The JAK2 V617F Mutation Identifies Specific Subgroups of Myelodysplastic Syndrome (MDS).

Abstract The myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal stem cell disorders characterised by ineffective erythropoiesis and peripheral blood cytopenias. However, specific subgroups of MDS with both myeloproliferative (MPD) and dysplastic features are recognized, which include chronic myelomonocytic leukaemia (CMML), refractory anaemia with ringed sideroblasts and thrombocytosis (RARS-T) and MDS/MPD unclassified. In addition cases of 5q- syndrome may present with a marked elevation in platelet count and a hypercellular bone marrow. The molecular events which determine the predominant morphological features however remain unclear. The JAK2 somatic point mutation (V617F) results in constitutive activation of the tyrosine kinase and culminates in both proliferation and differentiation signals to the cell and thus may contribute to the proliferative features identified in MDS as reported in the chronic MPDs. We analysed 194 patients from six centres with a diagnosis of MDS or MDS/MPD for the presence of the JAK2 mutation. 11 cases of RA; 70, RARS; 60, 5q- syndrome; 28, refractory anaemia with excess blasts (RAEB); 9, acute myeloid leukaemia with multi-lineage dysplasia (AML-MLD); 4, MDS/MPD-U; 1, CMML; and 1, hypoplastic MDS were analysed. DNA samples were obtained from bone marrow aspirate or peripheral blood. Ethical approval was obtained. Genomic DNA was prepared using standard methods (Qiagen). A modified allele specific PCR (AS PCR) was used to screen DNA samples. JAK2 mutated DNA samples were further subjected to pyrosequencing for confirmatory testing. The median age of the JAK2 mutant versus wild type (WT) cases was 66years (46–80years) v 67years (30–92years). The JAK2 mutation was detected in 13/194 (6.7%) cases overall. 4/70 (5.7%) RARS, 5/60 (8%) 5q- syndrome, 1/28 RAEB (associated with del 5q), 1/4 CMML, 1 MDS/MPD-U and 1/8 AML-MLD demonstrated the mutation. Within the RARS cohort, the prevalence of JAK2 mutation increased to 4/6 (67%) of cases with thrombocytosis (platelets >500x109/l). The presence of the mutation was confirmed in 8/13 cases. In cases analysed, the mutation was detected in CD34+, CD14+, CD15+ and CD61+ cell fractions but not CD3+ or CD19+ cell lineages. On analysis of the haemoglobin (Hb), platelet count and total white cell count (WCC) in the JAK2 mutant versus wild-type groups, there was no difference in median Hb (10.8 v 9.2 g/dl, p=0.275) but a significantly higher median platelet count (526 v 209 x109/l, p<0.001) and WCC (6.43 v 4.84x109/l, P<0.001) was observed in the JAK2 mutant cases. In addition, all JAK2 mutant cases were associated with marked increase in bone marrow cellularity. The JAK2 mutation identifies a subset of MDS patients with proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis. The presence of JAK2 mutation helps to identify a subgroup of MDS patients who may benefit from therapies that specifically target the mutant JAK2.

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Cultivation of Megakaryocytes On a Collagen Matrix: A More Sensitive and Reproducible Test for the Diagnosis of Patients with Essential Thrombocythaemia.

Blood ◽

10.1182/blood.v114.22.4970.4970 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4970-4970

Author(s):

Adrian Emanuel Schmidt ◽

Patricia Darlington ◽

Lucie Kopfstein ◽

Elisabeth Ischi ◽

Elisabeth Oppliger Leibundgut ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Negative Predictive Value ◽

Predictive Value ◽

Healthy Subjects ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Essential Thrombocythaemia ◽

