In this study we describe the morphologic and immunohistochemical evaluation of bone marrow biopsies from 14 patients with therapy-related myelodysplastic syndromes (t-MDS). We employed CD34, anti-HLA-Dr, anti-elastase, CD68, anti-glycophorin, CD61 monoclonal antibodies immunostaining, and enzyme histochemistry for chloroacetate esterase. Moreover, we used PC10, a MAb raised against the proliferating cell nuclear antigen, to study the proliferative capacity of these marrows. Our data suggest that diagnosis of refractory anemia with excess of blasts (versus chronic myelomonocytic leukemia), the abnormal localization of immature precursors, marrow fibrosis, and augmented CD34 expression in the bone marrow biopsy are ominous prognostic factors at a statistically significant level (p < 0.0005). A combined morpho-immunohistochemical analysis of bone marrow biopsy correctly classifies t-MDS cases according to the biologic and clinical aggressiveness.

Intercellular communication mediated by cell surface antigens is important in the maintenance of synovial tissue (ST) integrity. Chronic inflammation is a common feature of osteoarthritis (OA). Cellular attachment to and migration into ST is one of the critical aspects of chronic inflammation. This study was undertaken to examine the tissue distribution of a broad spectrum of monoclonal antibodies (mAbs) containing tetraspan antigens (CD9, CD63, CD151), endothelial cell antigens (CD31, CD36, CD105, CD106, CD146), integrins (CD49a-f, CD29, CD41, CD51, CD61), CD39, CD98, CD99, CD143 and, CD147 supplied from fifth and sixth international workshops and conferences on human leukocyte differentiation antigens in a comparative manner in human OA and normal synovium. Ten primary OA patients and six normal individuals were included in this study. The average age of the patients was 65.0 +/- 8.3 years and the average age of the controls was 31.8 +/- 5.3 years. Sections were screened using an indirect immunoperoxidase method. Tetraspan antigens and CD98 presented rather unique staining pattern in OA synovium suggesting special roles for each antigen on the synovial lining layer (SLL). Endothelial cells and type A synoviocytes expressed CD31 and CD36 in OA, but only endothelium in normal subjects. Integrins presented a uniform staining pattern in both groups. There was a positive reaction in some of the ST stromal elements for CD143 in all specimens. In conclusion, human normal and OA synovium were comparatively reviewed by a broad spectrum of mAbs with special attention being given to their functional aspects. This data suggests a significant difference in antigenic phenotype of SLL cells in OA and ST not to be considered at a normal-like state in OA. The fact that their activation was independent of the degree of lymphocyte infiltration further emphasizes the possible central importance of SLL.

Pulmonary extramedullary hematopoiesis is a rare manifestation of myelofibrosis. We encountered a unique case of pulmonary extramedullary hematopoiesis occurring in a 59-year-old white man, where in addition to the typical foci of interstitial hematopoietic cells, a surgical lung biopsy showed airspace and arterial wall involvement. Airspace foci were associated with acute and organizing alveolar hemorrhage, while within arteries the hematopoietic elements had a striking predilection for the vascular intima. The hematopoietic foci included erythroid precursors, myeloid precursors, and megakaryocytes, which were immunoreactive with hemoglobin, myeloperoxidase, and CD61, respectively. Whether extramedullary hematopoiesis represents in situ embryonic stem cell differentiation or a compensatory seeding of hematopoietic cells from the bone marrow remains to be elucidated. However, familiarity with these findings in the lung could be helpful in uncovering occult hematological disorders accompanied by extramedullary hematopoiesis. Extramedullary hematopoiesis should also be considered as a cause of pulmonary hemorrhage, especially in the setting of myelofibrosis.

