Myocardial ischaemia (MI) results in extensive cardiomyocyte death and reactive oxygen species (ROS)-induced damage in an organ with little or no regenerative capacity. Although the use of adult bone marrow mesenchymal stem cells (BMMSCs) has been proposed as a treatment option, the high cell numbers required for clinical use are difficult to achieve with this source of MSCs, and animal studies have produced inconsistent data. We recently demonstrated in small and large animal models of acute MI that the application of human term placenta-derived multipotent cells (PDMCs), a foetal-stage MSC, resulted in reversal of cardiac injury with therapeutic efficacy. However, the mechanisms involved are unclear, making it difficult to strategize for therapeutic improvements. We found that PDMCs significantly reduced cardiomyocyte apoptosis and ROS production through the paracrine factors GRO-α, HGF and IL-8. Moreover, culturing PDMCs on plates coated with laminin, an extracellular matrix (ECM) protein, resulted in significantly enhanced secretion of all three paracrine factors, which further reduced cardiomyocyte apoptosis. The enhancement of PDMC paracrine function by laminin was mediated through αvβ3 integrin, with involvement of the signalling pathways of JNK, for GRO-α and IL-8 secretion, and PI3K/AKT, for HGF secretion. Our results demonstrated the utility of PDMC therapy to reduce cardiomyocyte apoptosis through modulation of ECM proteins in in vitro culture systems as a strategy to enhance the therapeutic functions of stem cells.

Bevacizumab is a humanized monoclonal IgG1 antibody, which neutralizes vascular endothelial growth factor and is used for treating multiple cancer types. As a known and frequent adverse event, this therapy can lead to renal damage including proteinuria and nephrotic syndrome. In a retrospective approach, we analyzed 17 renal biopsies from patients receiving bevacizumab treatment. We observed a distinctive histopathological pseudothrombotic pattern different from the previously reported thrombotic microangiopathy. Since this pattern includes some features similar to acute and chronic thrombotic microangiopathy, focal segmental glomerulosclerosis and cryoglobulinemic membranoproliferative glomerulonephritis, biopsies with these diagnoses were included for comparison. Clinical, laboratory, light microscopic, immunohistochemical (including a proximity ligation assay), proteomic and electron microscopic features were assessed. Nephrotic syndrome was present in 15 of the 17 bevacizumab-treated patients. All 17 displayed a patchy pattern of variably PAS-positive hyaline pseudothrombi occluding markedly dilated glomerular capillaries in their biopsies. Mass spectrometry-based proteome analysis revealed a special protein pattern demonstrating some features of thrombotic microangiopathy and some of cryoglobulinemic glomerulonephritis, including a strong accumulation of IgG in the pseudothrombi. Proximity ligation assay did not show interaction of IgG with C1q, arguing for accumulation without classic pathway complement activation. In contrast to thrombi in thrombotic microangiopathy cases, the hyaline pseudothrombi did not contain clusters of CD61-positive platelets. Electron microscopy of bevacizumab cases did not show fibrin polymers or extensive loss of podocyte foot processes. Even though cases of bevacizumab-associated microangiopathy share some features with thrombotic microangiopathy, its overall histopathological pattern is quite different from acute or chronic thrombotic microangiopathy cases. We conclude that bevacizumab therapy can lead to a unique hyaline occlusive glomerular microangiopathy, likely arising from endothelial leakage followed by subendothelial accumulation of serum proteins. It can be diagnosed by light microscopy and is an important differential diagnosis in cancer patients with nephrotic syndrome.

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin αIIbβ3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin αIIbβ3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin αIIbβ3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit αIIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin αIIbβ3.

BACKGROUND

The antiphospholipid syndrome (APS) is the association of thrombosis and recurrent pregnancy loss and/or pregnancy morbidity with persistent antiphospholipid antibodies (aPL). Previous studies of microparticles in patients with APS/aPL have mainly been small and findings, contradictory.

OBJECTIVE

To quantify endothelial and platelet microparticle levels in patients with isolated antiphospholipid antibodies or primary antiphospholipid syndrome (PAPS).

