OBJECTIVE

This in vitro study investigated the effects of different arginine (Arg) concentrations on angiogenic protein expressions of HeLa cells and endothelial cells (ECs) after stimulation. In addition, an inducible nitric oxide (iNO) synthase inhibitor (1400W) was used to investigate the possible role of iNO in angiogenesis.

METHODS

Endothelial cells and HeLa cells were treated with different concentrations of Arg and 1400W: Arg 0, 50, 100, and 1000 micromol/L; Arg 100 micromol/L+1400W 10 micromol/L; and Arg 1000 micromol/L+1400W 10 micromol/L for 24 h. Then, ECs and HeLa cells were cocultured for 2 h, and the supernatant in the transwell was collected for analysis of angiogenic protein secreted. The expression of CD51/CD61 by ECs was also analyzed.

RESULTS

The productions of vascular endothelial growth factor, basic fibroblast growth factor, prostaglandin E(2), and matrix metalloproteinase-2 were higher with Arg 100 and 1000 micromol/L than with Arg 0 and 50 micromol/L Arg, and this was consistent with the expression of CD51/CD61 by ECs. Inhibition of iNO production resulted in lower angiogenic protein expressions comparable with groups with low Arg administration.

CONCLUSIONS

The findings of this study suggest that Arg administration at levels similar to or higher than physiologic concentrations enhance the production of angiogenic protein and iNO may partly play a role in promoting angiogenesis in the presence of HeLa cells.

Four human megakaryocytoid cell lines, namely MOLM-1, MOLM-7, MEG-01 and HEL, were treated with phorbol 12-myristate 13-acetate (PMA) and expression of the platelet-derived growth factor (PDGF) A chain, von Willebrand factor (vWF) and endothelial cell growth factor (ECGF) genes was examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers. The gene for PDGF A chain is constitutively weakly expressed by MEG-01 cells and strong expression is induced in MEG-01, MOLM-1 and MOLM-7 but not in HEL cells after treatment with PMA for three days. All four cell lines express the vWF gene both constitutively and after exposure to PMA. None of the cell lines constitutively express the gene for ECGF but MEG-01 cells can be induced to do so after treatment with the phorbol diester. Immunohistochemical staining after exposure to PMA showed that the expression of the platelet-associated markers CD41 and CD61 was enhanced in all cell lines indicating possible differentiation along the megakaryocyte lineage. Our results illustrate differential platelet-associated factor gene expression in different megakaryoblastic cell populations in response to treatment with PMA, and suggest that expressions of the PDGF A chain gene and the ECGF gene may be good markers for megakaryocyte maturation.

We have previously demonstrated that activation of prostacyclin (IP) receptors in human erythroleukemia (HEL) cells phosphorylates the signal transducer and activator of transcription 3 (STAT3) via Gα(s) and Gα(16) hybrid signalling. This current study was designed to determine if functional responses to cicaprost in HEL cells were dependent on STAT3 phosphorylation. Cicaprost significantly enhanced the rapid change in HEL cell morphology induced by phorbol-12-myristate-13-acetate (PMA), and this effect was inhibited by the IP receptor antagonist RO1138452 and a STAT3 inhibitory peptide. Other indicators of PMA-induced HEL cell differentiation, such as increased expression of CD41/CD61 and an increase in cell complexity/granularity, were inhibited by cicaprost in an IP receptor-dependent and STAT3-dependent manner. Although thrombopoietic cytokines promote megakaryocytic differentiation and platelet production via activation of STAT3, the predominant STAT3-dependent effects of cicaprost in HEL cells were inhibitory towards the process of PMA-induced megakaryocytopoeisis.

