Changes In Number Of Cells Research Articles

Inhibition of Cyclin Dependent Kinase 8(CDK8): A Novel Approach to Target the Leukemia Initiating Cells (LICs) in T-Cell Acute Lymphoblastic Leukaemia (T-ALL)

Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy characterized by an aberrant proliferation of immature thymocytes affecting preferentially children and adolescents. Approximately 70% of T-ALL cases have activating mutations of the NOTCH1 followed by oncogenic pathways PI3K/Akt, the anti-apoptotic BCL-2 family, and CDKN2A/2B cell cycle regulators 1, 2. Unlike other leukemias such as chronic myeloid leukemia (CML) and Philadelphia-positive ALL, which are kinase-driven malignancies 3, 4, the initiating events in T-ALL cause the ectopic expression of transcription factors that drive leukemogenesis, hence targeted therapies needed to focused on this field. Cyclin Dependent Kinase 8 (CDK8) and its paralog CDK19, restrain activation of super-enhancer-associated tumor suppressors and lineage commitment genes in leukemia. Moreover CDK8 phosphorylates intracellular domain (ICD) and regulates activation and turnover of NOTCH1 5, 6, indicating that targeting CDK8 with specific inhibitors may be considered as a novel therapeutic strategy for T-ALL. RVU120 is a first-in-class, specific and selective inhibitor of CDK8/CDK19, currently in phase Ib dose-escalation trial in patients with relapsed or refractory (R/R) AML and HR-MDS (NCT04021368).Objectives: The goals of these studies were to investigated the effectiveness of RVU120 and other non-related CDK8/19 inhibitors against T-ALL cell lines/PDXs, including GSI-resistant lines. Comprehensive analysis of target inhibition and apoptotic signatures were evaluated. The clinical relevance of RVU120 in LICs was confirmed in Patient derived xenografts (PDXs).Results:T-ALL cell lines and PDXs were treated with RVU120 and other chemically non-related CDK8/19 inhibitors in different concentration for 12-18 days, and the changes in cell number and apoptosis using flow cytometry analysis were determined. High differential efficacy in double-digit nM range has been observed for NOTCH1 PEST domain mutant cell lines including GSI-sensitive HPB-ALL cells and GSI-resistant cells PF-382, KOPT-K1, and MOLT4, where both inhibition of cell proliferation and induction of apoptosis were observed. GI-resistant cell lines SUP-T1, SUP-T11, MOLT16 and JURKAT were considered as moderately sensitive and LOUCY (early T-cell phenotype ALL), and CCRF-CEM devoid those phenotype, were considered as resistant to RVU120. Results form etsblished cell lines were further corroborated in patient-derived xenograft cells (PDX) treated with RVU120. In T-ALL PDX 6522288 (Notch1 HD /PEST mut, TET2 mut, U2AF1 mut, WT1 mut and CUL76- CDKN2A/B mut, PTEN-/-) treatment with RVU120 was followed by a reduction in cell number and induction of apoptosis in phenotypically defined leukemia initiating cells (LICs) CD34+CD7+CD19-, with calculated IC50s value for bulk and LICs as 92.8 nM and 141.8 nM, respectively. The clinical relevance of these findings and impact on LICs are under the evaluation in PDX mice model. Mechanistically, treatment with RVU120 reduced the phosphorylation of STAT5 and STAT1 as well as the level of MYC and anti-apoptotic proteins MCL1, but induced expression of BAX in sensitive cells. Hence, inhibition of CDK8 is a novel approach to target the LICs in T-ALL.Conclusion:Inhibition of CDK8 by RVU120 triggers inhibition of cell proliferation and apoptosis in cell lines and LICs in T-ALL. Further clinical study is warranted to study the efficacy of RVU120 and CDK8 inhibition in T-ALL.Keywords: CDK8, RVU120, apoptosis, T-ALL, LICs in PDX modelReference1 Girardi, T., Vicente, C., Cools, J. & De Keersmaecker, K. Blood129, 1113-1123, doi:10.1182/blood-2016-10-706465 (2017).2 Sanchez-Martin, M. & Ferrando, A. Blood 129, 1124-1133, doi:10.1182/blood-2016-09-692582 (2017).3 Braun, T. P., Eide, C. A. & Druker, B. J. Cancer cell 37, 530-542, doi:10.1016/j.ccell.2020.03.006 (2020).4 Gazeau, N. et al. Leukemia 34, 2230-2233, doi:10.1038/s41375-020-0715-2 (2020).5 Pelish, H. E. et al. Nature526, 273-276, doi:10.1038/nature14904 (2015).6 Fryer, C. J., White, J. B. & Jones, K. A. Molecular cell 16, 509-520, doi:10.1016/j.molcel.2004.10.014 (2004). DisclosuresGolas: Ryvu Therapeutics: Current Employment. Windak: Ryvu Therapeutics: Current Employment. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Andreeff: ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Glycomimetics: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Amgen: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Senti-Bio: Consultancy. Borthakur: GSK: Consultancy; Astex: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees.

