Decitabine-Induced Early Platelet Response, a Predictor of Favorable Outcome during Hypomethylating Treatment of MDS, Is Associated with In Vivo Megakaryocytic Differentiation

Introduction Despite broad use of hypomethylating agents (HMAs) in MDS/AML treatment, the number of established outcome predictors is still very limited (Stomper and Lübbert, Sem Hematol 2019). One of them, the early, isolated and sometimes massive increase of platelets, is recurrently observed in patients with MDS treated with HMAs. It is not observed with non-HMA cytidine analogs such as low-dose cytarabine. In HMA-treated MDS patients, an early platelet increase is a predictor of overall and leukemia-free survival (van den Bosch et al., Leuk Res 2004; van der Helm et al., Br J Haematol 2011). HMAs induce cellular differentiation in vitro, by gene reactivation in the malignant cells. However, evidence for HMA-induced in vivo differentiation is still very limited. We hypothesized that the megakaryocytic cell lineage in MDS is a target for HMA-induced cellular maturation in vivo. Methods We systematically analyzed the bone marrow morphology of 34 higher-risk MDS patients (median age: 71.5 years, range 51-79) before and after 1 cycle of treatment with the HMA decitabine (DAC). All patients had been treated at a single center within 3 prospective clinical trials (Wijermans et al., Ann Hematol 2005; Lübbert et al., J Clin Oncol 2011). One treatment cycle consisted of 45 mg/m2 DAC per day (15 mg/m2 intravenously over 4 hours every 8 hours) for 3 consecutive days, repeated 6 weeks later. The early platelet response was evaluated after 1 cycle of DAC treatment. Based on the criteria of the International Working Group, an absolute increase in platelet count of 30x109/l or more compared to the pre-treatment count was defined as a platelet response. The histological analysis of the bone marrow specimens was performed by an experienced hematopathologist blinded to the treatment timepoints. Up to 200 megakaryocytes (MK) per sample were quantified at a magnification of 400 x using chloroacetate esterase staining. Results Thirteen of 34 patients (38%) showed a platelet response already after 1 cycle of DAC treatment, 21 (62%) did not. The median pre-treatment platelet count did not differ in patients with or without an early platelet response (median of 34x109/l in both groups, range 7-169 and 8-265, respectively). After 1 cycle of DAC treatment, patients with a platelet response had a median platelet count of 117x109/l (range 78-281), patients without this response had a median platelet count of 32 x109/l (range 4-155). Overall survival (OS) was measured from the time of early platelet response assessment after completion of 1 treatment cycle, i.e. after 6 weeks. The presence of a platelet response after 1 DAC cycle was associated with a longer OS compared to the absence of this early platelet response: median of 26.6 versus 14.0 months (p=0.04). Both pre- and post-treatment bone marrow biopsies of patients with an early platelet response showed higher numbers of MK, as well as significant differences in MK morphology compared to biopsies from patients without an early platelet response. Regarding MK numbers, increased MK density in specimens of patients with an early platelet response was observed both before (mean MK number per high power field 6.2 vs. 2.6, p=0.02) and after the application of DAC (mean MK number 10.4 vs. 3.1, p=0.01). Regarding MK maturation stage, more pre-treatment juvenile MK (on average 32.4% vs. 20.5% of all MK, p=0.03) and MK with typical myeloproliferative stigmata (present in 5/13 vs. 2/21 biopsies) were observed in patients with an early platelet response, compared to patients without this response. Regarding the induction of megakaryocytic maturation during this early treatment phase, more post-treatment "naked", mature MK nuclei indicative of active platelet shedding (on average 9.5% vs. 3.8% of all MK, p=0.01), were noted in patients with an early platelet response than in patients without an early platelet response. Conclusions This is, to the best of our knowledge, the first systematic hematopathological analysis of changes in quantitative and morphological MK features in bone marrow specimens of MDS patients during HMA treatment. DAC, which has in vitro differentiation-inducing effects on megakaryoblastic cells, induced maturation also of dysplastic MK in vivo in higher-risk MDS patients with an early platelet response to this HMA. The predictive value of an early platelet increase, a very easy-to-determine parameter, during this type of treatment is confirmed. Disclosures No relevant conflicts of interest to declare.

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Notch Signaling-Dependant Expulsion of Parasites through Mast Cell-Mediated Immunity.