An immunohistochemical and morphometric study was performed on bone marrow biopsies in 604 patients with chronic myelogenous leukemia (CML) to compare morphological and clinical features and to evaluate effects of interferon (IFN) and chemotherapy. Following morphometry significant correlations were calculated between number of CD61(+) megakaryocytes, including their precursors with fiber density. This finding is in line with the close functional relationship between megakaryopoiesis and fibroblasts regarding the complex pathomechanism of myelofibrosis. The latter was observed in about 28% of patients already at diagnosis. In a similar way, the frequency of CD68(+) macrophages was correlated with the amount of Ret40f(+) nucleated erythroid precursors, implicating an involvement of this cell lineage in iron turnover, hemoglobin synthesis, and degradation of the expelled nuclei from normoblasts. The (alpha-D-galactosyl residue-expressing) Pseudo-Gaucher cells were detectable in 30% of pretreatment specimens. Moreover, significant associations were calculable between reduction in erythropoiesis or increase in fibers with clinical features such as hemoglobin level, percentages of myelo- and erythroblasts in the peripheral blood, and spleen size. These variables are in keeping with more advanced stages of CML. Based on our morphometric evaluations, a classification into three different histological subgroups: granulocytic, megakaryocytic, and myelofibrotic was carried out. This simplified staging system was correlated with corresponding sets of hematological data. Sequential biopsies in 173 patients with monotherapy by IFN, hydroxyurea (HU), or busulfan (BU) revealed a fibrogenic effect of IFN in contrast to a fiber-reducing property of HU. The dynamics of myelofibrosis and changes of major cell lineages during treatment were readily demonstrable by calculating corresponding indices. These included the ratios between quantitative differences of corresponding variables at repeated examinations and time. Thus, in patients with complete hematological remission following IFN administration, regeneration of erythropoiesis was found to be accompanied by an increase in the total number of CD68(+) macrophages, including activated subpopulations. Histological subgroups showed a transition from a (nonfibrotic) granulocytic and megakaryocyte pattern to the myelofibrotic subtype in about 40% of patients. This change was opposed to a numerical reduction in the myelofibrotic subtype which occurred in 17 patients (36%), but predominantly in those under HU therapy. In conclusion, the striking heterogeneity of bone marrow features in CML warrants a careful morphological evaluation of trephine biopsies and appropriate means of processing to achieve relevant correlations with clinical data and, thus, allows a more elaborate insight into the dynamics of the disease process.

OBJECTIVE

Overexpression of hNUDC, a member of the nuclear distribution protein family, reduces cell population growth in prostate cancer cell lines, concurrent with induced morphological change and enhanced polyploidization. These phenomena are also closely associated with terminal phases of megakaryocyte maturation.

METHODS

In Dami cells, MTT and trypan blue assays were used to investigate cell viability and proliferation effects of hNUDC, and flow cytometry was used to analyse cell cycle and DNA content. Real-time RT-PCR was employed to detect mRNA expression. Activations of caspase-3, ERK, Akt and Stat-5 were determined by immunoblotting. May-Grünwald-Giemsa staining was performed to reveal cell morphology.

CONCLUSIONS

Functional studies using adenovirus-mediated hNUDC overexpression led to inhibition of megakaryocyte proliferation via cell cycle arrest in G2/M transition phase. This process could have been be mediated by upregulation of p21 and downregulation of its downstream targets, including cyclin B1, cyclin B2 and c-myc. Enhanced apoptosis in turn ensued, characterized by increased caspase-3 activation, upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-2. Furthermore, hNUDC overexpression elevated the level of megakaryocyte maturation, associated with increased polyploidy, cell morphological changes and increased expression of cell surface differentiation markers, including CD10, CD44, CD41 and CD61. Our results further suggest that the ERK signalling pathway was involved in hNUDC overexpression-induced apoptosis. Taken together, this study provides experimental evidence for overexpression of hNUDC in Dami cells and suggests that activation of apoptotic machinery may be involved in megakaryocytic differentiation.