METHODS

We measured endothelial and platelet microparticle levels by flow cytometry in 66 aPL/PAPS patients and 18 healthy controls.

RESULTS

Levels of circulating platelet (CD41 and CD61) and endothelial microparticles (CD51 and CD105) were significantly increased in patients with PAPS and aPL compared to healthy controls. There were correlations between platelet and endothelial microparticles levels in all patients with aPL.

CONCLUSIONS

Platelet and endothelial microparticles are increased in all patient groups within this cohort of patients aPL. Whether they may have a role in the pathogenesis of APS merits further study.

Polyphenols are used as phlebotonic drugs, but their mechanism of action remains unknown. Since platelet activity and platelet-endothelial cell interactions are involved in the pathogenesis of cardiovascular disease, this work examines whether different flavonoid and coumarin drugs are able to inhibit platelet aggregation. This specific case of coumarins, the antiplatelet effect is not linked with a possible interaction over blood coagulation since this effect only dicoumarols have it. The antiplatelet capacity of polyphenols was assayed using peripheral blood platelets from healthy controls. The distribution of the different platelets subsets was quantified by flow cytometry, using the calcium ionophore as a pro-aggregant. The number of GPIIb/IIIa receptors occupied by the drugs was assayed by flow cytometry using two CD61 surface fluorescein antibodies. All the polyphenols tested inhibited platelet aggregation. A percentage antiplatelet activity of 88.91±7.98% was recorded for naringin, 48.43±8.84% for naringenin, 53.83±7.87% for esculetin, 54.65±6.91% for fraxetin, and 25.75±4.12% for coumarin. Naringin showed significantly greater percentage occupation of GPIIb/IIIa receptors than did naringenin (14.82±0.81% vs. 3.90±0.55%), and esculetin returned significantly higher values than fraxetin and coumarin (12.47±0.97 vs. 7.53±0.49 and 7.90±0.69 respectively). All drugs show important antiplatelet activity. Naringin was the best antiplatelet compound, showing the greatest antiplatelet activity and the highest percentage binding of GPIIb/IIIa receptors. However, any of the compounds used could be used in the prevention of cardiovascular disease.

Previous studies have shown that CD61 (integrin-β3) promotes the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into germ-like cells. However, the mechanism remains unclear. In this study, we showed that overexpression of CD61 in canine adipose-derived mesenchymal stem cells (cADMSCs) promotes their differentiation into primordial germ cell (PGC)-like cells. Quantitative real-time PCR, immunocytochemistry and western blot detected higher levels of PGC-specific markers in CD61-overexpressed cADMSCs compared with those in control cells. Moreover, phosphorylation of Smad2, a downstream mediator of transforming growth factor beta (TGF-β), was increased in CD61-overexpressed cADMSCs than that in control cells. However, the expression of PGC-specific markers was downregulated in cADMSCs treated with a TGF-β inhibitor. These results suggested that CD61 could induce cADMSCs to differentiate into PGC-like cells by relying on the activation of TGF-β pathway. ADMSCs possess a considerable potential in treating the infertility of rare animal species.

BACKGROUND

The use of tissue microarrays (TMAs) is now a generally accepted method for the investigation of solid tumours. However, little is known about the applicability of the TMA technique for analysis of patients with acute leukaemia. A bone marrow (BM)-TMA analysis with 15 different immunohistochemical markers was performed. The TMA was validated by comparison with the corresponding full tissue sections.

METHODS

A BM-TMA comprising 148 cases of acute leukaemia, including 115 acute myeloid leukaemia (AML) and 33 acute lymphoblastic leukaemia (ALL) cases, was constructed. Expression of CD3, CD10, CD15, CD20, CD34, CD61, CD68, CD79a, CD99, CD117, CD138, myeloperoxidase, haemoglobin A1, glycophorin and terminal deoxynucleotidyl transferase was immunohistochemically analysed. 50 cases of the TMA were directly compared with the corresponding full tissue section to validate the results.

