Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice

2021 ◽  
Vol 14 (669) ◽  
pp. eabd0536
Author(s):  
Christina Mundy ◽  
Lutian Yao ◽  
Sayantani Sinha ◽  
Juliet Chung ◽  
Danielle Rux ◽  
...  
Keyword(s):  
Heterotopic Ossification ◽  
Developmental Trajectories ◽  
Neutralizing Antibody ◽  
Inflammatory Cells ◽  
Activin A ◽  
Rna Seq ◽  
Antibody Treatment ◽  
Gene Encoding ◽  
Bone Accumulation

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals Blocking TLR2 in vivo Protects against Accumulation of Inflammatory Cells and Neuronal Injury in Experimental Stroke

2010 ◽  
Vol 31 (2) ◽  
pp. 757-766 ◽  
Author(s):  
Gina Ziegler ◽  
Dorette Freyer ◽  
Denise Harhausen ◽  
Uldus Khojasteh ◽  
Wilfried Nietfeld ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Neuronal Death ◽  
Focal Cerebral Ischemia ◽  
Neuronal Survival ◽  
Inflammatory Cells ◽  
Experimental Stroke ◽  
Wild Type ◽  
Antibody Treatment ◽  
Cell Counts

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy.

scholarly journals Chi3l1 is a modulator of glioma stem cell states and a therapeutic vulnerability for glioblastoma

2021 ◽  
Author(s):  
Charlotte Guetta-Terrier ◽  
David Karambizi ◽  
Bedia Akosman ◽  
Jia-Shu Chen ◽  
Suchitra Kamle ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Transition Probabilities ◽  
State Transitions ◽  
Glioma Stem Cells ◽  
Beta Catenin ◽  
Glioma Stem Cell ◽  
Rna Seq ◽  
Antibody Treatment ◽  
Blocking Antibody

Chi3l1 (Chitinase 3-like 1) is a secreted protein highly expressed in glioblastoma. Here, we show that exposure of glioma stem cells (GSCs) to Chi3l1 reduces the CD133+/SOX2+ cells and increases the CD44+/Chi3l1+ cells. Chi3l1 binds to CD44 and induces phosphorylation and nuclear translocation of beta-catenin, Akt and STAT3. Single cell RNA-seq and RNA velocity following incubation of GSCs with Chi3l1 show significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq reveals that Chi3l1 increases accessibility of promoters containing MAZ transcription factor footprint. Inhibition of MAZ directly regulates genes with highest expression in cellular clusters exhibiting significant cell state transitions. Finally, targeting Chi3l1 in vivo with a blocking antibody, resets the transcriptomic profile of glioblastoma and inhibits tumor growth. Our work implicates Chi3l1 as modulator of GSC cellular states and demonstrates pre-clinical efficacy of anti-Chi3l1 antibody treatment.

scholarly journals Regulation of contact hypersensitivity by interleukin 10.

1994 ◽  
Vol 179 (5) ◽  
pp. 1597-1604 ◽  
Author(s):  
T A Ferguson ◽  
P Dube ◽  
T S Griffith
Keyword(s):  
T Cells ◽  
Neutralizing Antibody ◽  
Inflammatory Cells ◽  
Interleukin 10 ◽  
Lymphoid Cells ◽  
Contact Hypersensitivity ◽  
Mrna Transcription ◽  
Antigenic Challenge ◽  
A Site

Contact hypersensitivity (CHS) responses require the participation of T cells, along with a variety of cytokines and adhesion molecules. In the classical CHS, antigen-specific T cells are recruited to a site of antigenic challenge, where they react with antigen, release cytokines, and attract other inflammatory cells. In the mouse model of CHS, this reaction is elicited in sensitized mice by application of the immunogen 4-7 d after immunization. The reaction peaks at 24 h, is slightly reduced by 48 h, and can return to normal by 72 h. This is in spite of the fact that some antigen is still present at the site of challenge. Here we examined the hypothesis that locally produced interleukin 10 (IL-10) regulates the duration of the response. Our data show that IL-10 protein peaked 10-14 h after antigenic challenge and returned to background by 24 h. The production of IL-10 protein corresponded with, and followed IL-10 mRNA transcription as detected by reverse transcriptase-polymerase chain reaction. During peak IL-10 production after antigenic challenge, it was not possible to transfer CHS with immune lymphoid cells, unless neutralizing antibody to IL-10 was given first. Additionally, when sensitized mice were given neutralizing anti-IL-10 antibody at the time of antigenic challenge, the duration of CHS was prolonged well beyond the natural course of the response. Finally, we demonstrate that rIL-10, when injected into the skin before antigenic challenge, prevented the elicitation of CHS in previously sensitized mice. Taken together, our data show an important role for IL-10 in the natural regulation of CHS responses in vivo.

scholarly journals Predicting in vivo escape dynamics of HIV-1 from a broadly neutralizing antibody

2021 ◽  
Vol 118 (30) ◽  
pp. e2104651118
Author(s):  
Matthijs Meijers ◽  
Kanika Vanshylla ◽  
Henning Gruell ◽  
Florian Klein ◽  
Michael Lässig
Keyword(s):  
Viral Load ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Broadly Neutralizing Antibodies ◽  
Antibody Treatment ◽  
Trade Off ◽  
Escape Dynamics ◽  
Resistance Evolution ◽  
Hiv 1

Broadly neutralizing antibodies are promising candidates for treatment and prevention of HIV-1 infections. Such antibodies can temporarily suppress viral load in infected individuals; however, the virus often rebounds by escape mutants that have evolved resistance. In this paper, we map a fitness model of HIV-1 interacting with broadly neutralizing antibodies using in vivo data from a recent clinical trial. We identify two fitness factors, antibody dosage and viral load, that determine viral reproduction rates reproducibly across different hosts. The model successfully predicts the escape dynamics of HIV-1 in the course of an antibody treatment, including a characteristic frequency turnover between sensitive and resistant strains. This turnover is governed by a dosage-dependent fitness ranking, resulting from an evolutionary trade-off between antibody resistance and its collateral cost in drug-free growth. Our analysis suggests resistance–cost trade-off curves as a measure of antibody performance in the presence of resistance evolution.