We examined the changes in the early postmortem platelet count in postmortem blood and the reasons for these changes by counting the platelets, by performing in vitro hypostatic tests, by estimating the percentage of erythrocytes by volume in postmortem blood samples, by immunohistochemistry (anti-CD61, anti-fibrinogen), and by immunoelectron microscopy (anti-CD62, anti-CD63, anti-thrombospondin). The apparent initial increase in the platelet count in postmortem blood was found to be caused by hypostatic phenomena. The subsequent discontinuous decrease in the platelet count despite continuing hypostasis in the corpse can be explained in part by postmortem thrombolysis and the development of reversible platelet-platelet aggregates. The main point is, that changes in the postmortem blood environment cause potentially reversible adhesion of platelets to pre-adsorbed fibrinogen on erythrocytes. Thus the decrease in the number of platelets in postmortem blood is not attributable to postmortem clotting but to a decrease in the number of countable platelets in postmortem blood.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa polypeptides and the platelet reactive antibody 17 kDa, 22 kDa plus two high molecular weight >> 100 kDa) polypeptides. Immunohistological studies of the mAbs were performed in order to localize the cellular distribution of the detected antigens in various organs. The T lymphocyte reactive antibody detected an antigen, which was widely distributed in the T cell area of lymph nodes and spleen and in the thymic medulla. We conclude that this antibody is an anti-CD3 mAb and suggest that the platelet reactive antibody reacts to the CD41/CD61 integrin molecule. In addition to our own mAbs, more than 100 mAbs against leukocytes of human and various animal species have been analysed for cross-reactivity to mink leukocytes. We found eight to cross-react with mink. Of particular importance was an anticanine CD11a mAb, an antihuman CD79a mAb and an antihuman bcl-2 mAb.

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Our objective was to evaluate, probably for the first time, the impact of CD34 subsets on engraftment kinetics in allogeneic PBSC transplantation (PBSCT). PBSC graft components were analyzed in 62 cases for the absolute count/kg of total CD34+ and the following subsets: DR- and +, CD71+/-, CD38+/-, CD33+/- and CD61+/-. Time to ANC >0.5 and >1 x 10(9)/l and platelets >20 and >50 x 10(9)/l was reported. The median value for each parameter was used to discriminate rapid from slow engraftment. Four parameters showed significant predictive power of early neutrophil engraftment, namely CD34+ /DR- (P = 0.002), CD34+/38- (P = 0.02), CD34+/CD61- (P = 0.04) and total CD34+ cell dose (P = 0.04). Four parameters showed significant predictive power of early platelet engraftment, namely CD34+/CD61+ (P = 0.02), CD34+ /CD38- and total CD34+ cell dose (P = 0.04) and CD34+ /CD71- (P = 0.05). Comparing patients who received>> to those who received < the threshold dose(s), only CD34+ /CD38- lost its significance for neutrophil engraftment; and only CD34+ /CD61+ retained its significance for platelet engraftment (P = 0.03); furthermore, the former group required significantly fewer platelet transfusions (P = 0.018). We concluded that in allogeneic PBSCT, the best predictor of early neutrophil engraftment is the absolute CD34+ /DR- and for early platelet engraftment is the absolute CD34+ /CD61+ cell dose.

Megakaryocytes (Mks) are found in the lungs and the blood stream as well as in the bone marrow. We modified a whole blood filtration method for Mks by immunostaining for CD61 using biotin streptavidin, and used this technique to study Mks and their morphology in the central venous and arterial circulations before, during and after cardiopulmonary bypass (CPB) in haematologically normal patients undergoing routine cardiac surgery. Blood samples were taken immediately after the insertion of central venous (V) and arterial (A) catheters and after thoracotomy, immediately before bypass. Further samples were taken after 60-90 min on-CPB and 180-240 min post-bypass. In comparison with the steady state before bypass, circulating Mk levels in blood on bypass increased dramatically, from (V) 10.93 +/- 3.94/ml (mean +/- SD) to 36.48 +/- 11.52/ml and from (A) 8.37 +/- 4.39/ml to 38.65 +/- 20.68/ml. This effect was still present, to a lesser extent, 180-240 min post-bypass. Circulating levels of Mks were consistently lower in the arterial circulation than in the venous circulation off bypass, but levels in the two circulations were comparable during CPB, confirming previous suggestions that the lungs are net removers of Mks from the circulation. Type 4 Mks, the largest and most normal morphologically, were rarely seen in arterial blood, but increased significantly during CPB, indicating that the lungs selectively remove large Mks. The lungs appear to play an active role in the regulation of Mk levels. This is lost during CPB and despite the extracorporeal 40 microm arterial line filter, large Mks enter the systemic circulation. More effective extracorporeal filtration of large Mks might reduce the neurological impairment seen in some patients who have undergone CPB.