RESULTS

Morphologically and immunohistochemically, 6 (4%) of 148 cases and 765 (11%) cores of 6912 individual analyses were not evaluable. A direct comparison of TMA cases with conventional full sections showed a concordance of the results of 100%.

CONCLUSIONS

The small size of bone-marrow biopsies and the presence of bony trabeculae do not preclude construction and analysis of acute leukaemia TMAs. Acute leukaemia cases on TMA displayed the characteristic phenotypic profiles expected in different AML and ALL subtypes. Therefore, the TMA technique is also a promising method for high-throughput analysis of combined marker expression and clinicopathological correlations in patients with leukaemia.

Immune thrombocytopenic purpura (ITP) is a multifactorial autoimmune disease characterized by both increased platelet destruction and/or reduced platelet production. Even though they are detected in ≤ 50% of ITP patients, auto-antibodies play a pivotal role in the pathogenesis of ITP. Recent experimental and clinical observations have revealed abnormal autophagy in ITP patients. Autophagy is a catabolic process responsible for the elimination and recycling of cytoplasmic constituents, such as organelles and macromolecules, in eukaryotic cells. Additionally, it triggers cell death or promotes cell survival following various forms of stress, and maintains the microenvironment and stemness of haematopoietic stem cells. The role of autophagy in megakaryopoiesis, thrombopoiesis, and platelet function is slowly being uncovered. The abnormal autophagy in ITP patients may be caused by deletion of autophagy-related genes such as ATG7 and abnormal signalling due to overexpression of mTOR. These changes are thought to affect markers of haematopoietic stem cells, such as CD41 and CD61, and differentiation of megakaryocytes, ultimately decreasing the function and quantity of platelets and leading to the onset of ITP. This review highlights recent evidence on the essential role played by autophagy in megakaryopoiesis, megakaryocyte differentiation, thrombopoiesis, and platelet production. It also discusses the potential of targeting the autophagy pathway as a novel therapeutic approach against ITP.

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.

BACKGROUND

Platelet-monocyte complex (PMC) formation is a marker of in vivo platelet activation and may be readily measured by flow cytometry. Due to the high frequency of free platelets relative to monocytes and PMCs, false-positive identification through coincidence remains a significant technical problem.To overcome this problem, we evaluated the use of a doublet-discriminator strategy (DDM) to allow faster sample acquisition whilst significantly reducing aberrant coincidence.

METHODS

Fourteen healthy volunteers and 20 patients with coronary artery disease (CAD) gave arterial and/or peripheral venous blood samples (NaCit). Whole blood was labelled in duplicate with anti-CD61 and anti-CD14 using a standard lyse/wash protocol. One of each paired sample was serially diluted before analysis; the second was analyzed at full concentration but using FL1-width to exclude co-incident platelet and monocyte events. Control experiments were performed with ex vivo thrombin activated samples.

RESULTS

With the DDM use PMC frequencies in the peripheral blood of healthy individuals and in CAD patients fell significantly [6.27% ± 1.77 (mean ± sd) to 2.57% ± 0.99 (P = 0.02)] and from 16.04% (± 11.26) to 7.66% (± 5.18) (P < 0.01), respectively. DDM use significantly reduced the percentage of PMCs in the ex vivo thrombin activated samples (P < 0.05).

CONCLUSIONS

Use of DDM effectively reduces the coincidence and enumerates true PMC in the samples of normal individuals and in patients with CAD and in ex vivo thrombin activated samples.