Jak2 Mutation ◽

Spontaneous Proliferation

Abstract Abstract 4970 Background Essential thrombocythaemia (ET) is one of the chronic myeloproliferative neoplasms (MPN), along with polycythaemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukaemia (CML). Their common feature is excessive proliferation of a certain stem or progenitor cell in the bone marrow; in the case of ET, the megakaryocytic lineage is affected. Clinical manifestations include thrombotic events and haemorrhage. Diagnosis of ET according to new WHO-criteria requires a sustained high platelet count, bone marrow biopsy showing proliferation of the megakaryocytic lineage with large and mature morphology, demonstration of JAK2 V617F (although only present in about 50% of patients with ET) or another clonal marker and explicit exclusion of other myeloid and myeloproliferative neoplasms as well as signs of reactive thrombocytosis. Additionally, spontaneous proliferation of megakaryocytes obtained from peripheral blood can be detected in in vitro culture assays. Presently, we use agar as a matrix for megakaryocyte cultivation, although this assay has never been validated in connection with ET. The identification of megakaryocytic colonies grown on agar can sometimes be quite difficult. Our aims were therefore to technically evaluate the use of a collagen based matrix and to investigate its suitability to identify patients with ET. Patients and Methods We have examined 63 patients (26 with ET, 21 with PV, 8 with myelofibrosis [MF; including PMF and post-ET/PV-MF], 6 with secondary or idiopathic erythrocytosis and 2 with secondary thrombocytosis; mean age=59.8, male=33, female=30, mean platelet count 457 G/l) and 5 healthy subjects. Following informed consent, both clinical and laboratory data was collected. Medication intake, phlebotomies, smoking habits and regular haemogram results were noted in order to recognise possible confounding factors influencing laboratory results. Results of megakaryocyte cultivation on both agar and collagen matrixes were recorded, considering both spontaneous growth and growth stimulated by megakaryocyte derived growth factor (MDGF). Results Based on our collagen culture results we were able to define 2 or more spontaneously grown megakaryocyte colonies as the most optimal cut-off for the identification of patients with MPN (sensitivity 71%, specificity 100% with positive and negative predictive values of 100% and 45%, respectively). Compared to the agar culture results (where a specificity and a positive predictive value of 100% were demonstrated at a cut-off value of ≥ 10 CFU-Mega) we found a higher accuracy and better reproducibility. In addition, we observed an improved negative predictive value (45% with collagen versus 25% with agar cultures) reducing false negative results. Healthy subjects and patients with secondary thrombocytosis showed no significant spontaneous megakaryocyte proliferation. In patients with MF, we observed strong spontaneous and MDGF-stimulated growth of megakaryocytic colonies. At a cut-off value of ≥ 50 CFU-Mega (after stimulation with MDGF), the collagen assay showed a sensitivity of 100% and a specifity of 70% for this special form of MPN, resulting in a negative predictive value of 100%. We found no confounding clinical or laboratory parameters such as medication intake (particularly cytoreductive treatment with hydroxyurea) or phlebotomies influencing our culture results, and no significant effect of the Jak2-V617F mutation on the growth behaviour of megakaryocytic colonies. Conclusion The results of this ongoing study imply that the collagen based assay is more sensitive, specific, time efficient and user friendly regarding the detection of spontaneous proliferation of megakaryocytes than the currently used agar based culture assay. In addition, the collagen based assay also has the great advantage that it allows isolation of single megakaryocytic colonies for further analyses, for example PCR-based identification of a JAK2 mutation. Furthermore, the collagen based assay facilitates the diagnosis of patients with MPN, especially in cases where conventional diagnostic criteria are lacking, such as in ET without a JAK2 mutation. Ultimately, the new assay may well be able to detect transformation from PV/ET to MF. Disclosures No relevant conflicts of interest to declare.

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A Novel Mutation in MPL (Y252H) Results in Increased Thrombopoietin Sensitivity and Moderate Thrombocytosis in a Pediatric Patient with Essential Thrombocytosis

Blood ◽

10.1182/blood.v118.21.2817.2817 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2817-2817

Author(s):