Blood ◽

10.1182/blood.v104.11.1473.1473 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1473-1473

Author(s):

Mamiko Sakata-Yanagimoto ◽

Etsuko Yamaguchi-Nakagami ◽

Toru Sakai ◽

Keiki Kumano ◽

Atsushi Kunisato ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Notch Signaling ◽

Cell Generation ◽

Pre Treatment ◽

And Function ◽

Tgf Β1

Abstract [Background] Notch signaling is known to be important in hematopoiesis, but very little information is available about its significance in mast cells. Here we provide direct evidence that notch signaling is critical for both development and function of mast cells in vitro and in vivo. [Methods] A Lin− fraction of mouse bone marrow cells was cultured on immobilized Delta1 in the presence of SCF and IL-3, and emerging Lin−FcεRI+c-Kit+ mast cells were characterized. Next, production of mouse mast cell protease-1 (mMCP-1), which is specific for nematode infection through locally expressed TGF-β1 in vivo, by bone marrow-derived mast cells (BMMC) was analyzed after the stimulation with Delta1 in the presence of TGF-β1. Finally, mice were infected with Strongyloides venezuelensis after pre-treatment with Delta1, and expulsion of the worms was examined. [Results] Lin−FcεRI+c-Kit+ mast cells developed remarkably earlier if stimulated with Delta1 (at one week, 15% vs. 3%). DAPT, a γ-secretase inhibitor, blocked the Delta1 effect, while it did not affect the regular time-course mast cell generation by SCF and IL-3. SB431542, a selective inhibitor of TGF-β1 signaling, also blocked early mast cell generation by Delta1. Delta1 augmented mMCP-1 expression and secretion from BMMC by 50 fold. Both DAPT and SB431542 showed a dose-dependent inhibition of Delta1 effect on mMCP-1 expression and secretion. Pre-treatment of the hosts with Delta1 promoted the expulsion of S. venezuelensis, (left/inoculated ratios of worms, 3% vs. 40%) while Delta1 had no effect in the mast cell-deficient W/Wv mice. [Discussion] Our observations reveal that notch signaling regulates both development and function of mast cells in vitro in conjunction with TGF-β1 signaling. In vivo, it is also likely that Delta1 facilitates the functional maturation of intestinal mast cells to eradicate parasites. More precise mechanism of Delta1 action on mast cells in vivo is under a study.

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Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

Blood ◽

10.1182/blood.v116.21.660.660 ◽

2010 ◽

Vol 116 (21) ◽

pp. 660-660

Author(s):

Mark J. Levis ◽

Amy Sexauer ◽

Trivikram Rajkhowa ◽

Donald Small ◽

Michael J. Borowitz

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Western Blotting ◽

Peripheral Blood ◽

Terminal Differentiation ◽

Flt3 Inhibitor ◽

Microscopic Evaluation ◽

Pre Treatment

Abstract Abstract 660 AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures: Levis: Ambit Biosciences, Inc: Consultancy.

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Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽

10.1182/blood.v120.21.5028.5028 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5028-5028 ◽

Cited By ~ 1

Author(s):

Deepak Sampath ◽

Elizabeth Punnoose ◽

Erwin R. Boghaert ◽

Lisa Belmont ◽

Jun Chen ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Lines ◽

Single Agent ◽

Employment Equity ◽

Bone Marrow Biopsies ◽

Xenograft Models ◽

Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

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In Vivo Effects Of Ibrutinib On The Migration Of Chronic Lymphocytic Leukemia Cells Differ Between Patients and Reduce The Ability Of The Bone Marrow Microenvironment To Attract The Tumor Cells

Blood ◽

10.1182/blood.v122.21.604.604 ◽

2013 ◽

Vol 122 (21) ◽

pp. 604-604

Author(s):

Sarah E. M. Herman ◽

Jade Jones ◽

Rashida Z. Mustafa ◽

Mohammed Farooqui ◽

Adrian Wiestner

Keyword(s):