Combinational Therapy of Lenalidomide with Activin A Neutralizing Antibody; Preclinical Rationale for a Novel Anti-Myeloma Strategy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1871-1871
Author(s):  
Tyler Scullen ◽  
Sonia Vallet ◽  
Loredana Santo ◽  
Mariateresa Fulciniti ◽  
Homare Eda ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Research Funding ◽  
Therapeutic Potential ◽  
Neutralizing Antibody ◽  
Positive Outcome ◽  
Activin A ◽  
Tumor Burden ◽  
Bone Architecture ◽  
Bone Lesions

Abstract Abstract 1871 Multiple myeloma (MM) is characterized by the vast majority of patients developing osteolytic bone lesions consequent to biochemical imbalances in bone resorption and formation. As such, compound combinations that succeed in selectively inhibiting processes relevant to both osteolysis as well as MM cell viability would be of great therapeutic potential to the MM patient. Here we investigated the benefits of combining lenalidomide with an Activin A neutralizing antibody. Lenalidomide is an immunomodulatory drug which displays both direct anti tumor activity as well as indirect inhibition of osteoclastogenesis but lacks an anabolic bone effect. Conversely, Activin A, secreted predominantly by bone marrow stromal cells (BMSCs), is an extracellular protein complex with apparent paracrine functions critical in MM tumor-induced osteolysis whose sequestration has been recently demonstrated by us to restore bone architecture and reduce tumor burden in vivo. Given these properties, we hypothesized that an alleviation of the abnormal processes associated with the MM bone microenvironment via Activin A neutralizing antibody, combined with a selective targeting of MM cell growth and viability with lenalidomide could yield a positive outcome for MM patients with bone disease. Interestingly, our studies revealed that lenalidomide stimulated Activin A secretion in BMSCs, leading us to search for potential underlying mechanisms. Due to previous implications in MM induced Activin A secretion, as well as the presence of a highly conserved c-Jun-binding sequence in the promoter of the constituent subunit of the peptide, INHβA, the JNK-dependent pathway was of particular interest. Accordingly, BMSCs treated with lenalidomide displayed an early activation of JNK signaling evidenced by increases in both JNK phosphorylation and c-Jun mRNA expression, an effect which was found to be AKT dependent. We next investigated and accordingly observed that lenalidomide had a deleterious effect on osteoblastogenesis as evidenced via significant decreases in alkaline phosphatase activity associated with a downregulation of DLX5 mRNA expression and an increase in SMAD2 signaling. Expectantly, further studies revealed that osteoblasts differentiated both alone as well as in co-culture with MM cells exhibited variable levels of extracellular Activin A concentrations which were inversely correlated with ALP activity when treated with lenalidomide. Furthermore, the addition of lenalidomide was in fact noted to augment existing Activin A secretion as well as the resultant ALP dysfunction. This could be overcome by the addition of Activin A neutralizing antibody, which efficiently sequestered Activin A without compromising the cytotoxic MM activity of lenalidomide. Collectively, our results illustrate an Activin A mediated inhibition of osteoblastogenesis by lenalidomide, in which lenalidomide stimulates a JNK dependent secretion of Activin A by BMSCs. This however can be reversed via the addition of Activin A neutralizing antibody. Our work therefore indicates the validity of combining Activin A neutralizing antibody with lenalidomide, and provides the basis for a currently accruing Phase I clinical trial in MM (NCT01562405). Disclosures: Raje: Celgene: Consultancy; Onyx: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
Sonali Arora ◽  
Anca Mihalas ◽  
John Bassett ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Single Cell ◽  
Developmental Trajectories ◽  
Expression Patterns ◽  
Experimental Models ◽  
Tumor Formation ◽  
Sequencing Analysis ◽  
Rna Seq ◽  
Human Glioblastoma

Abstract Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.

scholarly journals Predicting in vivo escape dynamics of HIV-1 from a broadly neutralizing antibody

2020 ◽  
Author(s):  
Matthijs Meijers ◽  
Kanika Vanshylla ◽  
Henning Gruell ◽  
Florian Klein ◽  
Michael Lässig
Keyword(s):  
Viral Load ◽  
Neutralizing Antibodies ◽  
Fitness Landscape ◽  
Neutralizing Antibody ◽  
Broadly Neutralizing Antibodies ◽  
Antibody Treatment ◽  
Escape Dynamics ◽  
Resistance Evolution ◽  
Hiv 1

ABSTRACTBroadly neutralizing antibodies are promising candidates for treatment and prevention of HIV-1 infections. Such antibodies can temporarily suppress viral load in infected individuals; however, the virus often rebounds by escape mutants that have evolved resistance. In this paper, we map an in vivo fitness landscape of HIV-1 interacting with broadly neutralizing antibodies, using data from a recent clinical trial. We identify two fitness factors, antibody dosage and viral load, that determine viral reproduction rates reproducibly across different hosts. The model successfully predicts the escape dynamics of HIV-1 in the course of an antibody treatment, including a characteristic frequency turnover between sensitive and resistant strains. This turnover is governed by a dosage-dependent fitness ranking, resulting from an evolutionary tradeoff between antibody resistance and its collateral cost in drug-free growth. Our analysis suggests resistance-cost tradeoff curves as a measure of antibody performance in the presence of resistance evolution.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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