Michele Lambert ◽

Jing Jiang ◽

Wei Tong

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Bone Marrow Cells ◽

Novel Mutation ◽

Missense Variant ◽

Jak2 V617f ◽

Jak2 Mutation ◽

Hematopoietic Malignancies ◽

Healthy Control ◽

Pediatric Populations

Abstract Abstract 2817 Myeproliferative neoplams (MPNs) constitute a group of hematopoietic malignancies that feature enhanced proliferation and/or survival of one or more myeloid lineage cells, including Essential Thrombocythemia (ET). MPN development is rare in children with an estimated annual incidence of ET of 0.09/106 children. The WHO defines ET as persistent platelet count >600 k/mcL in the absence of known cause of reactive/secondary thrombocytosis. The JAK2 V617F mutation is most commonly reported in both children and adults with ET although the reported frequency varies in pediatric populations from 0 to 36% of patients. Mutations in the thrombopoietin receptor (TPO receptor or MPL) intracellular domain, specifically W515K/L, have also been reported in both adult and pediatric ET patients. Here we report a novel mutation in the MPL extracellular domain, Y252H, causing mild thrombocytosis. The patient presented at 2 years of age with a platelet count of 765 k/mcL. During the 3-year follow-up period, she possessed platelet counts between 600–700k/mcL, without any obvious indication of reactive/secondary thrombocytosis. Because of the persistently increased platelet count, her bone marrow was evaluated and it demonstrated increased numbers of megakaryocytes with focal clustering. JAK2 mutation analysis was negative and cytogenetics did not show any clonal abnormalities. Sequencing of the MPL gene showed a missense variant at c.754 T>C resulting in a tyr252his amino acid substitution. To investigate if this Y252H mutation in MPL dysregulates TPO/MPL- mediated cell growth, we introduced it into cytokine-dependent BaF3 cells. Cells stably expressing the mutant MPL allele showed increased proliferation to TPO, in particular at low concentration, in comparison to cells expressing wildtype (WT) MPL. Upon cytokine withdrawal, BaF3 cells expressing the MPL Y252H mutant survived better than that of WT MPL. Primary bone marrow cells from this patient along with the healthy control were subjected to colony forming unit -megakaryocyte (CFU-meg) assays in response to a serial dose of TPO. The Y252H MPL bone marrow showed significantly increased megakaryocyte colonies at low dose of TPO when compared to control bone marrow (17.5 ± 2.5 colonies versus 4.75 ± 1.1 colonies at 15 ng/mL TPO, p<0.001). These results are consistent with the clinically mild thrombocytosis. In summary, our results suggest a novel MPL mutation, Y252H, results in pediatric ET. Further evaluation of the mechanisms of increased TPO sensitivity imparted by this mutation should contribute to our understanding of the molecular pathogenesis of ET. Disclosures: Lambert: Cangene: Honoraria.

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Relationship between JAK2 V617F Mutation Status and Constitutive Mobilization of CD34-Positive Cells into Peripheral Blood in Patients with Chronic Myeloproliferative Disorder.

Blood ◽

10.1182/blood.v106.11.2586.2586 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2586-2586

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Emanuela Boveri ◽

Daniela Pietra ◽

Laura Vanelli ◽

...

Keyword(s):

Bone Marrow ◽

Cell Count ◽

Peripheral Blood ◽

Jak2 V617f ◽

Myeloproliferative Disorder ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Mutation Status ◽

