scholarly journals Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-13
Author(s):  
Mariana Lazarini ◽  
Vanessa Bernusso ◽  
Karla P.V Ferro ◽  
Adriana Silva Santos Duarte ◽  
Cristina Pontes Vicente ◽  
...  
Keyword(s):  
Mouse Model ◽  
Conflicts Of Interest ◽  
Granule Size ◽  
Slight Reduction ◽  
National Council ◽  
Wild Type ◽  
Cell Extracts ◽  
Megakaryocyte Differentiation

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Efficacy of BB-83698, a Novel Peptide Deformylase Inhibitor, in a Mouse Model of Pneumococcal Pneumonia

2004 ◽  
Vol 48 (1) ◽  
pp. 80-85 ◽  
Author(s):  
E. Azoulay-Dupuis ◽  
J. Mohler ◽  
J. P. Bédos
Keyword(s):  
Mouse Model ◽  
Lung Tissue ◽  
Resistant Strain ◽  
Peptide Deformylase ◽  
In Vitro Activity ◽  
Wild Type ◽  
Virulent Strains ◽  
Resistant Strains

ABSTRACT The efficacy of BB-83698, a novel potent peptide deformylase inhibitor, was evaluated in a mouse model of acute pneumonia. The Streptococcus pneumoniae isolates tested included four virulent strains (one penicillin-susceptible wild-type strain, one macrolide-resistant strain, and two quinolone-resistant mutants [a mutant carrying mutations in ParC and GyrA and an efflux mutant] isogenic to the wild type) and two poorly virulent penicillin-resistant strains. Pneumonia was induced by intratracheal inoculation of 105 CFU (virulent strains) into immunocompetent mice or 107 CFU (less virulent strains) into leukopenic mice. Animals received three or six subcutaneous injections of antibiotics at 12- or 24-h intervals, with antibiotic treatment initiated at 3, 6, 12, or 18 h postinfection (p.i.). BB-83698 showed potent in vitro activity against all strains (MICs, 0.06 to 0.25 μg/ml). In the in vivo model, all control animals died within 2 to 5 days of infection. BB-83698 (80 mg/kg of body weight twice daily or 160 mg/kg once daily) protected 70 to 100% of the animals, as measured 10 days p.i., regardless of the preexisting resistance mechanisms. In contrast, the survival rates for animals treated with the comparator antibiotics were 30% for animals treated with erythromycin (100 mg/kg) and infected with the macrolide-resistant strain, 34% for animals treated with amoxicillin (200 mg/kg every 8 h) and infected with the penicillin-resistant strain, and 0 and 78% for animals treated with ciprofloxacin (250 mg/kg) and infected with the ParC and GyrA mutant and the efflux mutant, respectively. At 80 mg/kg, BB-83698 generated a peak concentration in lung tissue of 61.9 μg/ml within 1 h and areas under the concentration-times curves of 57.4 and 229.4 μg · h/ml for plasma and lung tissue, respectively. The emergence of S. pneumoniae isolates with reduced susceptibilities to BB-83698 was not observed following treatment with a suboptimal dosing regimen. In conclusion, the potent in vitro activity of BB-83698 against S. pneumoniae, including resistant strains, translates into good in vivo efficacy in a mouse pneumonia model.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

ABCG2 C.421C>A Is Associated with Higher Trough Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 110-110
Author(s):  
Naoto Takahashi ◽  
Masatomo Miura ◽  
Stuart A Scott ◽  
Kenichi Sawada
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Plasma Levels ◽  
Myeloid Leukemia ◽  
Drug Transport ◽  
Conflicts Of Interest ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Large Patient