Circulating extracellular vesicles (EVs) are a novel and emerging biomarker for nonalcoholic steatohepatitis (NASH). It has been demonstrated that total circulating EVs and hepatocyte-derived EVs are elevated in male mice with diet-induced NASH. How hepatocyte-derived EVs change over time and other cellular sources of EVs in NASH have not been determined. Our objective was to define the quantitative evolution of hepatocyte-derived, macrophage-derived, neutrophil-derived, and platelet-derived EVs in male and female mice with dietary NASH. Fluorescently labeled antibodies and a nanoscale flow cytometer were used to detect plasma levels of EVs. Asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) are markers of hepatocyte-derived EVs; galectin 3 is a marker of macrophage-derived EVs; common epitope on lymphocyte antigen 6 complex, locus G/C1 (Ly-6G and Ly-6C) is a marker of neutrophil-derived EVs; and clusters of differentiation 61 (CD61) is a marker of platelet-derived EVs. Nonalcoholic fatty liver disease activity score (NAS) was calculated using hematoxylin and eosin-stained liver sections, and magnetic resonance imaging (MRI) was used for measurement of the fat fraction and elastography. Hepatocyte-derived EVs increased in both male and female mice at 12 and 10 weeks of feeding, respectively, and remained elevated at 24 weeks in both male and female mice and at 48 weeks in male mice and 36 weeks in female mice. Macrophage- and neutrophil-derived EVs were significantly elevated at 24 weeks of dietary feeding concomitant with the histologic presence of inflammatory foci in the liver. In fat-, fructose-, and cholesterol- (FFC) fed male mice, platelet-derived EVs were elevated at 12, 24, and 48 weeks, whereas in female mice, platelet derived EVs were significantly elevated at 24 weeks. Hepatocyte-, macrophage- and neutrophil-derived EVs correlated well with the histologic NAS. Conclusion: Circulating cell-type-specific EVs may be a novel biomarker for NASH diagnosis and longitudinal follow up.

Osteoclasts (OCs) and other cells of the mononuclear phagocyte system possess receptors for adhesive proteins present in the extracellular matrix. The antigenic phenotype of OCs and foreign body giant cells (FBGCs) was investigated for the presence of several integrin molecules and other largely platelet-associated antigens involved in cell adhesion reactions. Both OCs and FBGCs expressed the alpha-chains of the vitronectin receptor (CD51) and of the VLA-2 (CDw49b) and VLA-4 (CDw49d) molecules as well as their respective beta-chains, gpIIIa (CD61) and CD29. OCs and FBGCs also expressed CD9 and CD55 (DAF-Decay Accelerating Factor) and strongly reacted with antibodies directed against fibrinogen, fibronectin and vitronectin; the latter are ligands for several of the above matrix protein receptors. The data suggest that cell-cell and cell-matrix interactions involving adhesive proteins may be important in OC and FBGC function.

Rodent incisors grow throughout the animal's lives, and the tooth-forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre-ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61(-/-) ), also known as integrin β3, were significantly shorter than those of the wild-type mice at 8-week-old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre-ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2-week-old CD61(-/-) mice. The proliferation of pre-ameloblasts was reduced in CD61(-/-) ameloblasts. The siRNA-mediated suppression of CD61 (siCD61) reduced the proliferation of pre-ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre-ameloblastic cells through Lgr5.

OBJECTIVE

The redistribution of CD9 in peripheral blood eosinophils was investigated with respect to the interaction with platelets during in vitro activation, and whether this interaction exerts influence on eosinophil adhesion properties.

METHODS

Flow cytometry was used to investigate the CD9 expression in purified eosinophils or eosinophils from different whole blood preparations, with or without platelets present. To confirm an eosinophil/platelet interaction fluorescence microscopy was used, and to demonstrate release/shedding of CD9 molecules a biosensor technique was performed.

RESULTS

Our results show that both intracellular and surface expression of CD9 decrease upon in vitro activation in the absence of platelets, a phenomenon probably caused by release/shedding of soluble forms of CD9 and not due to intracellular degradation. Increased expression of CD9 on eosinophils, stimulated in the presence of platelets, is partly a result of interacting platelets, judged by the increase in platelet specific marker CD61. In our adhesion assay a significant increase in eosinophil adhesion properties to fibronectin was obtained when eosinophils were PMA stimulated and interacting with platelets, as compared to activated eosinophils without platelets.

CONCLUSIONS

Our findings, that CD9 expression on eosinophils is dynamically regulated, support our previous suggestion that CD9 may be a useful activity marker and that platelet interaction acts on eosinophil adhesion.