Cell Counts ◽

V617f Mutation

Abstract A gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently reported in patients with polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Abnormal trafficking of CD34-positive cells with increased numbers in the peripheral blood is found in CIMF and in advanced stages of other myeloproliferative disorders. To determine whether the unique JAK2 V617F mutation affects the mobilization of CD34-positive cells into peripheral blood, we studied the relationship between JAK2 mutation status, bone marrow and circulating CD34-positive cells in 72 patients diagnosed according to the WHO criteria. A quantitative real-time polymerase chain reaction (PCR)-based allelic discrimination assay was used for the quantitative detection of the JAK2 V617F alleles in circulating granulocytes. Bone marrow CD34-positive cells were quantitatively assed on paraffin immunostained sections, while circulating CD34-positive cells were enumerated by flow cytometry using a single-platform assay. Overall, 57% of the patients studied carried the JAK2 V617F mutation. Within these patients, median values for JAK2 V617F alleles in circulating granulocytes were as follows: 29% in PV, 4% in ET, 12% in prefibrotic CIMF, 27% in fibrotic CIMF, and 99% in post-PV myelofibrosis. The vast majority of circulating granulocytes were homozygous for the mutation in all but one of patients with post-PV myelofibrosis. Decreased numbers of bone marrow CD34-positive cells and increased counts of circulating CD34-positive cells were detected in patients with fibrotic bone marrow. The higher the degree of fibrosis, the higher the circulating CD34-positive cell count (P<0.001) and the lower the bone marrow CD34-positive cell count (P<0.01). All patients with PV, ET and prefibrotic CIMF, and 7 out of 21 patients with fibrotic CIMF had circulating CD34-positive cell counts lower than 10 x 106/L. Conversely, all patients with post-PV myelofibrosis had counts higher than 10 x 106/L. In univariate analysis, there was an inverse relationship between percentage of JAK2 V617F alleles and bone marrow CD34-positive cells (r=−0.35, P<0.01), and a direct relationship between percentage of JAK2 mutant alleles and circulating CD34-positive cells (r=0.46, P=0.001). Multivariate analysis showed that disease category (P=0.0008) and percentage of JAK2 V617F alleles (P=0.03) were independently related to circulating CD34-positive cell counts. These observations suggest that the JAK2 V617F mutation might be involved in the constitutive mobilization of CD34-positive cells into peripheral blood that is found in patients with myeloproliferative disorder. Nonetheless, constitutive mobilization is present in a considerable portion of patients who do not carry the JAK2 mutation, pointing to additional pathogenetic mechanisms. Findings on patients with PV suggest that transition form heterozygosity to homozygosity for JAK2 V617F may represent an important step in the progression of PV to myelofibrosis. Thus, sequential evaluation of the percentage of JAK2 mutant alleles and enumeration of circulating CD34-positive cells may be useful for disease monitoring in PV.

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JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.

Blood ◽

10.1182/blood.v108.11.4833.4833 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4833-4833

Author(s):

Ling Zhang ◽

Saskia Gueller ◽

Sophie Raynaud ◽

Phillip H. Koeffler ◽

Stephen Lee

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Bone Marrow Aspiration ◽

K562 Cells ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Jak2 Mutation ◽

V617f Mutation

Abstract Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”. Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed. Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control. Results: PCR results showed clear wild type PCR patterns in all 21 cases. Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

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JAK2 V617F Mutational Status in Myelofibrosis at and before Disease Progression Including Leukemic Transformation: A Longitudinal Study in 68 Patients.

Blood ◽

10.1182/blood.v106.11.2575.2575 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2575-2575

Author(s):

Ruben A. Mesa ◽

Heather Powell ◽

Terra Lasho ◽

Ayalew Tefferi

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Peripheral Blood ◽

Jak2 V617f ◽

Wild Type ◽

Leukemic Transformation ◽

Myeloid Metaplasia ◽

Patient Cohort ◽

Mutational Status ◽

Over Time

Abstract Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML. i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status. ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively. Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.

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Bone marrow-derived NGFR+ cells regulate arterial remodeling and those poor mobilizations in peripheral blood in acute coronary syndrome predicts plaque progression at the non-targeted lesion

European Heart Journal ◽

10.1093/ehjci/ehaa946.1679 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

S Takashima ◽

S Usui ◽

S Matsuura ◽

C Goten ◽

O Inoue ◽

...

Keyword(s):

