scholarly journals Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5082
Author(s):  
Ludovica Arpinati ◽  
Naomi Kaisar-Iluz ◽  
Merav E. Shaul ◽  
Christopher Groth ◽  
Viktor Umansky ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Ex Vivo ◽  
Surface Marker ◽  
Normal Density ◽  
Low Density ◽  
Ros Production ◽  
Surface Marker Expression ◽  
Density State ◽  
Surface Receptor

Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.

scholarly journals Novel in Vitro and In Vivo Drug Screening Platform for ALL.

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1189-1189
Author(s):  
Bandana Ajay Vishwakarma ◽  
Amy Wesa
Keyword(s):  
Bone Marrow ◽  
Drug Testing ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Drug Efficacy ◽  
Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Acute Lymphoblastic leukemia (ALL) is a malignancy of bone marrow. Accumulation of mutations in lymphoid progenitor cells give rise to either B-ALL or T-ALL. Treatment for ALL has improved in recent years, yet relapse of the disease and development of resistance are observed in patients. Lack of suitable and robust in vitro and in vivo drug testing platforms for primary ALL cells along with the lack of rapid development of novel therapeutics drugs encompassing the heterogeneity of the disease contribute to the delay in approved patient treatments. We have developed a short-term culture system that supports the survival of primary B-ALL and T-ALL cells. Our ALL bank includes patient-derived specimens with complete cytogenetics and surface marker expression information. Different culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL and T-ALL specimens. Cell growth/viability was assessed using the Cell Titer-Glo ® assay. Primary B-ALL cells survived in the optimized media for 3 days and a heterogenous dose dependent response was observed across the models to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. BCR-ABl - B-ALL patient samples were found to be resistant to imatinib in contrast to BCR-ABL + samples which were sensitive to imatinib. Similarly, culture conditions optimized for T-ALL primary cells supported the survival until day 6 and displayed a diverse response to standard of care drugs like venetoclax, imatinib, vincristine, cytarabine and methotrexate, reflecting the heterogeneity of the patient derived specimens. Immunophenotypic characterization of ALL cells grown in culture displayed retention of the B and T cells surface marker expression. Further, a patient derived pre-clinical xenograft model was developed in NCG mice to study in vivo ALL drug efficacy. 100% engraftment was observed for B-ALL primary cells, with latency of engraftment (>3%) in peripheral blood varying from 15 days to 3.5 months. 30-90% of the bone marrow cells were occupied by human CD45 cells. Infiltration of human B-ALL cells were observed in the spleen causing splenomegaly. 8 out of the 14 models having high penetrance were passaged until P3. Flow analysis at each passage demonstrated surface marker expression displaying low divergence from the primary samples. Additionally, evaluation of ex vivo drug response from B-ALL PDX splenocytes was largely concordant with the primary specimen ex vivo data in three of the models evaluated. In an in vivo drug efficacy study administration of venetoclax, CHOP and R-CHOP inhibited the proliferation of B-ALL cells. Significant reduction of B-ALL cells was observed while on treatment with Venetoclax. At termination of the study, up to 80% reduction of human B-ALL cells was observed in whole blood, bone marrow, and spleen after treatment with CHOP and R-CHOP in comparison to the vehicle cohort. Similarly, patient derived T-ALL pre-clinical xenograft model development is in progress. Thus, we have developed a robust in vitro drug testing platform for B-ALL and T-ALL to evaluate drug efficacy. We also demonstrate that NCG mice support the growth and proliferation of primary B-ALL cells and have successfully developed an in vivo platform that will facilitate the testing of clinically relevant chemotherapeutic drugs for ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Insulin Resistance Promotes Parkinson’s Disease through Aberrant Expression of α-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 740 ◽  
Author(s):  
Chien-Tai Hong ◽  
Kai-Yun Chen ◽  
Weu Wang ◽  
Jing-Yuan Chiu ◽  
Dean Wu ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Dysfunction ◽  
Ex Vivo ◽  
Proteinase K ◽  
Ros Production ◽  
Aberrant Expression ◽  
In Vivo Models