Bone Marrow ◽

Cell Migration ◽

Chronic Lymphocytic Leukemia ◽

Tumor Cells ◽

Lymphocytic Leukemia ◽

Dual Chamber ◽

Bone Marrow Plasma ◽

Pre Treatment

Abstract The Bruton’s tyrosine kinase inhibitor ibrutinib has recently been shown to be well tolerated, and to induce objective clinical responses in the majority of patients, irrespective of adverse prognostic markers (Byrd et al., NEJM 2013). Despite the demonstrated clinical activity, ibrutinib also leads to a transient lymphocytosis that is thought to reflect a redistribution of cells from tissue compartments into the peripheral blood. The mechanisms contributing to this lymphocytosis are not well understood. To date, two groups have demonstrated that in vitro treatment with ibrutinib inhibits migration of CLL cells in chemokine gradients (de Rooji et al., Blood 2012 and Ponader et al., Blood 2012). Here we sought to assess the in vivo effect of ibrutinib on cellular migration. To validate our assay we first treated CLL cells with 1uM ibrutinib for 1 hour in vitro and measured migration of CLL cells to a mix of SDF-1 (at 200ng/mL) and CCL19 (at 100ng/mL), two chemo-attractants known to induce migration of CLL cells. Migration was assayed in a dual chamber system separated by a membrane with 5µm pores after 3 hours of incubation. Confirming published data we found a significant reduction in the migration index (ratio of migration to chemokines divided by migration to media alone) of ibrutinib treated cells compared to untreated cells (mean reduction 24%; P = 0.04). Next, we analyzed the migration of CLL cells obtained from patients (n = 9) enrolled on a clinical trial with single agent ibrutinib that were sampled pre-treatment and after 4 weeks on drug. We observed highly variable responses; in about half of the patients treated cells showed increased migration, while in the other half there was decreased migration to the SDF-1/CCL19 mix. Interestingly, patients showing a decrease in migration on treatment often had del17p but there was no difference in regards to IGHV mutation status and no correlation to the degree of lymphocytosis observed in the patient. T-cell migration was not affected by ibrutinib. In order to extend the analysis to a mix of chemo-attractants that the tumor cells may encounter in vivo we used the supernatant harvested from bone marrow aspirates and found that it efficiently induced migration of CLL cells in the dual chamber assay (mean fold increase 5.2 compared to control). Comparing CLL cells from patients sampled pre-treatment to those obtained on treatment day 28 we again found the same mixed effects of ibrutinib on the ability of CLL cells to migrate to bone marrow plasma as we had observed with the SDF-1/CCL19 mix. Thus, direct inhibition of CLL cell migration can account for only a subset of patients with treatment-induced lymphocytosis. Given reports that ibrutinib can inhibit cytokine and chemokine secretion from CLL cells and T-cells (Ponader et al., Blood, 2012; Herman et al., Blood, 2011), we hypothesized that ibrutinib treatment might change the content of chemo-attractants in the bone marrow We therefore compared the ability of the bone marrow plasma obtained pre-treatment and after 2 months on ibrutinib to attract CLL cells (these cells were obtained from the peripheral blood pre-treatment from the same patient donating the marrow). We found that in 4/4 patients evaluated there was a significant reduction in the migration of CLL cells to the on-treatment bone marrow plasma compared to the matching pre-treatment sample (mean decrease 20%; P < 0.05). In conclusion, migration of CLL cells from patients on ibrutinib can be inhibited or increased, with most del17p patients showing decreased migration. Intriguingly, these patients tend to have slower resolution of the treatment induced lymphocytosis, raising the question whether inhibition of homing to tissue sites could affect the time to resolution of the lymphocytosis. In addition, we provide evidence that bone marrow plasma on ibrutinib therapy has a reduced capacity to attract CLL cells, suggesting that ibrutinib may alter the composition of the bone marrow microenvironment This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

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Miniaturized 3D bone marrow tissue model to assess response to Thrombopoietin-receptor agonists in patients

eLife ◽

10.7554/elife.58775 ◽

2021 ◽

Vol 10 ◽

Author(s):

Christian A Di Buduo ◽

Pierre-Alexandre Laurent ◽

Carlo Zaninetti ◽

Larissa Lordier ◽

Paolo M Soprano ◽

...

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Response Prediction ◽

Bone Marrow Niche ◽

Platelet Response ◽

Tissue Model ◽

Bone Marrow Tissue ◽

Thrombopoietin Receptor ◽

Pre Treatment

Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.

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A New Flow-Based Assay to Assess Treatment Response in Congenital Thrombotic Thrombocytopenic Purpura

Blood ◽

10.1182/blood-2018-99-115965 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2441-2441

Author(s):

Ferras Alwan ◽

Chiara Vendramin ◽

Alice Taylor ◽

Mari Thomas ◽

Ri Liesner ◽

...