Cytological analysis of the lung material of C57BL/6Y mice in the simulation of acute respiratory distress syndrome

The work is devoted to the study of changes in the cellular composition of bronchoalveolar lavage over time in the modeling of acute respiratory distress syndrome (ARDS) in mice. ARDS was modeled by administering α-galactosylceramide and a mixture of lipopolysaccharide with a complete Freud’s adjuvant. After euthanasia, bronchoalveolar lavage was taken for analysis. On this basis, changes in the total number of white blood cells, the percentage of neutrophils and macrophages were assessed. It was found that the percentage of neutrophils in the ARDS group shows a statistically significant difference from that in the intact group, starting from 3 hours after modeling ARDS. Further, a statistically significant decrease in macrophages was observed.

The Influence of Low-Dose Occupational Radiation Exposure on Peripheral Blood Cell in a Cohort Chinese Medical Radiation Workers

The study aims to assess the change of peripheral blood cell number following protracted low-dose radiation exposure among medical radiation workers.A cohort of 185 Chinese radiation workers which divided into 4 groups by different occupational categories which include Interventional radiologists group (IRG); nuclear medicine doctors group (NMD); medical diagnostic radiographers group (MDR); radiologists group (RG) were followed by 5 years (2015-2019). This study observed and recorded the changes in the cumulative dose and peripheral blood cell counts consecutively for five years. While exploring the changes in blood cells and cumulative dose, the correlation between dose and the quantity of peripheral blood cells was also discussed. Individual doses were recorded by dose monitoring system. The counts of peripheral blood cell were based on the physical examination data of the workers of the annual physical examination in the hospitality-test, SD-T test and Variance analysis were used to analyze the radiation dose-response changes of the peripheral blood cell. Correlation analysis was used to analyze the correlation between the number of peripheral blood cells and the cumulative dose.The results of this study showed that compared with the control group, the number of white blood cells (WBCs), the number of hemoglobin (Hb) and the ratio of eosinophils (EO%) in the study group decreased (P < 0.05). Correlation analysis between the average cumulative dose in different years and the number of peripheral blood cells in different groups showed that the average cumulative dose of nuclear medicine group in 2016 was negatively correlated with WBC (r = -0.602, P < 0.05) and NE% (r = -0.596, P < 0.05). There was a significant positive correlation between the average cumulative dose of the radiological diagnosis group in 2015 and MO% (r = 0.530, P < 0.01). For the radiologists' group, there was a significant negative correlation between the average cumulative dose and the number of WBCs in 2016 (r = -3.37, P < 0.05), and there was a significant negative correlation between the average cumulative dose and RBC in 2017 (r = -4.12, P < 0.05).The results of the study suggest that the blood cell levels of workers exposed to low-dose ionizing radiation for a long time can show dynamic changes, and relevant workers should take appropriate protection measures and regularly monitor peripheral blood cell indicators.

Aglycone flavonoid brachydin A shows selective cytotoxicity and antitumoral activity in human metastatic prostate (DU145) cancer cells.

In prostate cancer, flavonoids possess a wide variety of anticancer effects, focused on the antioxidant/pro-oxidant activity, inactivation of the androgen receptor, cell cycle arrest, apoptosis induction, metastasis inhibition, among others. This current research investigated the antitumoral in vitro activity of Brachydin A (BrA), a dimeric flavonoid isolated from Fridericia platyphylla, in human castration-resistant prostate cancer DU145. It was compared BrA selective effects in tumor prostate DU145 cells with non-tumor prostate epithelial PNT2 cells. Cell viability experiments (resazurin, neutral red, MTT, and LDH release assays) showed that BrA was sevenfold more cytotoxic to tumor cells than non-tumor prostate cells, with IC50 values of 77.7 µM and 10.7 µM for PNT2 and DU145 cells, respectively. Furthermore, BrA induced necrosis and apoptosis (triple fluorescence staining assay) without interfering with oxidative stress (CM-H2DCFDA) in DU145 cells. Also, BrA (15.36 µM) reduced cell proliferation on clonogenic assay (DU145 cells) but no change in cell number and protein content was observed when cell growth curve assay was used. Wound healing and transwell assays were used for checking the effects of BrA on cell migration and invasion, and BrA impaired these processes in PNT2 (wound healing) and DU145 cells (transwell). Our results inspire further studies to test BrA as a novel chemotherapeutic drug and to evaluate its effects on drug-resistant metastatic cancer cells.

Robust innate responses to SARS-CoV-2 in children resolve faster than in adults without compromising adaptive immunity.

SARS-CoV-2 infection in children is less severe than it is in adults. We perform a longitudinal analysis of the early innate responses in children and adults with mild infection within household clusters. Children display fewer symptoms than adults do, despite similar initial viral load, and mount a robust anti-viral immune signature typical of the SARS-CoV-2 infection and characterized by early interferon gene responses; increases in cytokines, such as CXCL10 and GM-CSF; and changes in blood cell numbers. When compared with adults, the antiviral response resolves faster (within a week of symptoms), monocytes and dendritic cells are more transiently activated, and genes associated with B cell activation appear earlier in children. Nonetheless, these differences do not have major effects on the quality of SARS-CoV-2-specific antibody responses. Our findings reveal that better early control of inflammation as observed in children may be key for rapidly controlling infection and limiting the disease course.