We have evaluated a flow cytometric screening method for identification of HPA la negative individuals, using a commercially available monoclonal anti-CD61 antibody specific for the HPA 1a allotype, and compared the method with an ELISA based method for HPA la phenotyping and two methods for PCR genotyping. HPA 1a phenotyping by fluorochrome conjugated monoclonal anti-HPA la and analysis by flow cytometry is a rapid, reliable and inexpensive technique, suitable for screening purposes.

Neonatal blood (NB) contains substantial numbers of stem and progenitor cells which decline rapidly after birth. Using a combination of cord blood (CB) and NB, we performed a successful, sibling transplant for a thalassaemia patient, leading to the proposal that NB could be used as an adjunct to CB for transplantation. This study was aimed at addressing the feasibility of expanding NB and thus minimizing the volume needed from a NB collection. In the presence of early acting cytokines interleukin-1beta (IL-1beta), IL-3, IL-6, stem cell factor (SCF), flt-3 ligand with and without thrombopoietin (Tpo), we compared the expansion capacity of CD34+ enriched cells from CB, NB and bone marrow (BM). Flow cytometry and colony-forming unit (CFU) analyses show that Tpo significantly increased the expansion of CD34+ cells from CB and NB to early and committed progenitors. No significant difference was observed between the expansion of CB and NB at 7, 14 or 21 days of culture in terms of CFU, CD34+ and CD61+ cell subsets. The expansion capacity of BM was significantly lower than that of NB or CB, possibly related to the low proportion of CD34+ CD38- cells observed at day 0. There was a relatively rapid expansion of NB which was evident at day 7 whilst the expansion of CB and BM remained low, suggesting a speedy maturation process in the postnatal infant. The expanded cells, being heterogeneous in their morphology and cell surface marker expression, were mostly of the myeloid lineage (CD45+, CD33+ and HLA-DR+). Our results showed that the expansion capacity of NB is comparable to that of CB and if transplanted, the expanded products of NB might contribute to the engraftment kinetics of the neutrophil and megakaryocyte lineage.

Individuals with Down syndrome have an increased incidence of leukemia compared to the general population. In addition, Down syndrome children may acquire a myeloproliferation that resembles acute leukemia that undergoes a spontaneous, durable remission. To clarify the relationship between these two disorders, the morphologic, immunophenotypic and cytogenetic characteristics of 28 patients with Down syndrome and the morphologic manifestations of acute leukemia were examined. Three cytomorphological groups were discerned. The first two groups consisted of five patients with acute lymphoblastic leukemia (group I) and three patients with acute myeloid leukemia (group II). These leukemias resembled those of non-Down individuals. The third and largest group (group III) consisted of 20 cases of acute myeloid leukemia that showed prominent megakaryocytic and/or erythroid differentiation and occurred in children under 6 years of age. The blasts in this group were non-reactive for myeloperoxidase or non-specific esterase and expressed CD7, CD34 and CD36 with variable expression of CD61, CD13 and CD33. Four patients in this group had an acquired trisomy 8. Four group III leukemias underwent a durable, spontaneous remission within 2 months of diagnosis. There were no morphologic differences between those leukemias in this group that progressed and those that remitted; however, all remissions occurred in newborns. It is concluded that Down syndrome children acquire a characteristic acute myeloid leukemia that has prominent megakaryocytic and/or erythroid differentiation and an unusual immunophenotype. This group of leukemias may undergo a durable, spontaneous remission in the newborn period.

METHODS

Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation.

RESULTS

In both types of PC, levels of glucose decreased during storage but were not totally depleted >> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at>> 6.8 in all units. Hypotonic shock response scores were>> 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was>> 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII.

CONCLUSIONS

Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.