Bone Marrow ◽

Acute Coronary Syndrome ◽

Peripheral Blood ◽

Funding Source ◽

Arterial Remodeling ◽

Plaque Progression ◽

Wild Type ◽

Plaque Volume ◽

Coronary Syndrome

Abstract Background In our previous 5-year cohort study, we demonstrated that low gene expression of nerve growth factor receptor (NGFR) in peripheral leucocytes in acute coronary syndrome (ACS) predicted repetitive coronary interventions at the de novo lesions. An NGFR-positive cell has been demonstrated to reside in bone marrow (BM) stromal fraction and to be increased in peripheral blood mononuclear cell (MNCs) fraction in patients with ischemic heart disease. Purpose To investigate whether the BM-NGFR+ cell is associated with arterial remodeling and the relationship between the levels of peripheral NGFR+ cells after ACS and coronary plaque progression in an experimental and prospective clinical study. Methods and results In an experimental study, 8-week-old C57B6/J wild type male mice were subjected to irradiation with 9.6 Gy and transplantation with BM (BMT) isolated from GFP-transgenic NGFR wild type (WT) or knock-out (KO) mice at day 1. Four weeks after BMT, the right carotid artery was ligated for 4 weeks. Induced neointimal area was increased (p<0.05), where cells under apoptosis were decreased (p<0.05) in NGFR-KO-BMT group compared to WT-BMT group (n=4). NGFR+ cells were not detected in wild type sham-operated artery, whereas in the ligated artery in WT-BMT group NGFR+ cells assembled in the developed neointima and exclusively presented double positive with GFP, but absent in NGFR-KO-BMT group (p<0.05, n=4). In a clinical study, thirty patients with ACS who underwent primary percutaneous coronary intervention (PCI) were enrolled. The peripheral blood sample was collected on days 0, 3 and 7, and 9 months follow-up and the number of NGFR+MNCs were measured by flowcytometric analysis. The plaque volume at non-targeted coronary lesion (non-TL:>5 mm proximal or distal to the implanted stents) were quantitatively analysed using gray-scale intravascular ultrasound (IVUS) and Q-IVUS™ software at the acute phase and 9 months follow-up. The number of NGFR+MNCs in peripheral blood was 1.5-fold increased at day 3 (0.064±0.056%) compared to day 0 (0.042±0.030%) (p<0.05). The change in normalized total plaque volume (TAVN) at non-TL at 9 months was negatively correlated with the number of NGFR+MNCs at day 0 (r=−0.51), day 3 (r=−0.51) and 9 months (r=−0.59) after ACS (p<0.05). Multiple regression analysis showed that NGFR+MNCs at day 0 (β=−0.48, p=0.01) and CRP (β=−0.53, P<0.01) are independent factors associating with TAVN change at non-TL at 9 months, regardless of LDL-cholesterol control level. ROC analysis revealed that NGFR+MNCs <0.049 at day 0 predicted the increase of TAVN with AUC 0.78; sensitivity 0.82 and specificity 0.67. Conclusions Bone marrow-derived peripheral NGFR+ cells negatively regulate arterial remodeling through appropriate apoptosis of neointimal cells and the peripheral level of NGFR+ cells in ACS predicts plaque progression at the non-targeted lesion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): KAKENHI

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Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽

10.1182/blood.v90.6.2148 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2148-2159 ◽

Cited By ~ 118

Author(s):

Harshal H. Nandurkar ◽

Lorraine Robb ◽

David Tarlinton ◽

Louise Barnett ◽

Frank Köntgen ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

White Blood Cells ◽

Null Mutation ◽

Wild Type ◽

Normal Numbers ◽

Interleukin 11 ◽

Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

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Evidence for a Vessel Wall Defect in Immuno-thrombocytopenic Hamsters

Blood ◽

10.1182/blood.v15.5.675.675 ◽

1960 ◽

Vol 15 (5) ◽

pp. 675-680 ◽

Cited By ~ 5

Author(s):

RAJENDRA G. DESAI ◽

GEORGE P. FULTON

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Negative Pressure ◽

Peripheral Blood ◽

Vessel Wall ◽

Bleeding Time ◽

Red Cell ◽

Clot Retraction ◽

Wall Defect ◽

Pressure Tests

Abstract Experimental purpura was produced in the hamster by administration of anti-platelet serum obtained from rabbits previously injected with hamster platelets. Spontaneous petechiae and generalized bleeding were observed. The derangement in the hemostatic mechanism has been analyzed by study of the changes in blood, bone marrow and vessel walls. The platelet count in peripheral blood fell from 9.02 ± 0.85 (x 105) to 0.66 ± 0.32 (x 105) at 24 hours after 2.0 ml. intravenous injection of antiplatelet serum. The red cell and hemoglobin values dropped to 50 per cent before death related to generalized bleeding occurred. Significant changes were seen in the megakaryocytes of the bone marrow. The bleeding time and clot retraction were extended. Evidence for a defect in the vessel wall has been shown by the microelectrode, moccasin snake venom and negative pressure tests. The cause of bleeding has been postulated as a double defect resulting from a decrease of platelets in the circulation and an alteration in the integrity of the vessel wall or perivascular supporting sheath.