Evaluating effects of electromagnetic fields on the total number and percentage of white blood cells in mice

To evaluate the effects of electromagnetic waves on the total number and percentage of white blood cells and also antioxidant effects of vitamin C on the effects radiation, 24 male mice (Balb/c) were used: control under the influence of low frequency electromagnetic waves and under the influence of waves with vitamin C. Total number of white blood cells in under the influence waves group significantly increased compared to control group, and also under the influence waves group with vitamin C because its antioxidant property is able to prevent the increasing impact of electromagnetic waves. The percentage of white blood cells in the under influence waves group did not significantly change compared to the control group and also, the under influence waves group with vitamin C had no significant change compared to the other group. The percentage of neutrophils in samples of the under influence waves group had significant decrease compared to control group but in the under influence waves group with vitamin C compared to the other group, this vitamin C could prevent a significant reduction in percentage of neutrophils. Our findings indicated that low electromagnetic fields have caused significant changes in the total number of white blood cells and percentage of neutrophils in mice. In the group that received vitamin C injection, significant changes were observed in the total number of white blood cells and percentage of neutrophils relative to the group under the influence of low electromagnetic waves, which indicates that vitamin C could restore the mean total number of white blood cells and percentage of neutrophils to normal value.

Scanning Electron Microscopic Study of the White Pulp of Spleen in Adult Goats

The white pulp of the spleen in adult goats was thoroughly screened under the scanning electron microscope (VEGA3 TESCAN). The study revealed the presence of lymphoid and non-lymphoid cells. The T lymphocytes and B lymphocytes could morphologically be differentiated under the magnification of 8000 to 20000 times by Scanning electron microscopy. The changes in number of cells in relation to the age was also recorded. The peri arterial lymphatic sheath and its structural connections with Reticular cells and other non-lymphoid cells were clearly demonstrated. The types of lymphoid cells and their arrangement around the central arteries upto the marginal zones were observed in detail. A conclusion about the type of circulation in the spleen of goats was arrived after three dimentional observation of the tissue under scanning electron microscope.

Abstract CT168: Changes in mast cell (MC) numbers and phenotype in patients (pts) with indolent systemic mastocytosis (ISM) treated with avapritinib

Abstract Purpose: The selective tyrosine kinase inhibitor avapritinib has been shown to improve skin lesions in pts with ISM (Hartmann K et al. EACCI 2020. Abstract 1832). We assessed numbers and immunophenotypic changes of MCs in bone marrow (BM) and skin biopsies from lesional tissue (LT) and non-lesional tissue (NLT) in 39 pts with ISM from Part 1 of the placebo-controlled PIONEER (NCT03731260) study. Methods: BM biopsies and aspirates were obtained at Screening; skin biopsies from LT and NLT were obtained at Screening and end of Week 12 (W12). Immunohistochemistry was performed on formalin-fixed sections using the Ventana Benchmark assay with CD117, tryptase, CD25, CD30, and CD34 antibodies. Samples were centrally reviewed by three pathologists. Statistical significance of MC numbers and immunophenotypic changes were assessed. Data cut-off was December 4, 2020. Results: Screening BM biopsies had median 10% (range, 1-60%) MCs, of which 90% (10-100%) were spindled. BM aspirates had median 1% (0-10%) MCs, of which 5% (2-6%) were immature. Median number of MCs/mm2 was 355 (53-4300) in LT and 90 (10-540) in NLT. At W12, avapritinib reduced median number of MCs/mm2 in LT (143 [33-837]) but not NLT (102 [32-207]). BM biopsies had higher median rates of CD25+ and CD30+ MCs (90% CD25+/50% CD30+) compared with skin LT (2% CD25+/10% CD30+) and NLT (1% CD25+/1% CD30+) (Table). Avapritinib produced significant reductions in the proportion of CD30+ MCs in LT at W12 versus placebo (P=0.0053) and non-significant reductions in CD30+ MCs in NLT (P=0.0988). Conclusions: CD25+ and CD30+ MCs were observed in both LT and NLT skin biopsies; however, at markedly lower levels than seen in BM. Treatment with avapritinib decreased the total number of MCs as well as the number of CD30+ and CD25+ MC in both LT and NLT with statistically significant decreases in the CD30+ MC fraction in LT. This is the first study to examine changes in MCs in LT and NLT or ISM during precision therapy of ISM. Table. Immunophenotypic changes of MCs in BM and skin biopsies at screening and W12MC PhenotypeBone MarrowSkin Lesional TissueSkin Non-Lesional TissueAvapritinibPlaceboAvapritinibPlaceboScreening (n=39)Screening (n=25)W12 (n=17)Screening (n=8)W12 (n=7)Screening (n=25)W12 (n=21)Screening (n=8)W12 (n=7)CD25+, median % (range)90 (0-100)2 (1-40)2 (1-10)3 (1-50)5 (2-5)1 (1-5)2 (1-10)2 (1-5)5 (2-5)CD30+, median % (range)50 (0-100)10 (1-100)1 (1-5)13 (1-80)5 (1-30)1 (1-20)1 (1-2)2 (1-30)1 (1-10)Abbreviations: BM, bone marrow; MC, mast cell; W12, Week 12. Citation Format: Tracy George, Sigurd Broesby-Olsen, David Wada, Scott Florell, Karin Hartman, Frank Siebenhaar, Cem Akin, Kate Newberry, Hongliang Shi, Maria Roche, Marcus Maurer, Hanneke Oude Elberink. Changes in mast cell (MC) numbers and phenotype in patients (pts) with indolent systemic mastocytosis (ISM) treated with avapritinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT168.