Our prior study demonstrated that neonatal blood (NB) contained hematopoietic stem and progenitor cells that declined rapidly after birth. To validate that NB is a source of functional stem cells, we characterized this population in terms of cobblestone area-forming cells (CAFC), long-term culture-initiating cells (LTC-IC) and NOD/SCID mouse repopulating cells (SRC) in NB and umbilical cord blood (CB). Our data demonstrated that the frequencies of CAFC (30.2 vs 37.1, P = 0.14) and LTC-IC (28.6 vs 31.0, P = 0.49) in 1 x 10(5) mononuclear cells (MNC) of NB and CB were similar, suggesting that these cells were preserved in the circulation of the neonates shortly after birth. Sublethally irradiated NOD/SCID mice were transplanted with CD34(+) cells enriched from thawed NB and CB. At 6 weeks post transplant, human (hu)CD45(+) cells were detected in the bone marrow (BM), spleen and peripheral blood (PB) of the mice as demonstrated by flow cytometric and DNA analysis. Levels of huCD45(+)cells and colony forming units (CFU) appeared to be dependent on the infusion cell dose and were higher in animals receiving CB cells when compared with those of the NB group. The transplanted cells were capable of differentiation into multi-lineage progenitor cells (CD34(+) cells and differential CFU), as well as mature myeloid (CD14(+), CD33(+)), B lymphoid (CD19(+)) and megakaryocytic (CD61(+)) cells in the recipients. NB cells, subjected to ex vivo culture in an optimized preclinical condition, were significantly expanded to early and committed progenitor cells. Expanded NB contained SRC at a reduced quantity but with high proportions of CD14(+) cells and CD33(+) cells. Our study confirms that NB contains pluripotent hematopoietic stem and progenitor cells capable of homing and engrafting the NOD/SCID mice.

In the past, studies on CD34+ cells have been based on the use of monoclonal antibodies conjugated with different fluorochromes that show different fluorescence intensity and yield variable results. Moreover, most of these studies have neither specifically focused on adult human BM samples nor have they used combinations to explore specifically the phenotype of myeloid committed CD34+ cells. The aim of the present study has been to characterize the normal human CD34+ precursor cells from adult BM in order to identify missing or extremely rare phenotypes that can be used for detecting minimal residual disease (MRD) in patients with AML. For this purpose we have utilized the fluorochrome conjugates that provide the most sensitive signals for identifying low antigenic expression, and the technique has been adapted to the characterization of cells present at very low frequencies. Normal human BM samples from 13 adult healthy volunteers have been analyzed using triple stainings at flow cytometry. The mean percentage of CD34+ cells detected was 0.72 +/- 0.33%; these cells displayed an heterogeneous light-scatter distribution. Most CD34+ cells coexpressed CD38 (96.7 +/- 5.7%), HLADR (81.6 +/- 14.0%), CD33 (84.7 +/- 18.3%), CD13 (84.6 +/- 16.2%) and CD71 antigens (65.5 +/- 9.1%). In addition, almost half of CD34+ cells were CD117+ (60 +/- 26.8%). Only a small proportion of CD34+ cells coexpressed CD4 (15.5 +/- 11.7%, CD36 (31.7 +/- 6.2%), CD61 (16.3 +/- 12.9%), CD41 (6.5 +/- 5.5%) or the lymphoid associated markers CD10 (18.6 +/- 11.8%) and CD19 (12.3 +/- 13.2%). Reactivity for the CD15 antigen was observed in a small population of CD34+HLADR+ cells (11.6 +/- 11.2%) although its intensity of expression was lower than that of the more mature granulocytic cells. No CD34+ cells displayed CD14, CD65, CD20, strong CD22, CD3 and CD56 antigens. Accordingly, most adult bone marrow CD34+ cells appeared to be committed to the myeloid lineage (CD13+/CD33+) and displayed an intermediate-to-large FSC/SSC while the lymphoid-committed CD34+ cells (CD19+, CD10+) were in a minority with low FSC/SSC values. By triple marker stainings several phenotypes of CD34+ precursor cells were found to be either undetectable or present at very low frequencies (< 1 x 10(-3)) in the normal human adult bone marrow. These data may be of great value for defining leukemia 'associated' phenotypes used to detect minimal residual disease in adult acute leukemia patients.

