, MSI-H/dMMR) in these tumors. We performed genomic and transcriptomic analysis to confirm associations of mutant RAS with immune infiltration of the TME conducive to metastasis vs. potential response to immunotherapies. Methods: A total of 7,801 CRC were analyzed using next-generation sequencing on DNA (NextSeq, 592 Genes and WES, NovaSEQ), RNA (NovaSeq, whole transcriptome equencing) and IHC (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by FA, IHC and NGS. TMB-H was based on a cut-off of > 10 mutations per MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q) was <0.05. Results: Mutant KRAS was seen in 48% of mCRC tumors; NRAS in 3.7%, HRAS in 0.1%. The distribution was similar in patients < or >= than 50 yrs. In MSS tumors, there was a significantly higher neutrophil infiltration in KRAS MT (median cell fraction 6.6% vs. 5.9%) and NRAS MT (6.9%) overall and also when individual codons were studied. B cells, M2 macrophages, CD8+ T cells, dendritic cells and fibroblasts were lower in KRAS mutant tumors; B cells and M1 macrophages are lower in NRAS (q<0.05). dMMR/MSI-H was significantly more prevalent in RAS WT (9.1%) than in KRAS (2.9%) or NRAS MT (1.8%) tumors, and highest in HRAS MT tumors (60%, q<0.05).TMB-H was more prevalent in RAS WT (11%) than KRAS (5.8%) or NRAS (5.1%) MT, and highest in HRAS MT tumors (70%, all q<0.05). In MSS tumors, KRAS MT tumors showed more TMB-H than WT (3.1% vs. 2.1%, q<0.05), especially in KRAS non 12/13/61 mutations (5.5%, vs. 2.1%, q<0.05) and G12C (4.4%, p<0.05). PD-L1 expression was studied: in MSS tumors, KRAS-G12D (10.4%) and G13 MT (11.8%) showed higher mutation rates than RAS WT tumors (q<0.05). Conclusions: KRAS & NRAS mutations are associated with increased neutrophil abundance, with codon specific differences, while HRAS shows no difference. Overall CD8+ T cells and B cells are less abundant in KRAS & NRAS mutants; substantial variability was seen amongst different protein changes. RAS mutations were more prevalent overall than generally reported, but did not vary by age. These results demonstrate significant differences in the TME of RAS mutant CRC that identify variable susceptibilities to immuno-oncologic agents, and provide further detailed characterization of heterogeneity between RAS variants, at the molecular as well as immunogenic levels.


2021 ◽  
Author(s):  
Gryte Satas ◽  
Simone Zaccaria ◽  
Mohammed El-Kebir ◽  
Benjamin J. Raphael

AbstractMost tumors are heterogeneous mixtures of normal cells and cancer cells, with individual cancer cells distinguished by somatic mutations that accumulated during the evolution of the tumor. The fundamental quantity used to measure tumor heterogeneity from somatic single-nucleotide variants (SNVs) is the Cancer Cell Fraction (CCF), or proportion of cancer cells that contain the SNV. However, in tumors containing copy-number aberrations (CNAs) – e.g. most solid tumors – the estimation of CCFs from DNA sequencing data is challenging because a CNA may alter the mutation multiplicity, or number of copies of an SNV. Existing methods to estimate CCFs rely on the restrictive Constant Mutation Multiplicity (CMM) assumption that the mutation multiplicity is constant across all tumor cells containing the mutation. However, the CMM assumption is commonly violated in tumors containing CNAs, and thus CCFs computed under the CMM assumption may yield unrealistic conclusions about tumor heterogeneity and evolution. The CCF also has a second limitation for phylogenetic analysis: the CCF measures the presence of a mutation at the present time, but SNVs may be lost during the evolution of a tumor due to deletions of chromosomal segments. Thus, SNVs that co-occur on the same phylogenetic branch may have different CCFs.In this work, we address these limitations of the CCF in two ways. First, we show how to compute the CCF of an SNV under a less restrictive and more realistic assumption called the Single Split Copy Number (SSCN) assumption. Second, we introduce a novel statistic, the descendant cell fraction (DCF), that quantifies both the prevalence of an SNV and the past evolutionary history of SNVs under an evolutionary model that allows for mutation losses. That is, SNVs that co-occur on the same phylogenetic branch will have the same DCF. We implement these ideas in an algorithm named DeCiFer. DeCiFer computes the DCFs of SNVs from read counts and copy-number proportions and also infers clusters of mutations that are suitable for phylogenetic analysis. We show that DeCiFer clusters SNVs more accurately than existing methods on simulated data containing mutation losses. We apply DeCiFer to sequencing data from 49 metastatic prostate cancer samples and show that DeCiFer produces more parsimonious and reasonable reconstructions of tumor evolution compared to previous approaches. Thus, DeCiFer enables more accurate quantification of intra-tumor heterogeneity and improves downstream inference of tumor evolution.Code availabilitySoftware is available at https://github.com/raphael-group/decifer


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