The Role of Smad2 in Adult Neuroplasticity as Seen through Hippocampal-Dependent Spatial Learning/Memory and Neurogenesis.

Adult neural plasticity is an important and intriguing phenomenon in the brain, and adult hippocampal neurogenesis is directly involved in modulating neural plasticity by mechanisms that are only partially understood. We have performed gain-of-function and loss-of-function experiments to study Smad2, a transcription factor selected from genes that are demethylated after exercise through the analysis of an array of physical activity-induced factors, and their corresponding gene expression, and an efficient inducer of plasticity. In these studies, changes in cell number and morphology were analyzed in the hippocampal dentate gyrus (cell proliferation and survival, including regional distribution, and structural maturation/differentiation, including arborization, dendritic spines, and neurotransmitter-specific vesicles) of sedentary male mice, after evaluation in a battery of behavioral tests. As a result, we reveal a role for Smad2 in the balance of proliferation versus maturation of differentiating immature cells (Smad2 silencing increases both the proliferation and survival of cycling cells in the dentate granule cell layer), and in the plasticity of both newborn and mature neurons in mice (by decreasing dendritic arborization and dendritic spine number). Moreover, Smad2 silencing specifically compromises spatial learning in mice (through impairments of spatial tasks acquisition both in long-term learning and working memory). These data suggest that Smad2 participates in adult neural plasticity by influencing the proliferation and maturation of dentate gyrus neurons.SIGNIFICANCE STATEMENT Smad2 is one of the main components of the transforming growth factor-β (TGF-β) pathway. The commitment of cell fate in the nervous system is tightly coordinated by SMAD2 signaling, as are further differentiation steps (e.g., dendrite and axon growth, myelination, and synapse formation). However, there are no studies that have directly evaluated the role of Smad2 gene in hippocampus of adult animals. Modulation of these parameters in the adult hippocampus can affect hippocampal-dependent behaviors, which may shed light on the mechanisms that regulate adult neurogenesis and behavior. We demonstrate here a role for Smad2 in the maturation of differentiating immature cells and in the plasticity of mature neurons. Moreover, Smad2 silencing specifically compromises the spatial learning abilities of adult male mice.

Coronary Artery Bypass Grafting Is Associated with Significant Pertubation of Circulating Dendritic Cell Populations

IntroductionCoronary artery bypass grafting (CABG) with sternotomy is a complex procedure that triggers an overall systemic inflammatory response as a result of exposing patient blood to the bypass circuitry which is hypothesised to contribute to adverse patient outcomes including infection and prolonged ICU length of stay. Dendritic cells (DC) are antigen presenting cells that link the innate and adaptive immune response and can be divided into two main subsets; myeloid DC (mDC; HLA-DR+CD11c+) and plasmacytoid DC (pDC; HLA-DR+CD11c-CD123+). Circulating immature DC have a high phagocytosis and antigen-processing capacity and mature in response to inflammation as they migrate to lymphoid organs to regulate T cells. Extracorporeal circuitry associated with CABG has been reported to trigger a complex inflammatory reaction where pro- and anti- inflammatory cytokines are produced, leucocyte subsets such as monocytes become activated and changes to the composition of circulating cell populations occur which may be associated with adverse patient outcomes following surgery. Given the central role DC plays in the patient immune response, this study aimed to address the limited understanding of how DC function changes during cardiac surgery by assessing changes in absolute cell number of DC populations.MethodsPatient whole blood (EDTA) was collected from CABG patients (n=33) at 5 time-points (Admission, Peri-operative, ICU, Day 3, Day 5). The absolute count of DC subsets (mDC Lineage-HLA-DR+CD11c+; pDC Lineage-HLA-DR+CD11c-CD123+) and the number of DC expressing specific activation and adhesion markers (CD9, CD38, CD40, CD80, CD83, CD86) was assessed using Trucount tubes (BD Biosciences, Australia). A repeated measure one-way ANOVA with Dunnett's post-test was used for the analysis of absolute cell counts with the admission sample used as the comparator baseline. P values <0.05 were considered significantResultsA significant decrease in circulating mDC was observed during CABG surgery. A recovery in mDC numbers towards baseline was evident by day 5 post-surgery. The number of mDC expressing CD38, CD40, CD80, CD83 and CD86 were also reduced, suggesting mDC in circulation maintained an immature phenotype. Of note, CD40 expressing mDC numbers were reduced during surgery before a dramatic increase during the ICU period followed by a return towards baseline levels at day 5. In contrast to mDC, circulating pDC numbers significantly increased during surgery before decreasing during the ICU period and regressing towards normal baseline levels by day 5. The number of pDC expressing CD38 increased during surgery before regressing towards baseline at day 5.ConclusionThese data demonstrate that CABG is associated with significant perturbation of circulating DC populations. While numbers of mDC decreased, numbers of pDC increased suggesting CABG resulted in differential modulation of these two DC subsets. Of note both DC subsets demonstrated a toleregenic phenotype rather than an activatory profile. Decreased numbers of mDC during surgery suggest impaired functional capacity potentially increasing the risk of infection post-surgery. Normalisation towards baseline levels was observed by day 5 post-surgery for both mDC and pDC. We are currently assessing whether changes in the DC cell count and phenotype is associated with patient outcomes and may serve as a predictive biomarker for adverse patient outcomes. DisclosuresNo relevant conflicts of interest to declare.