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JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽

10.1182/blood.v106.11.3503.3503 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3503-3503

Author(s):

Ruben A. Mesa ◽

Ayalew Tefferi ◽

Heather Powell ◽

Terra Lasho ◽

David Loegering ◽

...

Keyword(s):

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Neutrophil Apoptosis ◽

Wild Type ◽

Jak2 Mutation ◽

Myeloid Metaplasia ◽

Mutation Status ◽

Stat3 Phosphorylation ◽

V617f Mutation ◽

Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

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Contribution of HGF to Liver Regeneration by Strong Mobilization of Bone Marrow Stem Cells.

Blood ◽

10.1182/blood.v108.11.1680.1680 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1680-1680

Author(s):

Fumihito Tajima ◽

Hiroyuki Tsuchitya ◽

Kenichi Nishikawa ◽

Toshirou Okazaki ◽

Goushi Shiota

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Liver Regeneration ◽

Peripheral Blood ◽

Wild Type Mouse ◽

Bone Marrow Stem Cell ◽

Bone Marrow Stem Cells ◽

Wild Type ◽

Cd34 Cells ◽

Marrow Stem Cell

Abstract Cell plasticity of bone marrow stem cells to hepatocytes is known, however, the details are still unclear. hepatocyte growth factor (HGF) promotes an increase in liver stem cells in severely injured liver, but intervention to bone marrow stem cell is unclear. We examined a role of HGF in bone marrow stem cell-mediated liver regeneration in order to obtain effective liver regeneration. First, we found that the phenotype of stem cells, which can differentiate into hepatocytes, is Lin−c-kit+Sca-1+CD34− in bone marrow or Lin−c-kit+Sca−1+CD34+ in peripheral blood mobilized by G-CSF. We transplanted single Lin−c-kit+Sca-1+CD34− bone marrow cell harvested from male EGFP mouse to female wild type mouse, and then, using this GFP-chimera-mouse, we found that bone marrow origin GFP+ and Y chromosome+ hepatic cells were present in liver after acute liver damage. Next, single Lin−c-kit+Sca-1+CD34+ peripheral blood cell, which was mobilized in peripheral blood by administration of G-CSF, was transplanted into the portal vein of the wild type mouse which was given hepatic damage. We found that the GFP-positive cells also expressed albumin. Second, we investigated whether the HGF can mobilize stem cells from bone marrow to peripheral blood. In peripheral blood of HGF transgenic mice, 1.1% developed CD34+ cells and 20±3 colony forming cells of 1X106 peripheral blood mononuclear cells were shown. Colony forming cells were found in the mouse into which an HGF-expressing adenovirus was administered. After injection of rHGF to mice, a significant time-dependent increase of percentage of CD34+ cells in the PB was noted at the first 3 hours and CD34+ cells were increased in dose-dependent manner of rHGF and reached plateau level at 100 m/kg. The mice having transplantation with PB cells from 100 mg HGF-treated mice for 4days showed engraftment 2 months after transplantation. Upon activation of rHGF in mouse MS-5 stromal cells, phosphorylation c-Met and SCF were up-regulated, while VCAM -1, MMP-9, SDF -1 or CXCR4 were not changed. SCF level in conditioned media was also increased after the HGF stimulation. Finally, we examined whether the bone marrow stem cells in PB mobilized by HGF transdifferentiate into hepatocytes. Using GFP-chimera-mice given acute liver injury after administration of retorolusine and CCl4, the levels of GFP+ cells in liver of GFP-chimera-mice 2 months after treatment by PBS and HGF were 2.2±1.4% and 12.7±3.6%, respectively (p<0.01). In conclusion, HGF can mobilize stem cells with long-term engraftment capabilities from bone marrow to peripheral blood, resulting in contribution to liver regeneration.

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