An 18-year-old male was admitted to our hospital for fever, and was diagnosed with acute megakaryoblastic leukemia (AML M7) based on the presence of CD42a and CD61 positive myeloblasts in peripheral blood (PB). Induction chemotherapy at our hospital resulted in complete remission (CR). Subsequently, he underwent unrelated HLA-DR one locus-mismatched allogeneic bone marrow (BM) transplantation. Although CR was maintained without development of graft-versus-host disease (GvHD), the WT1 mRNA level in PB was elevated on post-transplant day 134. As BM aspiration performed 1 week later confirmed maintenance of CR, and because the WT1 mRNA level in BM was not high in comparison with PB, we suspected extramedullary relapse. PET-CT demonstrated a thymic tumor and a gastric tumor with abnormal accumulation of FDG, and biopsy confirmed both to be extramedullary relapse of AML M7. Induction chemotherapy following local radiation therapy achieved a second CR, following which he received HLA haploidentical peripheral blood stem cell transplantation on day 256 after the first transplant. The patient is currently surviving free from both relapse and GvHD. High WT1 mRNA levels in PB as compared with BM should raise suspicion of extramedullary relapse, and PET-CT is very useful for whole body evaluation in such cases.

In chronic myeloid leukemia following therapy with Imatinib (STI571) hematologic and cytogenetic response is associated with conspicuous changes of bone marrow morphology. However, it is not known to which extent these alterations are accompanied by a loss of the bcr/abl translocation. To study regression of the leukemic cell population we recruited 14 patients lacking pretreatment. Therapy resulted in a reduction of CD61(+) megakaryopoiesis. Dwarf megakaryocytes characteristic for this disorder were replaced by large, normally appearing cells of this lineage. Morphometric analysis confirmed the significant decrease in the number of micromegakaryocytes and yielded planimetric parameters in keeping with normalization. Moreover, a fluorescence in-situ hybridization study in five patients of this cohort revealed that before therapy 70% of all myeloid cells exhibited the bcr/abl gene. Regarding megakaryopoiesis about 65% of the micromegakaryocytes displayed positive signals. Following treatment these bcr/abl(+) cell populations decreased significantly while the emerging large megakaryocytes lacked a proper labeling. Because cytogenetic response and reduction of atypical micromegakaryocytes are linked, this feature may be useful to monitor therapeutic efficacy.

OBJECTIVE

In a previous study, we demonstrated that in vitro cell growth stimulated by human placental conditioned medium distinguished between transient leukemia (TL) and congenital acute myeloid leukemia (AML) in neonates. We then sought to determine whether the application can be expanded if in vitro cell growths are stimulated by recombinant human cytokines including granulocyte-macrophage colony-stimulating factor (rhGM-CSF), interleukin-3 (rhIL-3), stem cell factor (rhSCF) and thrombopoietin (rhTPO).

METHODS

Eight neonates with features indistinguishable from AML were studied. Seven patients had Down syndrome and the eighth a normal phenotype. Bone marrow or peripheral blood mononuclear cells (MNC) were cultured in the presence of rhGM-CSF+rhIL-3+rhSCF or of rhTPO alone. After incubation, granulocyte-macrophage colony-forming units (CFU-GM)-derived colonies and clusters were scored on an inverted microscope. Colony-forming units-megakaryocyte (CFU-MK)-derived colonies were counted with an in situ CD61 immunostained dish. Liquid suspension cultures of MNC were stimulated by rhGM-CSF and/or rhTPO.

RESULTS

CFU-GM-derived colonies and clusters from bone marrow and peripheral blood MNC revealed normal patterns in seven patients. RhTPO-stimulated megakaryocyte colony formation was normal in one patient. Cytospin smears of liquid suspension cultures all showed good myeloid or megakaryocytic maturation consistent with TL rather than AML. One neonate died on the 2nd day of life, but in the seven remaining patients, blasts disappeared from the peripheral blood within 10 months. Among four patients followed long-term, one developed myelodysplastic syndrome at 21 months. This child was given tailored chemotherapy and had a disease-free survival>20 months.

CONCLUSIONS

In vitro cell growth stimulated by recombinant human cytokines can help to diagnose TL in neonates.

An 11-year-old spayed-female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2-3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P-selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.