Repurposing Itraconazole As an Anti-Cancer Agent Against Diffuse Large B-Cell Lymphoma

Introduction: Results from the Retrospective Non-Hodgkin Lymphoma Research (SCHOLAR-1) study demonstrated that relapsed/refractory Diffuse Large B cell Lymphoma (r/r DLBCL) have a low response rate to third-line therapy (20-30%) and an overall survival of 6 months. It stresses the need to develop novel therapeutic strategies for patients with r/r lymphoma. Itraconazole (ITZ) is a common widely clinical used orally bioavailable azole antifungal drug, with a more than thirty years well-documented safety record. Recently, scientific evidence has highlighted ITZ anti-tumor effects at clinically achievable doses in patients with variety of solid tumor malignancies. However, little is known about ITZ activity in B-cell lymphoma, especially in chemotherapy resistant lymphoma. Here we are the first group to report that potent anti-tumor activity of ITZ as a single agent or combined with other chemotherapeutic drugs in both sensitive and resistant lymphoma pre-clinical models. We also further explored the mechanism of action of ITZ to overcome drug resistance.Methods: A panel of rituximab-chemotherapeutic therapy-sensitive (TSCL), -resistant (TRCL) cell lines, developed in our laboratory, and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of ITZ (0-20uM). IC50 values were calculated using the GraphPad Prism6 software. Changes in cell viability, cell number and cellular ATP levels were evaluated using the Presto Blue, Trypan Blue and Titer Glo assay, respectively. Mitochondrial respiration rate indicated as oxygen consumption rate (OCR) were measured by an XFe96 flux analyzer. Mitochondrial membrane potential (MMP) were quantified by DiOC6 staining and flow cytometry. Subsequently, TSCL and TRCL cells were exposed to ITZ alone or in combination with various chemotherapy agents or small inhibitors and differences in cell viability was determined. Coefficient of synergy was calculated using the CalcuSyn software. HKII-VDAC complex disruption by ITZ was identified by co-immunoprecipitation and western blot. HKII knockdown was generated by using ON-TARGET plus SMART pool siRNA containing a mixture of 4 siRNAs designed to specifically target HKII. Standard 51Cr release assays were conducted to study the effects of ITZ on the anti-tumor activity of monoclonal antiCD20 antibodies against TSCL and TRCL cell lines. For in vivo studies, severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose), obinotuzumab (at 10mg/kg/dose) with or without ITZ. Anti-tumor activity was evaluated by functional imaging and length of survival.Results: We found that ITZ induced a dose-dependent cell death in TSCL, TRCL and primary patient lymphoma cells. Single agent of ITZ decreased cell growth, reduced ATP production, lower the mitochondrial respiration rate and MMP in both TSCL and TRCL. ITZ exhibited additive/synergistic effects when combined with chemotherapeutic drugs (dexamethasone and cisplatin), proteasome inhibitors (bortezomib, carfilzomib, or ixazomib), targeted inhibitors (ABT199 and A-1210477 [an MCL-1 inhibitor]). At the molecular level, we found that, ITZ disrupted HKII/VDAC complex formation. Silencing HKII in TRCL attenuated the killing effect of ITZ. More importantly, ITZ in combination with rituximab or obinutuzumab led to reduced tumor intensity by functional imaging than rituximab or obinotuzumab single agent in vivo.Conclusion: Our findings support the potential benefits for using ITZ as an anti-cancer drug are: 1) reduce costs, 2) clear defined toxicity profile, 3) availability, and 4) pre-clinical activity in rituximab-chemotherapy resistant lymphomas. ITZ can be an inexpensive and effective agent to enhance standard chemotherapy in B-cell lymphoma. Repurposing of ITZ can result in novel therapeutic options for lymphoma patients. (Research supported by Roswell Park Cancer Institute Alliance Grant, National Natural Science Foundation of China (NSFC) 81573063) DisclosuresNo relevant conflicts of interest to declare.