An immunohistochemical and morphometric analysis was performed on trephine biopsy specimens of the bone marrow in 40 patients (23 men and 17 women, mean age 62 years) with different subtypes of myelodysplastic syndromes (MDS) to determine dysmegakaryopoiesis, but particularly precursor cells--that is, pro- and megakaryoblasts. In 31 of the 40 patients the numbers of megakaryocytes were increased which was associated with a predominance of smaller cell forms (micromegakaryocytes). Compared with periodic acid Schiff, immunostaining with a formalin resistant monoclonal antibody against glycoprotein IIIa (Y2/51(CD61) showed a clinically important proportion of immature elements. These could be designated pro- and megakaryoblasts by taking morphometric measurements on smears and bone marrow sections. There was a relevant increase in the number of promegakaryoblasts in 32 patients, consistent with uncontrolled expansion of the precursor pool. Seventeen repeated bone marrow biopsy specimens taken after chemotherapy largely showed a decrease in the numbers of megakaryocytes including the precursor cell population. Moreover, morphometric evaluation disclosed that micromegakaryocytes in MDS differ significantly from those in chronic myeloid leukaemia (CML) due to distinctive nuclear features and a disturbed nuclear:cytoplasmic ratio. These changes generate a more pleomorphic or atypical appearance of this cell population in MDS, compared with micromegakaryocytes in CML. It is concluded that the disproportionate increase in megakaryocyte precursors and the grossly abnormal aspects of micromegakaryocytes in MDS are characteristics of the severe defect involving haematopoiesis in this disorder.

Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet-binding sensor (PDI-sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s(-1) was correlated with the PDI-sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice.

Background Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 μg mL(-1) corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s(-1) ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion PDI-sAb is a sensitive and real-time reporter of platelet- and vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.

Human fetal livers contain progenitor cells that become mast cells after 4 weeks of culture with recombinant human stem cell factor. Expression of cell surface CD29 (beta 1), CD18 (beta 2), CD61 (beta 3), and beta 5 integrins was investigated on such cells by flow cytometry and adhesion measurements. High surface expression of CD49e, CD51, and CD61 along with kit was apparent by 4 weeks of culture, whereas expression of each at day 0 was low to undetectable. CD29 and CD49d were detected on cells from day 0 to 4 weeks of culture; CD49b, CD49c, CD49f, CD18, and CD54 expression was negligible. The fetal liver-derived mast cells spontaneously adhered to vitronectin. No evidence for degranulation was found during vitronectin-dependent adhesion. Adhesion occurred in part through the CD61/CD51 receptor. No evidence for adhesion to vitronectin through CD29 and beta 5 integrins was obtained. Almost all of the vitronectin-adherent cells expressed CD51, CD61, kit, and tryptase, and exhibited metachromasia with toluidine blue. Thus, among the fetal liver-derived cells, developing mast cells were selectively adherent to vitronectin. These mast cells and the other cell types present also adhere spontaneously to fibronectin and to laminin, this adhesion being partially inhibited by antibodies against CD61 and CD29 integrins. In conclusion, human mast cells acquire functional vitronectin receptors as they develop from fetal liver progenitors under the influence of rhSCF. This may be important for the recruitment, localization, and retention of developing mast cells.

OBJECTIVE

Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC).

METHODS

MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™).

RESULTS

PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05).

CONCLUSIONS

Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load.

Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbβ3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin αIIbβ3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin αIIbβ3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.

BACKGROUND

Platelets play an important role in the natural history of ischemic stroke, and are known to be activated in the acute phase. Although aspirin reduces risks of myocardial infarction, stroke and cardiovascular death, the extent of platelet action and the effect of aspirin on platelet function in patients recovering from stroke remain unclear.

METHODS

We studied 120 individuals divided into three equal groups: aspirin-free patients after ischemic stroke, post-stroke patients receiving aspirin (81-650 mg/daily), and aspirin-free subjects with multiple risk factors for vascular disease. Conventional platelet aggregation induced by 5 microM ADP and 5 microM epinephrine, cartridge-based analyzers (Ultegra, and PFA-100) readings, and expression of CD31, CD41a, CD42b, GPIIb/IIIa activity, CD51/CD61, CD62p, CD63, CD107a, CD154, CD165, formation of platelet-monocyte aggregates, intact (SPAN12), and cleaved (WEDE15) PAR-1 thrombin receptors by flow cytometry were analyzed.