Thrombocytopenia is a common occurrence in sick newborn babies. Despite this, platelet production in the newborn has rarely been assessed, principally because of the difficulties of obtaining bone marrow, especially on a serial basis. We have developed two miniaturized assay systems to study megakaryocyte (MK) progenitor cell differentiation, from BFU-MK and CFU-MK to mature MK, by culturing mononuclear cells purified from 0.5-1 ml of neonatal peripheral blood. BFU-MK and CFU-MK were assayed in agar, whilst total cultured MK precursors and mature MK were assessed in liquid culture. In both systems, MK lineage cells were identified morphologically and by an anti-IIb/IIIa antibody (CD61). Normal ranges for MK precursors in term neonates were established from cord blood studies of 40 healthy term babies and compared with cord blood studies in 16 non-thrombocytopenic pre-term babies (gestational age range 24-36 weeks). Pre-term babies had greater numbers of all MK precursors than term babies: BFU-MK 414 +/- 61 v 151 +/- 18 colonies/ml (mean +/- SEM); CFU-MK 2444 +/- 337 v 869 +/- 64 colonies/ml; total MK precursors 213 +/- 36 v 54 +/- 6 x 10(3) cells/ml and mature MK 20 +/- 4 v 7 +/- 1 x 10(3) cells/ml. In addition, in newborn babies (n = 22), with no evidence of platelet consumption, circulating MK progenitor numbers at birth correlated with platelet numbers. These data indicate that in the newborn the measurement of circulating MK precursors provides a good indicator of megakaryocytopoiesis, and hence platelet production, and therefore is a useful and practical way of investigating neonatal thrombocytopenia.

Early plasmacytoid dendritic cell (pDC) leukemia/lymphoma has recently been described as a CD4(+)CD56(+) lineage negative malignancy with characteristic clinical, morphologic, immunophenotypic, and biological features. We present a case of a 72-year-old man who was diagnosed with isolated skin involvement 30 months ago and received numerous chemotherapy cycles that did not prevent three relapses of the disease, the last two involving the bone marrow. The bone marrow was nearly completely infiltrated with small- to medium-sized blasts displaying a high nuclear to cytoplasmic ratio, a cytoplasm with faint basophilia lacking granulations or Auer rods. Small vacuoles surrounding the nucleus were frequently observed. Flow cytometry showed CD4(+), CD56(+), CD45(+), CD38(+), HLA-DR(+), CD33(+), CD123(+), CD2(-), cyCD3(-), CD7(-), CD10(-), CD11b(-), CD13(-), CD14(-), CD16(-), CD19(-), cyCD22(-), CD24(-), CD34(-), CD57(-), CD61(-), CD64(-), CD65(-), cyCD79a(-), CD117(-), MPO(-), and TdT(-) population. At the second bone marrow relapse, CD117 was also positive. Our patient was initially treated with acute myeloid leukemia-type chemotherapy, later he was given acute lymphoblastic leukemia-type treatment, and at the last relapse he received CHOP chemotherapy. Each treatment led to rapid response of tumor manifestations with disease-free intervals of 7 months, 9 months, and 8 months, respectively. Although patients usually have an ominous prognosis, with only 25% living more than 24 months, our patient is alive after 30+ months and has again achieved complete remission after the last chemotherapy.

The identification of vascular invasion in follicular thyroid neoplasms is essential for categorizing lesions as benign (follicular adenomas) or malignant (follicular thyroid carcinomas). Among the histologic criteria diagnostic of true vascular invasion is tumor-cell associated thrombosis, including fibrin deposition and platelet clumping. This study aims to evaluate whether an immunohistochemical stain for the platelet-associated protein CD61 could assist in identifying tumor-associated thromboses and thereby confirm vascular invasion in follicular thyroid neoplasms. Histologic review and CD61 immunostaining of 19 atypical follicular adenomas, 13 non-metastatic follicular thyroid carcinomas, and 11 metastatic follicular thyroid carcinomas was performed. Linear arrays or clustered groups of CD61-expressing intravascular platelets were present in 51% of cases overall, including 54% of follicular thyroid carcinomas and 47% of follicular adenomas, mostly within intracapsular or peritumoral vessels. In three follicular thyroid carcinomas (all with distant metastases), CD61-expressing platelets were present in association with intravascular tumor cells. This finding was not present in adenomas. CD61 staining alone did not distinguish between atypical follicular adenomas, non-metastatic carcinomas, and metastatic carcinomas. When present in association with intravascular tumor cells, however, CD61-expressing platelets may serve as a marker for vascular invasion and aid in the diagnosis of follicular thyroid carcinoma.