Probing the peripheral immune response in mouse models of oxaliplatin-induced peripheral neuropathy highlights their limited translatability.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of various chemotherapeutic agents, including oxaliplatin. It is highly prevalent amongst cancer patients, causing sensory abnormalities and pain. Unfortunately, as the underlying mechanisms remain poorly understood, effective therapeutics are lacking. Neuro-immune interactions have been highlighted as potential contributors to the development and maintenance of CIPN, however, whether this is the case in oxaliplatin-induced peripheral neuropathy (OIPN) is yet to be fully established. Methods: In this study we used flow cytometry to examine the peripheral immune response of male C57BL/6 mice following both single and repeated oxaliplatin administration. In animals exposed to repeated dosing, we also undertook mechanical and thermal behavioural assays to investigate how oxaliplatin alters phenotype, and conducted RT-qPCR experiments on bone marrow derived macrophages in order to further inspect the effects of oxaliplatin on immune cells. Results: In contrast to other reports, we failed to observe substantial changes in overall leukocyte, lymphocyte or myeloid cell numbers in dorsal root ganglia, sciatic nerves or inguinal lymph nodes. We did however note subtle, tissue-dependant alterations in several myeloid subpopulations following repeated dosing. These included a significant reduction in MHCII antigen presenting cells in the sciatic nerve and an increase in infiltrating cell types into the inguinal lymph nodes. Though repeated oxaliplatin administration had a systemic effect, we were unable to detect a pain-like behavioural phenotype in response to either cold or mechanical stimuli. Consequently, we cannot comment on whether the observed myeloid changes are associated with OIPN. Conclusions: Our discussion puts these results into the wider context of the field, advocating for greater transparency in reporting, alignment in experimental design and the introduction of more clinically relevant models. Only through joint concerted effort can we hope to increase our understanding of the underlying mechanisms of CIPN, including any immune contributions.

Probing the peripheral immune response in mouse models of oxaliplatin-induced peripheral neuropathy highlights their limited translatability

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of various chemotherapeutic agents, including oxaliplatin. It is highly prevalent amongst cancer patients, causing sensory abnormalities and pain. Unfortunately, as the underlying mechanisms remain poorly understood, effective therapeutics are lacking. Neuro-immune interactions have been highlighted as potential contributors to the development and maintenance of CIPN, however, whether this is the case in oxaliplatin-induced peripheral neuropathy (OIPN) is yet to be fully established. Methods: In this study we used flow cytometry to examine the peripheral immune response of male C57BL/6 mice following both single and repeated oxaliplatin administration. In animals exposed to repeated dosing, we also undertook mechanical and thermal behavioural assays to investigate how oxaliplatin alters phenotype, and conducted RT-qPCR experiments on bone marrow derived macrophages in order to further inspect the effects of oxaliplatin on immune cells. Results: In contrast to other reports, we failed to observe substantial changes in overall leukocyte, lymphocyte or myeloid cell numbers in dorsal root ganglia, sciatic nerves or inguinal lymph nodes. We did however note subtle, tissue-dependant alterations in several myeloid subpopulations following repeated dosing. These included a significant reduction in MHCII antigen presenting cells in the sciatic nerve and an increase in infiltrating cell types into the inguinal lymph nodes. Though repeated oxaliplatin administration had a systemic effect, we were unable to detect a pain-like behavioural phenotype in response to either cold or mechanical stimuli. Consequently, we cannot comment on whether the observed myeloid changes are associated with OIPN. Conclusions: Our discussion puts these results into the wider context of the field, advocating for greater transparency in reporting, alignment in experimental design and the introduction of more clinically relevant models. Only through joint concerted effort can we hope to increase our understanding of the underlying mechanisms of CIPN, including any immune contributions.

Small Intestinal Tuft Cell Activity Associates With Energy Metabolism in Diet-Induced Obesity.

Little is known about the involvement of type 2 immune response-promoting intestinal tuft cells in metabolic regulation. We here examined the temporal changes in small intestinal tuft cell number and activity in response to high-fat diet-induced obesity in mice and investigated the relation to whole-body energy metabolism and the immune phenotype of the small intestine and epididymal white adipose tissue. Intake of high fat diet resulted in a reduction in overall numbers of small intestinal epithelial and tuft cells and reduced expression of the intestinal type 2 tuft cell markers Il25 and Tslp. Amongst >1,700 diet-regulated transcripts in tuft cells, we observed an early association between body mass expansion and increased expression of the gene encoding the serine protease inhibitor neuroserpin. By contrast, tuft cell expression of genes encoding gamma aminobutyric acid (GABA)-receptors was coupled to Tslp and Il25 and reduced body mass gain. Combined, our results point to a possible role for small intestinal tuft cells in energy metabolism via coupled regulation of tuft cell type 2 markers and GABA signaling receptors, while being independent of type 2 immune cell involvement. These results pave the way for further studies into interventions that elicit anti-obesogenic circuits via small intestinal tuft cells.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Epac1 regulates TLR4 signaling in the diabetic retinal vasculature