The development of reagents against leukocyte differentiation antigens in veterinary species is delayed compared to mouse and men and therefore also the number of existing reagents for the characterisation of leukocytes derived from species with importance in veterinary medicine is restricted. Cross-reactive studies with existing well defined monoclonal antibodies directed against leukocyte differentiation antigens derived from other species are an alternative approach to enhance the panel of reagents in veterinary immunology. This study describes the activities of the animal homologue section in frame of human leukocyte differentiation antigen 8-workshop (HLDA8) were 376 monoclonal antibodies, mainly directed against human leukocytes had been tested for their reactivity with 17 different animal species including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round 182 mAb were selected based on there reactivity in FCM analyses with at least one species for further studies, including multi-colour FCM, and molecular analyses of the antigens. Interesting was the species-overlapping reactivity of mAb directed against distinct clusters: 11 out of 17 species reacted with CD9, 11 of 17 with CD11a, CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of cross-reactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.

OBJECTIVE

The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced thrombocytopenia. Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for an optimum production of megakaryocytic cells.

METHODS

CD34(+) PBPC were cultured in serum-free medium comparing different (n = 23) combinations of stem cell factor (SCF) (S), IL-1beta (1), IL-3 (3), IL-6 (6), erythropoietin (EPO) (E), thrombopoietin (TPO) (T) and promegapoietin (PMP, a novel chimeric IL-3/TPO receptor agonist). Ex vivo-generated cells were assessed by flow cytometry, morphology, and progenitor cell assays.

RESULTS

Addition of TPO to cultures stimulated with S163E, a potent progenitor cell expansion cocktail previously described by our group, effectively induced the generation of CD61(+) cells (day 12: 31.4 +/- 7.9%). The addition of PMP tended to be more effective than TPO +/- IL-3. Whereas EPO was not required to maximize TPO- or PMP-induced megakaryocytic cell production, the use of IL-6 and IL-1beta augmented cellular expansion as well as CD61(+) cell production rates in the majority of cytokine combinations studied. Thus, the most effective CD61(+) cell expansion cocktail consisted of S163 + PMP which resulted in 65.9 +/- 3.0% CD61(+) at day 12 and an overall production of 40.7 +/- 4.5 CD61(+) cells per seeded CD34(+) PBPC. However, the basic 2-factor combination S + PMP also allowed for an effective CD61(+) cell production (day 12 CD61(+) cell production: 15.1 +/- 1.6). Moreover, maximum amplification of CFU-Meg was observed after 7 days using this two-factor cocktail (12.9 +/- 2.6-fold). The majority of CD61(+) cells generated in TPO- or PMP-based medium were low-ploidy 4N and 8N cells, and ex vivo-generated CD61(+), CD41(+), and CD42b(+) cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (vWF) (76.7 +/- 3.3%, 58.8 +/- 4.4%, and 82.7 +/- 2.5%, respectively).

CONCLUSIONS

Taken together, this study demonstrates that megakaryocytic cells can be effectively produced ex vivo with as little as two-factors (SCF + PMP), an approach that might be favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia.

Apart from qualitative flags, that are typically inefficient and uninformative, haematology instruments provide little meaningful information about lymphocyte populations or the lineage of atypical or immature elements, The CELL-DYN Sapphire haematology analyser uses integrated optical and fluorescence (488 nm) measurements, with FL1 (FITC) and FL2 (PE) detectors being configured for fluorescent analysis. As monoclonal antibodies (Mab) are widely used as cellular probes, and are likely to constitute the future basis for immunodifferentials, we explored the feasibility of implementing immunofluorescence on this routine haematology analyser. An extensive series of Mab (CD2, CD3, CD4, CD8, CD11b, CD13, CD14, CD16, CD19, CD22, CD33, CD34, CD41, CD42b, CD45, CD56, CD61, CD64, CD235a and HLA-DR) were tested singly or in FITC/PE combinations. Analyser processing and data acquisition was achieved using CD-Sapphire automated CD61 immunoplatelet or CD3/4/8 assay procedures and, apart from mixing EDTA-blood and antibody, no further sample manipulation was required. Downloaded raw files were processed with cytometry software, and all evaluated reagents showed population discrimination analogous to flow cytometry. Practical procedures were straightforward and required minimal operator training. Extended information that can be obtained from monoclonal antibodies with a routine haematology analyser has the potential to extend haematology laboratory practices and positively impact laboratory and clinical efficiency.