OBJECTIVE

To define the immune phenotype of colon cancer cells.

METHODS

Using a panel of 40 anti-human monoclonal antibodies (MoAbs), the cells of colon cancer HR8348 were analyzed with three-color flow cytometry after direct immunofluorescent staining.

RESULTS

HR8348 cell line did not express CD2, CD3, CD4, CD5, CD7, CD8, TCR, CD10, CD11b, CD14, CD16, CD19, CD22, CD25, CD28, SmIg, CD33, CD35, CD36, CD41a, CD45, CD45RA, CD45RO, CD56, CD61, CD64, CD66b, CD69, CD71, CD117, CD122 and P-glycoprotein but expressed CD13, CD15, CD20, CD38, CD95 and HLA-DR.

CONCLUSIONS

The results demonstrate that colon cancer cell line HR8348 shares some antigenic determinants with leucocyte lineage.

Pluripotential haematopoietic stem cells and their progeny, the so-called committed precursor cells, i.e., progenitor cells which are already lineage-restricted, may be identified by the membrane-bound expression of CD34. In accordance with this peculiar property it became possible to enrich and characterize primitive precursor cells by using different methods of cell separation techniques, which involved fluorescence staining or ferro-magnetic particles bound to CD34 antibodies. Recently conducted studies demonstrate that CD34-positive (CD34+) stem cells of the peripheral blood represent a relatively uniform cell population with almost round nuclei, a finely dispersed chromatin pattern and a small portion of weakly basophilic cytoplasm. From the cytological viewpoint they resemble so-called large stimulated lymphocytes (virocytes). Ultrastructural studies are compatible with a paucity of organelles and a lymphoid character of these progenitors. In comparison, the stem cell population, derived from the bone marrow consists of more heterogeneous elements. These are generally larger and reveal an admixture of fairly immature as well as more differentiated cells, sharing bean-shaped or indented nuclei with prominent nucleoli and a more extended cytoplasm. CD34+ progenitors from the peripheral blood and those from the bone marrow display a co-expression of CD43 (MT1) and CD45 (LCA). Furthermore, different subpopulations exhibit--dependent on their origin (blood/bone marrow) and to a various extent--lineage-restricted markers like CD33, CD38, CD61, CD20, CD11a/c, glycophorin C und CD15 (LeuM1). The recently developed immuno- and ferromagnetic enrichment methods for CD34+ progenitor cells are considered innovative tools for modern oncology. These techniques play an important role in the treatment of haematological malignancies and advanced tumours in the context of autologous and, although so far rarely applied, heterologous stem cell transplantation procedures.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