Toll-like receptor 4 (TLR4) polymorphisms occur in diabetic patients. Previous work showed that TLR4 is in the retina of diabetic mice, as well as in retinal endothelial cells (REC) and Müller cells. Since we have shown that exchange protein activated by cAMP 1 (Epac1) can reduce inflammatory mediators, we hypothesized that Epac1 would inhibit TLR4 signaling. We also hypothesized that direct TLR4 inhibition would protect the diabetic retina. Human REC in normal and high glucose were treated with an Epac1 agonist to explore the actions of Epac1 on TLR4 signaling in vitro. Subsequently, 2-month diabetic endothelial cell specific knockout mice for Epac1 (Cdh5Cre-Epac1) and Epac1 floxed mice retinas were used for Western blotting for TLR4 signaling pathways. We also used direct inhibition of TLR4 via Tak242 to investigate diabetes-induced changes in retinal permeability and neuronal loss in the mice. The Epac1 agonist reduced TLR4 signaling in REC grown in high glucose. TLR4 levels and both MyD88-dependent and -independent signaling pathways are increased in Cdh5Cre-Epac1 mice compared to Epac1 floxed mice. Tak242 reduced TLR4 signaling in diabetic mice and reduced diabetes-induced increases in permeability and cell loss in the ganglion cell layer in the Epac1 floxed and Cdh5Cre-Epac1 mice. In conclusion, Epac1 reduced TLR4 signaling in the retina and in REC. Direct inhibition of TLR4 was able to protect the retina against diabetes-induced changes in permeability and cell numbers in the ganglion cell layer.

Effects of Maternal Nutrient Restriction During Gestation on LH Production in the Female Bovine Fetus

Abstract Gestational nutrient restriction causes epigenetic and phenotypic changes that affect multiple physiological processes in the offspring. Gonadotropes, the cells in the anterior pituitary that secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are particularly sensitive to nutritional changes during fetal development. Our objective herein was to investigate the effects of gestational nutrient restriction on LH protein content and number of gonadotropes in the fetal bovine pituitary. We hypothesized that moderate nutrient restriction during mid to late gestation decreases pituitary LH production, which is associated with a reduced number of gonadotropes. Embryos were produced in vitro with X-bearing semen from a single sire then split to generate monozygotic twins. Each identical twin was transferred to a virgin dam yielding four sets of female twins. At gestational d 158, the dams were randomly assigned into two groups, one fed 100% NRC requirements (control) and the other fed 70% of NRC requirements (restricted) during the last trimester of gestation, ensuring each pair of twins had one twin in each group. At gestational d 265, the fetuses (n = 4/group) were euthanized by barbiturate overdose, and the pituitaries were collected. Western blots were performed using an ovine LH-specific antibody (Dr. A.F. Parlow, NIDDK). The total LH protein content in the pituitary tended to be decreased in the restricted fetuses compared to controls (P &amp;lt; 0.10). However, immunohistochemistry analysis of the pituitary did not reveal any significant changes in the total number of LH-positive cells (control = 460±23 cells/0.5 mm2; restricted = 496±45 cells/0.5 mm2, P = 0.58). In conclusion, while maternal nutrient restriction during gestation resulted in a trend of reduced LH content in the fetal pituitary, immunohistological findings suggest that these changes are likely related to the individual potential of each gonadotrope to produce LH, rather than alterations in cell differentiation during fetal development.

TGF‐b receptor II plays a vital role in neural crest‐derived osteogenesis but not in limb osteogenesis during postnatal development

It is widely accepted that TGF-b signaling enhances osteogenesis via osteoprogenitor enrichment. However, removing TGF-b receptor II (Tgfbr2, a key receptor for TGF-b signaling) in bone cells by osteocalcin-Cre resulted in a controversial long bone phenotype: an increase in trabecular bone but a decrease in cortical bone. To address the role of Tgfbr2 in postnatal mandibular development, we first generated a mouse line in which Tgfbr2 was conditionally deleted in early mesenchymal progenitors by crossing Gli1-CreERT2; Tgfbr2fl/fl; and R26RtdTomato (for cell lineage tracing) compound mice (early cKO). This early cKO line was induced at P5 and harvested at P28. Compared to the control littermates, BV/TV and BMD were significantly reduced in both the cortical bone and alveolar bone of the mandible based on uCT analysis (n = 4; p<0.05). Masson trichrome and polarized light images showed a loss of periodontal ligament fibers and bone mass in the early cKO, whereas there was no apparent change in TRAP positive cells in the cKO bone, excluding the impact of osteoclasts on bone phenotypes. The cell lineage tracing data reveals a great reduction in red bone cell numbers in early cKO. Immunostain images revealed a 70% reduction of OSX in the early cKO bone cells (p < 0.001). Immunostain images also displayed a sharp reduction of bone matrix markers such as MEPE and SOST in the cKO, supporting a requirement of Tgfbr2 in the early osteogenesis. Next, we generated a mouse line in which Tgfbr2 was conditionally deleted in osteoblasts by crossing 3.2 Col1-CreERT2; Tgfbr2fl/fl; R26RtdTomato compound mice (late-cKO). Both uCT and histological data showed mild reduction of bone volume in the late cKO compared to the control littermates. The tracing data revealed little change in bone cell number, and immunostain images showed no apparent alternations in OSX or other bone markers. Importantly, in both cKO mice, there is no apparent change in postnatal long bone development, which suggests that TGF-b signaling plays a more critical role in the neural-crest derived mandible growth. Based on the above data, we conclude that Tgfbr2 in mandible bone formation is mainly required for early bone progenitor cells, and deletion of Tgfbr2 has little impact on the late bone-forming cells during postnatal development of alveolar bone.