Keyword(s):

Treatment Response ◽

Research Funding ◽

Surface Coverage ◽

Thrombus Formation ◽

Post Treatment ◽

Normal Range ◽

Plasma Infusion ◽

Pre Treatment

Abstract Introduction Congenital TTP (cTTP) is an ultra-rare disorder in which deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) results in circulating ultra large Von Willebrand Factor (VWF) multimers and subsequent microthrombi formation. Regular prophylactic therapy aims to improve outcomes from long-term complications, but also ongoing symptoms, including lethargy, headaches and abdominal pain, despite normal blood counts. Existing methods of quantifying ADAMTS13 activity lack the sensitivity to enable their use for evaluating treatment response in patients with cTTP. We present a novel flow-based assay with the aim of assessing treatment response, novel therapeutic options and analyzing the impact of different mutations on disease severity. Method A VenaFlux semi-automated microfluidic system was used to provide shear flow to mimic in vivo flow rates. Using whole blood, we analyzed platelet adhesion, aggregation and thrombi formation on microchannels coated with type I collagen and mounted onto the stage of an inverted epifluorescence microscope. Fresh, citrated whole blood was treated with DiOC6 to achieve platelet fluorescence and a macro on Image-Pro Premier was designed for automated calculation of total surface coverage. Surface coverage represented increasing thrombus formation with total coverage by thrombus within 180 seconds quantified as 100% coverage. Results were compared to a normal range developed using 43 normal controls (26=female, 17=male) with normal hemoglobin, platelet count and hematocrit. The surface coverage normal range was 6-39%. cTTP samples were analyzed for complete blood count, ADAMTS13 activity, VWF antigen, VWF activity and percentage surface coverage. Samples were taken 30 minutes before and after prophylactic treatment, either plasma infusion or BPL-8Y. Recombinant ADAMTS13 was added in-vitro on all pre-treatment samples with 15 minutes incubation time. Further re-measurement was undertaken after initiation of aspirin for at least ten days. Results Eighteen patients with cTTP confirmed by genetic analysis and ADAMTS13 levels <5 IU/dl were included (16 = female, 2 = male) with a median age of 33 (range: 15-69 years). Median VWF antigen levels: 114% (range: 54% - 276%, NR: 50-160%) and median VWF activity levels: 173% (range: 83% - 338%, NR: 50-187%). The median pre-treatment surface coverage was 90% (range 47% - 100%). There was no significant difference in surface coverage considering genetic mutation type (median coverage for homozygous patients 88%, heterozygous 67%, p=0.99), mutation location (pre-spacer mutation surface coverage 67%, post spacer mutation surface coverage 84%, p=0.84), or age of first symptom onset (childhood onset surface coverage 59%, adult onset 86%, p=0.19). Plasma infusion improved surface coverage results with pre treatment coverage of 90% compared to 44% post plasma infusion (p=0.0003). In vivo recombinant ADAMTS13 administration on pre prophylaxis samples, resulted in normalization of surface coverage in all patients (p<0.0001)(median post rADAMTS13 coverage 28%, range 3-39%). In patients initiated on aspirin, surface coverage had improved both pre and post prophylaxis. The median pre treatment surface coverage for patients on aspirin was 51% (vs. 90% pre treatment and no aspirin, p=0.004). This improvement persisted after treatment with post treatment surface coverage of 18% (vs. 44% post treatment but not on aspirin, p=0.003). 100% of patients who received aspirin saw surface coverage return to the normal range post treatment compared to 82% with plasma infusion alone (p=0.0195). Conclusion Plasma infusion and aspirin synergistically reduce surface coverage by thrombus in patients with cTTP, demonstrated on peak and trough samples. Furthermore, in vitro addition of recombinant ADAMTS13 completely normalized thrombus formation. There were no major differences in surface coverage by genetic mutation. The newly developed flow-based assay presented can be used to assess treatment options and efficacy in cTTP in addition to demonstrating cTTP disease pathophysiology that has not previously been identified. In combination with clinical symptoms it offers potential to improve and personalize treatment for patients with cTTP. Figure. Figure. Disclosures Liesner: Bayer: Consultancy, Research Funding; Sobi: Speakers Bureau; Roche: Research Funding; Baxalta: Consultancy, Research Funding; Novo Nordisk: Research Funding, Speakers Bureau; Octapharma: Consultancy, Other: Clinical study investigator for NuProtect Study (Octapharma sponsored), Research Funding, Speakers Bureau. Scully:Novartis: Honoraria, Other: Member of Advisory Board, Speakers Bureau.