Abstract Abstract 110 [Background] Despite the excellent efficacy of imatinib for the treatment of chronic myeloid leukemia (CML), trough imatinib plasma levels can vary widely among patients. This may be due, in part, to inter-individual variation in imatinib metabolism and drug transport efficacy. To investigate the role of genetic variation in the pharmacokinetics of imatinib, we analyzed common single nucleotide polymorphisms within important imatinib pathway genes including ABCG2 (BCRP), ABCB1 (MDR1), ABCC2 (MRP2), CYP3A5, and SLC22A1 (OCT1) in 67 CML patients treated with imatinib. In addition, trough imatinib plasma levels were determined using high-performance liquid chromatography-tandem mass spectrometry. [Results] Distinct imatinib pharmacokinetics were identified in association with ABCG2 c.421C>A (p.Q141K; rs2231142) genotype. Specifically, the presence of the variant c.421A allele was significantly (p=0.024) associated with higher imatinib concentrations [median Cmin/Dose 2.70 (range: 1.50-8.30) ng/ml/mg; n=25] compared to patients with the wild-type ABCG2 (c.421C/C) genotype [median Cmin/Dose 2.27 (range: 0.37-5.30) ng/ml/mg; n=42]. ABCG2 is an efflux transporter for many xenobiotics, including imatinib, and is expressed at high levels in the human liver. Previous studies indicate that c.421A causes a 40% reduction in imatinib transport in vitro when compared to the wild-type genotype. Our data suggest that CML patients with ABCG2 c.421A allele may have deficient ABCG2 activity in vivo, resulting in reduced hepatic excretion of imatinib. Of note, although less common among Africans and individuals of European decent, the ABCG2 c.421C>A allele occurs at a high frequency in the Japanese (0.311) and Han Chinese (0.289) populations. [Conclusion] The association of ABCG2 c.421C>A with imatinib pharmacokinetics may explain why some Japanese CML patients administered less than 400 mg/day of imatinib have clinically sufficient trough imatinib plasma levels. Prospective studies are warranted to confirm the association between ABCG2 genotype and imatinib pharmacokinetics in large patient populations. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 111-111
Author(s):  
Meghedi N Aghourian ◽  
Catherine A Lemarie ◽  
Francois-Rene Bertin ◽  
Mark D Blostein
Keyword(s):  
Endothelial Cells ◽  
Venous Thromboembolism ◽  
Microarray Analysis ◽  
Conflicts Of Interest ◽  
Prostaglandin E ◽  
Wild Type ◽  
Prostaglandin E Synthase ◽  
Murine Lung

Abstract Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation

2006 ◽  
Vol 74 (12) ◽  
pp. 6907-6919 ◽  
Author(s):  
Andrea Hamilton ◽  
Carl Robinson ◽  
Iain C. Sutcliffe ◽  
Josh Slater ◽  
Duncan J. Maskell ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mutant Strain ◽  
Natural Host ◽  
Wild Type ◽  
Animal Suffering ◽  
Organ Cultures ◽  
Mucus Production ◽  
Streptococcus Equi

ABSTRACT Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM 138 - 213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt 190 - 685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt 190 - 685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt 190 - 685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM 138 - 213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt 190 - 685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

scholarly journals A Novel BCL2-Driven Compound Mutant Mouse Model for In Vivo Research on BH3 Mimetics

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Ismini Halmer ◽  
Alexandra da Palma Guerreiro ◽  
Laura Beckmann ◽  
Christian Reinhardt ◽  
Hamid Kashkar ◽  
...  
Keyword(s):  
Overall Survival ◽  
B Cells ◽  
Mouse Model ◽  
Mouse Models ◽  
Clinical Symptoms ◽  
Conflicts Of Interest ◽  
Bh3 Mimetics ◽  
Normal Ranges

Introduction: Eµ-TCL1-transgenic mouse models are often applied to discover and observe the development and kinetic of chronic lymphocytic leukaemia (CLL), as they develop diseases most similar to human CLL with a very high penetrance. To gain a better understanding on new therapy options and their effect on disease regression it is very important to observe therapy response, overall survival and symptoms during treatment of the disease not only in vitro but also in vivo in a suitable mouse model. However, application of BH3 mimetics like venetoclax is limited in the classical Eµ-TCL1 mouse model, since these mice are resistant towards venetoclax treatment. Therefore, we have generated a novel mouse model with Eµ-TCL1 as back bone and conditional overexpression of BCL2. Methods and results: We established a new mouse model (TBC) by crossbreeding mice expressing Eµ-TCL1tg/wtwith mice containing a B-cell specific conditional Bcl-2Rosa26/wt; Cd19CreCre/wtoverexpression and compared the disease kinetics to classical Eµ-TCL1 mice and to BC mice. TBC animals exhibit a severe leukocytosis at very early stages of disease development (12 weeks; mean 96.000/µl) in comparison to TC (15.100/µl) and BC (81.900/µl) mice. TBC mice develop CD23low/CD21neg leukemic B cells as they are known from TC mice with CD19+/CD5+ expression. Indeed, these mice show a significantly shortened overall survival of ~300 days (n=43) compared to TC mice (n=106; ~350 days; p&lt;0.001) and BC mice (n=28; ~410 days; p&lt;0.001) with severe clinical symptoms such as splenomegaly and cachexia. Strikingly, in contrast classical TC mice, which are resistant towards venetoclax, isolated B-cells of TBC mice are 10-times more sensitive towards venetoclax in vitro (0,02 µM) and can also be killed by the MCL1 inhibitors in nanomolar ranges, but not by BCL-xl inhibitors (&gt;2µM). Based on our in vitro data, we have treated TBC mice with venetoclax and observed an early and dramatic drop of leukocytes to normal ranges within the first two weeks of treatment. Leukocyte reduction lasted for the whole period of treatment. When investigating the spleens after sacrificing the mice they showed high amounts of dead cells inside the spleens, indicating that venetoclax was also efficient in lymphatic tissues as we know it from human trials. Conclusions: Autochthonous mouse models on which BH3 mimetics can be tested are rare. In our mouse model apoptosis screening in vitro we can show good results for BH3 mimetics with a high sensitivity already in low dosing. The BCL2-driven TCL1 mouse model enables the investigation of treatment with venetoclax in vivo to gain a better understanding of this frequently on patients applied therapy. Moreover, this model will help us to test other drugs (like MCL1 inhibitors) in combination with venetoclax to identify synergistic drugs in vivo in a timely manner. Furthermore, this model will offer us the opportunity to identify treatment strategies to overcome venetoclax resistance in vivo. Disclosures No relevant conflicts of interest to declare.