Exposure to Hog Dust Extract Decreases Barrier Integrity in Caco‐2 Cells, Induces Airway Inflammation and Increases Intestinal Permeability in Mice

Agricultural enterprises, such as concentrated animal feeding operations (CAFOs), are responsible for the production of 10% of global greenhouse gases and harmful environmental pollutants including hydrogen sulfide, ammonia and particulate matter. Seasonal farm workers, especially swine farmers, are frequently exposed to organic dust that is pro-inflammatory in the lung and are thus at greater risk of developing pneumonia, asthma and other respiratory conditions. In addition to respiratory disease, these air pollutants are directly associated with altered gastrointestinal (GI) physiology and the development of GI diseases. Therefore, the aim of the current study was to identify mechanisms by which extracts of dusts derived from hog CAFOs (HDE) can disrupt intestinal homeostasis. Caco-2 BBE monolayers were seeded at 0.5x106cells/well in a Transwell system and exposed to a single dose of either 0.25%, 0.5%, 0.75%, 1% or 5% HDE. A decreasing trend was seen in monolayer integrity, determined by transepithelial electrical resistance (TEER), at 24-and 48-hours following treatment with 5% HDE (Two-Way ANOVA, p=0.1824 and p=0.2017, respectively, n=3). Moreover, 8-week-old male or female C57BL/6 mice (n=12) were intranasally exposed to saline (n=6) or 12.5% HDE (n=6) for 3 weeks. Bronchoalveolar lavage fluid from HDE-treated mice showed no changes in macrophage cell numbers (unpaired t-test, p=0.2993), but exhibited elevated total cell and neutrophil levels (unpaired t-test, p=0.0027 and p=0.0061, respectively) in comparison to saline controls. Furthermore, no significant differences were seen among the colon lengths of HDE-treated mice (8.32 ± 0.13cm SEM) to that of saline controls (9.2 ± 0.67cm SEM). Additionally, HDE increased permeability to FITC-dextran 4kDA and rhodamine B-dextran 70kDA (unpaired t-test, p<0.001). Moreover, histological changes observed by hematoxylin and eosin staining indicated epithelial cell loss and minimal-mild inflammation in the proximal and distal colon of HDE-treated mice. Together, these findings suggest that airway exposure to CAFOs dusts promotes airway inflammation and can access the gastrointestinal tract to increase regulated and/or unrestricted intestinal permeability, possibly due to intestinal epithelial cell damage. Further identification of mechanistic interactions between the respiratory and gastrointestinal systems is imperative to understand the complex physiological consequences of chronic exposure to organic dusts derived from CAFOs on the gut-lung axis.

An in-silico Investigation Into the Role of Strain and Structure on Vascular Smooth Muscle Cell Growth.

The orientation of vascular cells can greatly influence the in vivo mechanical properties and functionality of soft vascular tissues. How cell orientation mediates the growth response of cells is of critical importance in understanding the response of soft tissues to mechanical stimuli or injury. To date, considerable evidence has shown that cells align with structural cues such as collagen fibers. However, in the presence of uniaxial cyclic strain on unstructured substrates, cells generally align themselves perpendicularly to the mechanical stimulus, such as strain, a phenomenon known as “strain avoidance.” The cellular response to this interplay between structural cues and a mechanical stimulus is poorly understood. A recent in vitro experimental study in our lab has investigated both the individual and collective response of rat aortic smooth muscle cells (RASMC) to structural (collagenous aligned constructs) and mechanical (cyclic strain) cues. In this study, a 2D agent-based model (ABM) is developed to simulate the collective response of RASMC to varying amplitudes of cyclic strain (0–10%, 2–8%, 4–6%) when seeded on unstructured (PDMS) and structured (decellularized collagenous tissue) constructs. An ABM is presented that is fit to the experimental outcomes in terms of cellular alignment and cell growth on PDMS substrates, under cyclic strain amplitudes of (4–6%, 2–8%, 0–10%) at 24 and 72 h timepoints. Furthermore, the ABM can predict RASMC alignment and change in cell number on a structured construct at a cyclic strain amplitude of 0–10% after 10 days. The ABM suggests that strain avoidance behavior observed in cells is dominated by selective cell proliferation and apoptosis at these early time points, as opposed to cell re-orientation, i.e., cells perpendicular to the strain increase their rate of proliferation, whilst the rate of apoptosis simultaneously increases in cells parallel to the strain direction. The development of in-silico modeling platforms, such as that presented here, allow for further understanding of the response of cells to changes in their mechanical environment. Such models offer an efficient and robust means to design and optimize the compliance and topological structure of implantable devices and could be used to aid the design of next-generation vascular grafts and stents.