Background: Insulin resistance (IR), considered a hallmark of diabetes at the cellular level, is implicated in pre-diabetes, results in type 2 diabetes, and negatively affects mitochondrial function. Diabetes is increasingly associated with enhanced risk of developing Parkinson’s disease (PD); however, the underlying mechanism remains unclear. This study investigated the probable culpability of IR in the pathogenesis of PD. Methods: Using MitoPark mice in vivo models, diabetes was induced by a high-fat diet in the in vivo models, and IR was induced by protracted pulse-stimulation with 100 nM insulin treatment of neuronal cells, in vitro to determine the molecular mechanism(s) underlying altered cellular functions in PD, including mitochondrial dysfunction and α-synuclein (SNCA) aberrant expression. Findings: We observed increased SNCA expression in the dopaminergic (DA) neurons of both the wild-type and diabetic MitoPark mice, coupled with enhanced degeneration of DA neurons in the diabetic MitoPark mice. Ex vivo, in differentiated human DA neurons, IR was associated with increased SNCA and reactive oxygen species (ROS) levels, as well as mitochondrial depolarization. Moreover, we demonstrated concomitant hyperactivation of polo-like kinase-2 (PLK2), and upregulated p-SNCA (Ser129) and proteinase K-resistant SNCA proteins level in IR SH-SY5Y cells, however the inhibition of PLK2 reversed IR-related increases in phosphorylated and total SNCA. Similarly, the overexpression of peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC)-1α suppressed ROS production, repressed PLK2 hyperactivity, and resulted in downregulation of total and Ser129-phosphorylated SNCA in the IR SH-SY5Y cells. Conclusions: These findings demonstrate that IR-associated diabetes promotes the development and progression of PD through PLK2-mediated mitochondrial dysfunction, upregulated ROS production, and enhanced SNCA signaling, suggesting the therapeutic targetability of PLK2 and/or SNCA as potential novel disease-modifying strategies in patients with PD.

scholarly journals Functional disruption of α4 integrin mobilizes bone marrow–derived endothelial progenitors and augments ischemic neovascularization

2006 ◽  
Vol 203 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Gangjian Qin ◽  
Masaaki Ii ◽  
Marcy Silver ◽  
Andrea Wecker ◽  
Evelyn Bord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Cell Surface Receptor ◽  
Ischemic Disease ◽  
Angiogenic Therapy ◽  
Ischemic Tissue ◽  
Surface Receptor

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Different protein of Echinococcus granulosus stimulates dendritic induced immune response

Parasitology ◽  
2015 ◽  
Vol 142 (7) ◽  
pp. 879-889 ◽  
Author(s):  
YANA WANG ◽  
QIANG WANG ◽  
SHIYU LV ◽  
SHENGXIANG ZHANG
Keyword(s):  
Immune Response ◽  
Cell Proliferation ◽  
T Cell ◽  
Echinococcus Granulosus ◽  
Surface Marker ◽  
T Cell Proliferation ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Dc Maturation

SUMMARYCystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

A High-Throughput Screen to Identify Compounds Preserving Primary Human AML Stem Cells Ex-Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3587-3587
Author(s):  
Caroline Pabst ◽  
Josee Hebert ◽  
Guy Sauvageau
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Surface Marker ◽  
Leukemic Cells ◽  
High Throughput Screen ◽  
Self Renewal ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Abstract 3587 Acute myeloid leukemia (AML) is the second most frequent leukemia in adults with still high relapse rates supposed to be due to a rare population of leukemia stem cells (LSC) within the leukemic bulk not responding to conventional anti-cancer drugs. Engraftment of human leukemic cells in immunocompromised mice mimicking the human disease is considered to prove the existence of stem cells in the sample; however, the engraftment potential of primary human AML cells is usually quickly lost upon ex-vivo culture indicating that LSC have undergone differentiation or apoptosis and thus lost their stem cell properties. This lack of appropriate culture conditions enabling ex-vivo maintenance of primary human LSC is a major obstacle in the development of LSC targeted therapies. Our aim is, therefore, to identify conditions which allow the maintenance and expansion of these rare cells ex-vivo to understand their unique properties and to further elucidate cellular pathways underlying the self-renewal program of human primary LSC. We established a flow cytometry based high-throughput screen in which we tested more than 6,000 selected compounds with regards to their potential to expand primary human AML cells while preserving their phenotypic and functional characteristics. We considered alteration of surface marker expression, i.e. loss of CD34 and acquisition of markers of myeloid maturation initially not expressed on the cells, as indicator of differentiation implicating at the same time a loss of stem cells in the culture. We hypothesized that compounds that would be able to prevent these alterations in surface marker expression could have the potential to promote self-renewal of primary human LSC ex vivo. A normal-karyotype AML representing the most frequent cytogenetic group of AML in adults with FAB M1 morphology was chosen to perform the screen. Moreover, the initial CD34+CD15- phenotype allowed us to monitor changes in surface marker expression. Cells were plated in 384 well plates with a density of 5,000 cells per well. 6,160 compounds were tested with and without an inhibitor to aryl hydrocarbon receptor (AhR) and the vehicle DMSO was used as negative control resulting in a final DMSO concentration of ∼0.1% in each well, which had been proven to be not toxic to the cells. After 5 days of incubation the cells were analyzed on a BD LSRII high-throughput flow cytometer with regards to CD34 and CD15 surface expression. Compounds showing an increase of ≥45% of CD34+ and CD34+CD15- cells over DMSO compared to the AhR antagonist were selected for further evaluation. In a secondary screen we retested 32 compounds in 5 different concentrations ranging from 1:34 to 3x based on the initial screen concentration after 5 and 9 days of incubation and confirmed an increase ≥50% of relative and absolute CD34+CD15- leukemic cells after 5 days for 13 compounds and after 9 days for 7 compounds. We have initiated functional in-vitro and in-vivo experiments to validate these results and to prove the existence of LSC in cultures where compounds have been added. The ability of the newly identified compounds to preserve human LSC ex-vivo will be further evaluated using a collection of primary AML samples comprising different morphological and cytogenetic groups. Together, our observations document the feasibility of finding anti-differentiation (pro-self-renewal?) compounds of primary human AML cells that can be expanded ex-vivo. These studies represent a first key step in the identification of specific LSC targeting compounds. Disclosures: No relevant conflicts of interest to declare.

Ex vivo low-density lipoprotein oxidizability and in vivo lipid peroxidation in patients on CAPD

2001 ◽  
Vol 59 (s78) ◽  
pp. 128-136 ◽  
Author(s):  
Johannes M. Roob ◽  
Thomas Rabold ◽  
Marianne Hayn ◽  
Gholamali Khoschsorur ◽  
Ulrike Resch ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Ex Vivo ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density

scholarly journals The Antimicrobial Peptide, Bactenecin 5, Supports Cell-Mediated but Not Humoral Immunity in the Context of a Mycobacterial Antigen Vaccine Model

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 926
Author(s):  
Tulika Munshi ◽  
Adam Sparrow ◽  
Brendan W. Wren ◽  
Rajko Reljic ◽  
Samuel J. Willcocks
Keyword(s):  
Antimicrobial Peptide ◽  
Ex Vivo ◽  
Mycobacterial Antigen ◽  
Vaccine Adjuvants ◽  
Human Tonsil ◽  
Mhc I ◽  
Surface Marker Expression ◽  
Bacillus Subtilis Spore

Bactenecin (Bac) 5 is a bovine antimicrobial peptide (AMP) capable of killing some species of bacteria through the inhibition of protein synthesis. Bac5 and other AMPs have also been shown to have chemotactic properties and can induce inflammatory cytokine expression by innate immune cells. Recently, AMPs have begun to be investigated for their potential use as novel vaccine adjuvants. In the current work, we characterise the functionality of Bac5 in vitro using murine macrophage-like cells, ex vivo using human tonsil tissue and in vivo using a murine model of vaccination. We report the effects of the peptide in isolation and in the context of co-presentation with mycobacterial antigen and whole, inert Bacillus subtilis spore antigens. We find that Bac5 can trigger the release of nitric oxide from murine macrophages and upregulate surface marker expression including CD86, MHC-I and MHC-II, in the absence of additional agonists. When coupled with mycobacterial Ag85 and B. subtilis spores, Bac5 also enhanced IFNγ secretion. We provide evidence that B. subtilis spores, but not the Bac5 peptide, act as strong adjuvants in promoting antigen-specific immunoglobulin production in Ag85B-vaccinated mice. Our findings suggest that Bac5 is an important regulator of the early cell-mediated host immune response.

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