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Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

Author(s):

Hiroh Yamazaki ◽

Itsuro Kobayashi ◽

Tadahiro Sano ◽

Takio Shimamoto

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

The Other ◽

Endothelial Surface ◽

Important Interaction ◽

Blood Coagulability ◽

The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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CONTRACEPTION BY AN INJECTABLE LONG-ACTING OESTROGEN-PROGESTOGEN AGENT. III

Acta Endocrinologica ◽

10.1530/acta.0.0690067 ◽

1972 ◽

Vol 69 (1) ◽

pp. 67-76

Author(s):

Rolf Plesner

Keyword(s):

Treatment Period ◽

Treatment Cycle ◽

Post Treatment ◽

Mixed Phase ◽

Discontinuation Of Treatment ◽

Phase Type ◽

Secretory Type ◽

Pre Treatment ◽

Long Acting ◽

Fertile Women

ABSTRACT Twenty-two fertile women were treated cyclically in from 4–30 cycles (mean 15.5) with a total of 341 injections of Deladroxate®, an injectable, long-acting oestrogen-progestogen. The injections were administered on the 8th (7th–9th) day of each cycle. Before treatment, the last pre-treatment cycle was controlled by means of daily recordings of the basal body temperature (BBT), urinary excretion of pregnanediol and total pituitary gonadotrophins at certain intervals, and by endometrial biopsies obtained late in the cycle. The effects of Deladroxate® on ovulation, on pituitary gonadotrophic function, and on the endometrium were controlled by the above mentioned parameters during cycles 1, 3, and 6, and all assessments were repeated after discontinuation of treatment. During treatment, there was a statistically significant fall in gonadotrophin excretion values (as compared with the pre-treatment values), and the fall was found to be gradually progressive during treatment. After discontinuation of treatment, there seemed to be a tendency towards an increase in the excretion values. Suppression of ovulation as determined by means of the pregnanediol excretion during treatment, was effective in nearly all of the treatment cycles checked. The fall in pregnanediol excretion was also gradually progressive during treatment, while there was a slight increase in excretion values in the post-treatment period. During treatment, 79 BBT curves were recorded. Nearly 50 % were monophasic, indicating anovulatory cycles, 17 curves were biphasic, but with the rise in temperature occurring at non-characteristic times in the cycles, 18 curves were classified as thermogenic because of a rise in temperature occurring within 24 hours after the injection, and 5 curves were not assessable. During the first month after discontinuation of treatment, 8 out of 10 recorded curves were monophasic. Out of 53 endometrial biopsies obtained around the 23rd day of the cycle, 31 were of the mixed phase type, but showing a predominance of proliferative patterns, 15 were of the secretory type, and 7 were purely proliferative. Out of 15 biopsies obtained in the post-treatment period, only two were of the mixed phase type, 12 were proliferative and one was purely secretory.

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Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190123161447 ◽

2019 ◽

Vol 14 (4) ◽

pp. 305-319 ◽

Cited By ~ 4

Author(s):

Marietta Herrmann ◽

Franz Jakob

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Progenitor Cell ◽

Adult Stem Cells ◽

Current Knowledge ◽

Cell Populations ◽

Surface Markers ◽

Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

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Experimental Type 2 Diabetes Differently Impacts on the Select Functions of Bone Marrow-Derived Multipotent Stromal Cells

Cells ◽

10.3390/cells10020268 ◽

2021 ◽

Vol 10 (2) ◽

pp. 268

Author(s):

Jonathan Ribot ◽

Cyprien Denoeud ◽

Guilhem Frescaline ◽

Rebecca Landon ◽

Hervé Petite ◽

...

Keyword(s):

Type 2 Diabetes ◽

Bone Marrow ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Therapeutic Modality ◽

Multipotent Stromal Cells ◽

Cell Therapies

Bone marrow-derived multipotent stromal cells (BMMSCs) represent an attractive therapeutic modality for cell therapy in type 2 diabetes mellitus (T2DM)-associated complications. T2DM changes the bone marrow environment; however, its effects on BMMSC properties remain unclear. The present study aimed at investigating select functions and differentiation of BMMSCs harvested from the T2DM microenvironment as potential candidates for regenerative medicine. BMMSCs were obtained from Zucker diabetic fatty (ZDF; an obese-T2DM model) rats and their lean littermates (ZL; controls), and cultured under normoglycemic conditions. The BMMSCs derived from ZDF animals were fewer in number, with limited clonogenicity (by 2-fold), adhesion (by 2.9-fold), proliferation (by 50%), migration capability (by 25%), and increased apoptosis rate (by 2.5-fold) compared to their ZL counterparts. Compared to the cultured ZL-BMMSCs, the ZDF-BMMSCs exhibited (i) enhanced adipogenic differentiation (increased number of lipid droplets by 2-fold; upregulation of the Pparg, AdipoQ, and Fabp genes), possibly due to having been primed to undergo such differentiation in vivo prior to cell isolation, and (ii) different angiogenesis-related gene expression in vitro and decreased proangiogenic potential after transplantation in nude mice. These results provided evidence that the T2DM environment impairs BMMSC expansion and select functions pertinent to their efficacy when used in autologous cell therapies.

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