In this report an attempt has been made to discuss some of the issues pertinent to myelofibrosis complicating chronic myeloproliferative disorders (CMPDs) that are significantly associated with megakaryocyte function. In this context, biochemical, clinical and particularly morphological features were reviewed. Morphological findings based on elaborate techniques were in keeping with the assumption that in chronic myeloid leukemia (1) the number of CD61-positive megakaryocytes, and in particular their precursors were the parameters most closely associated with myelofibrosis (2) an increased content of reticulin fibers in follow-up biopsies significantly correlated with laboratory data indicative of a high tumor burden (anemia, peripheral blasts, hepatosplenomegaly) and thus a more advanced stage of the disease process (3) even a slight increase in reticulin, i.e. doubling of the normal fiber density was associated with a worse prognosis independent of therapeutic regimens given (4) Dynamics of myelofibrosis was significantly influenced by treatment. In this context, calculation of the myelofibrosis progression index (MPI) revealed a higher score following interferon therapy compared with busulfan. In addition, in idiopathic myelofibrosis (5) the evolution of myelofibrosis was unpredictable and according to the MPI, progression occurred at a relatively low rate (6) proliferation and dilatation of sinusoids accompanying intravascular hematopoiesis and collagen type IV deposits were predominant features in later (fibro-osteosclerotic) stages in the course of disease (7) transmural migration of megakaryocytes demonstrated by three dimensional reconstruction revealed a mole-like tunneling through the thickened sinusoidal wall. A very careful assessment of the numerous correlations between bone marrow features and laboratory data will allow clinicians and pathologists to gain a better insight into the mutual relationships between hematological and morphological findings in CMPDs.

OBJECTIVE

To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma.

METHODS

Citrate-anticoagulated whole blood from 30 healthy adult horses.

METHODS

Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead-calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples.

RESULTS

Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP.

CONCLUSIONS

Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.

OBJECTIVE

To evaluate the influence of treatment with ultralow-dose aspirin (ULDAsp) on platelet aggregation, P-selectin (CD62P) expression, and formation of platelet-leukocyte aggregates in clinically normal dogs.

METHODS

18 clinically normal dogs.

METHODS

Studies were conducted before and 24 hours after ULDAsp administration (0.5 mg/kg, PO, q 24 h, for 2 days). Whole blood impedance aggregometry for the assessment of platelet function was performed with sodium citrate-anticoagulated blood and aggregation agonists (ADP at 20, 10, and 5 μmol/L; collagen at 10, 5, and 2 μg/mL). Onset, maximum response, and rate of platelet aggregation were recorded. Flow cytometric assays were configured to detect thrombin-induced CD62P expression and platelet-leukocyte aggregates in EDTA-anticoagulated whole blood. Externalized platelet CD62P and constitutive CD61 (GPIIIa) were labeled with antibodies conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. Red blood cell-lysed paraformaldehyde-fixed EDTA-anticoagulated whole blood was dual labeled with CD61-FITC and a panleukocyte antibody (CD18-PE) to characterize platelet-leukocyte aggregates.

RESULTS

ULDAsp significantly delayed platelet aggregation onset with ADP at 20 μmol/L by 54% to 104%, attenuated maximum aggregation with various concentrations of ADP and collagen by ≥ 41%, and slowed aggregation rate with the highest ADP and collagen concentrations by ≥ 39%. Depending on the parameter tested, up to 30% of dogs failed to have an ULDAsp effect. Thrombin stimulation significantly increased CD62P expression in platelets and platelet-leukocyte aggregates, but ULDAsp did not alter basal or thrombin-stimulated CD62P expression.

CONCLUSIONS

ULDAsp treatment of clinically normal dogs impaired platelet aggregation in most dogs, but did not influence CD62P platelet membrane expression.

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90⁺ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90⁺/CD45^- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1⁺ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1⁺ and TLR4⁺ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25⁺ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61⁺ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14⁺ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.

OBJECTIVE

Mutations in CALR (calreticulin) have been discovered in 50% to 80% of JAK2 (Janus kinase 2) and MPL (myeloproliferative leukemia protein) wild-type patients with Philadelphia-negative myeloproliferative neoplasm (MPNs). We evaluate the performance of a monoclonal antibody for immunohistochemical detection of CALR mutations.

METHODS

A computerized archival search was performed for cases of non-chronic myeloid leukemia (CML) MPNs with available CALR and JAK2 V617F mutational analysis data. Bone marrow biopsy specimens were stained with monoclonal antibody CAL2, and the percentage of stained megakaryocytes was calculated. In select cases, double immunofluorescence staining was done with CAL2 and each of the following: CD61, myeloperoxidase, CD34, and glycophorin A.