In Vitro and In Vivo Characterization of a Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FXRoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2697-2697
Author(s):  
Elise Roy ◽  
Paris Margaritis ◽  
Harre D. Downey ◽  
Katherine A. High
Keyword(s):  
Mouse Model ◽  
Thrombus Formation ◽  
Bleeding Tendency ◽  
Factor X ◽  
Wild Type ◽  
Intrinsic Pathway ◽  
Vessel Occlusion ◽  
Tail Clip

Abstract The complex and dynamic interplay between the intrinsic and extrinsic pathways of blood coagulation is incompletely understood. The mediator of prothrombin cleavage, Factor X (FX), plays a pivotal role as part of both the extrinsic and intrinsic tenase complexes. Moreover, the existence of naturally occurring Factor X mutations that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., 1988), is due to a missense mutation (Thr318←Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3% wild-type) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50% wild-type) in the extrinsic pathway as measured by PT. We chose to generate a mouse model of FX asymmetric activation to further probe the extrinsic-intrinsic pathway physiological relationship in hemostasis and thrombosis. For this, we used both an in vitro and an in vivo approach. We first constructed and purified the mouse homolog of FXRoma (mFXRoma) as well as wild-type mFX. Using a clotting-based assay, mFXRoma exhibited intrinsic and extrinsic activity comparable to that reported for the human mutation (5% and 18%, respectively). The reduced intrinsic and extrinsic activity of mFXRoma was not due to a secretion defect, based on Western blot analysis of supernatant and cell extracts from mFXRoma and mFX stably-transfected human embryonic kidney (HEK-293) cell lines. Mice homozygous for the analogous mutation (Thr315←Met) in exon 8 of the murine FX gene were generated by using a plug-and-socket approach. This resulted in the endogenous mFX exon 8 sequence being replaced with the mutated one, thus affording gene expression under the endogenous promoter. Analysis of mFXRoma homozygous mice showed a 6.4% and 19.2% intrinsic and extrinsic activity relative to wild-type littermates, respectively, confirming our in vitro data. The reduced activity in these mice resulted in a slight reduction in levels of the thrombin-antithrombin (TAT) complex. To determine any physiological defect of this mutation on the two pathways of coagulation, we performed two hemostatic challenges of the macrocirculation (tail clip and FeCl3-induced thrombus formation). In the tail-clip assay, blood loss showed no statistical difference between wild-type (n=5) and mFXRoma (n=6) mice. In contrast, following FeCl3-induced injury on the carotid artery (larger vessel diameter that in the tail), mFXRoma mice (3/3) failed to result in vessel occlusion (up to 30 min of observation), whereas wild-type littermates showed stable vessel occlusion (3/4) within ∼6 min of FeCl3 application. Although the type of injury was different, these data suggest that an impeded intrinsic activity of FX does not appear to affect hemostasis of the macrocirculation at relatively small diameter vessels but is essential for thrombus formation in large diameter vessels, and a relatively normal extrinsic activity does not compensate for this defect. This mouse model will aid in determining the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway of coagulation.

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