RESULTS

We studied 38 bone marrow biopsy specimens of non-CML MPNs (primary myelofibrosis, n = 21; essential thrombocythemia, n = 15; and n = 2 post-polycythemia vera myelofibrosis) from 31 patients. All eight bone marrow biopsy specimens from patients with mutant CALR showed strong cytoplasmic staining of the megakaryocytes (83.5%; range, 50%-98%; median, 87%) with the CAL2 antibody. Double immunofluorescence staining of the small mononuclear cells seen in CALR mutant cases revealed them to be myeloid blasts.

CONCLUSIONS

Immunohistochemistry in routinely processed bone marrow biopsy specimens for mutated CALR is feasible and accurately identifies mutated cases, including rare cases with additional driver mutations.

Recent studies emphasize the paramount significance of beta 3 integrin in cell adhesion and homing, which may be particularly relevant in cancer progression and metastasis. In contrast, the presence and potential role of beta 3 on human T cells is practically unknown. We show that T cells can express significant amounts of alpha-beta 3 integrin (CD41/CD61), and the expression of alpha(v)-beta 3 (CD51/CD61) remains very low. T-cell beta 3 integrin is probably transferred by platelet-derived microparticles.

Many studies have documented faster engraftment after transplantation with peripheral blood stem cells (PBSC) compared to bone marrow (BM) stem cells. Most comparisons, however, have been between unprimed BM and primed PBSC. We have collected engraftment data on 39 patients from 4 Danish centres and compared G-CSF primed BM with G-CSF primed PBSC in malignant lymphoma and solid tumours. In the lymphoma group 6 BM transplants were compared with 8 PBSC transplants, whereas in the testicular cancer group 16 BM transplants were compared with 9 PBSC transplants. In the lymphoma group, the time to platelet engraftment (platelets >20x10(9)/l unsupported) was median 15 d in PBSC transplants and median 34 d in BM transplants (p=0.003). In the solid tumour patients the difference in time to platelet engraftment was 11 and 18 d in PBSC and BM transplants, respectively (p<0.0001). In an attempt to explain this difference we performed CD34+ subset analysis of BM and PBSC. This analysis revealed a higher content of lineage restricted cells (CD34+CD61+ and CD34+GlyA+) in PBSC compared to BM. In conclusion, G-CSF mobilized PBSC seems to result in faster engraftment than G-CSF primed BM, which could be explained by an increased number of lineage specific progenitors in PBSC compared to BM.

We describe a case of acute myeloid leukemia in a 2 1/2-year-old boy presenting with a mediastinal tumor causing respiratory distress, and lymph node enlargement in the cervical and inguinal regions. Apart from myeloid markers CD13 and CD33, blast cells also expressed stem cell marker CD34 and megakaryocytic marker CD61. Cytogenetically, inv(7)(p21q31) was found in 9/25 and 15/25 analyzed metaphases from short-term cultures of lymph node and bone marrow cells, respectively. The patient is in continued complete remission 26 months post diagnosis. The case demonstrates that chromosome aberrations other than inv(16), t(8;21), and t(9;11) may be associated with extramedullary disease, and that not all chromosome 7 aberrations are prognostic adverse findings.

A monoclonal antibody (JM2E5) specific for the integrin beta3 chain, or CD61 or GPIIIa subunit, has been employed to determine the expression of the canine homologue CD41/CD61 or CD51/CD61 complex on different canine cells in peripheral blood lymphocytes, monocytes, granulocytes, platelets, erythrocytes, lymph-node cells, spleen cells and breast tumour cells). The canine homologue CD41/CD61 or CD51/61 was present on peripheral blood lymphocytes, monocytes, granulocytes, breast tumour cells and spleen cells as well as on platelets and it was absent from erythrocytes and lymph-node cells. An antigen with components of molecular masses of 25/100/120 kDa (under reducing conditions) was immunoprecipitated from canine peripheral lymphocytes and platelets, but not from granulocytes or monocytes. Expression on canine lymphocytes of the canine homologue of the human beta3 integrin chain was unexpected, based on the expression pattern of this molecule in human tissue.