RESULTS

There were no differences between aspirin-free post-stroke patients and aspirin-free controls. Although aggregation was slightly higher, 12 out of the 14 receptor analyses, were surprisingly lower in the post-stroke cohort. Aspirin-treated patients exhibited highly significant inhibition of epinephrine-induced aggregation (p=0.0001), prolongation of the closure time (p=0.03), and reduction of the aspirin reactive units (p=0.02) measured by the Ultegra device. In addition, surface platelet expression of thrombospondin (p=0.001), GPIIb/IIIa activity (p=0.04), P-selectin (p=0.03), CD40-ligand (p=0.04), CD165 (p=0.02), the formation of the platelet-monocyte aggregates (p=0.01), and intact epitope of PAR-1 thrombin receptor (p=0.03) were significantly lower in the aspirin-treated group.

CONCLUSIONS

Platelets are not activated in aspirin-free patients after ischemic stroke. Platelet function is significantly inhibited in those treated with aspirin when compared with healthy subjects with risk factors for vascular disease. Bleeding complications and hemorrhagic transformations after aggressive antiplatelet regimens could be related to the decreased or normal baseline platelet characteristics in such patients. Further analysis of platelet heterogeneity and its clinical significance remains to be determined in randomized trials.

OBJECTIVE

Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3-mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis.

METHODS

CD34(+) cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA.

RESULTS

In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61(+)GPA(-) megakaryocytic and a CD61(+)GPA(+) mixture of megakaryocytic and erythroid precursors from CD34(+) hematopoietic precursors at a pharmacological concentration (100 microg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61(-)GPA(+) erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61(-)GPA(+) erythroid precursors. In serum-containing cultures, only low numbers of CD61(+) or GPA(+) cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34(+) cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF.

CONCLUSIONS

These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease.

OBJECTIVE

Anagrelide is an agent with a significant platelet-lowering activity in humans. Contrasting the wealth of clinical data, bone marrow (BM) changes during therapy have been relatively rarely studied; information is particularly lacking regarding specific features of megakaryocytopoiesis.

METHODS

A study was performed on 15 patients with essential thrombocythemia and early stage chronic idiopathic myelofibrosis presenting with an elevated platelet count. These patients received anagrelide for 25 months resulting in a significant improvement of thrombocytosis. Evaluations were carried out by morphometry on sequential BM biopsies following enzyme- and immunohistochemical stainings that also included those for proliferative capacity and apoptosis.

RESULTS

No significant change in proliferation or apoptosis was recognizable during anagrelide treatment. The most conspicuous alterations were those of the CD61+ megakaryocytopoiesis. Megakaryocytopoiesis revealed an increase in promegakaryoblasts together with an enhancement of proliferating cell nuclear antigen activity. This feature is in keeping with an inhibitory effect on endoreduplication implying an arrest of polyploidization and maturation into platelet-shedding large megakaryocytes. On the other hand, a significant increase in the number of megakaryocytes was not detectable. Anagrelide failed to exert a stimulating influence on the progression of myelofibrosis or on the amount of CD34+ progenitor cells. Regarding angiogenesis, there was no increase in the density of BM vessels, but distension of the vascular lumina corresponded with a vasodilatory effect.

CONCLUSIONS

Anagrelide exerts a significant effect on endoreduplicative activity of megakaryocytes consistent with an inhibition of maturation and therefore, generates a relative predominance of precursor cells but fails to stimulate myelofibrosis.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4 weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

OBJECTIVE

This study assessed whether the increased numbers of platelet-monocyte aggregates observed in patients with venous stasis ulceration (VSU) represent a response to dermal ulceration or if it is a condition associated with underlying chronic venous insufficiency (CVI). We also analyzed the expression of CD11b in patients with CVI to determine whether leukocyte activation, known to occur in VSU, is a precursor of or a response to ulceration.

METHODS

Patients with varying classes of CVI (n = 24) and healthy control subjects (n = 15), whose status was documented by means of duplex scanning, stood upright and stationary for 10 minutes. Two aliquots of blood, drawn from a distal leg vein and an antecubital fossa vein, were incubated with either buffer or one of three platelet agonists. After fixation, these samples were further incubated with fluorescent-labeled monoclonal antibodies (f-MoAb) specific for CD14 (monocytes) and CD61 (platelets). The activated leukocyte assay was performed by incubating another aliquot of the blood samples with f-MoAb specific for CD11b and CD14. All samples were evaluated by means of flow cytometry.

RESULTS

We observed significantly more platelet-monocyte aggregates throughout the circulation in patients with CVI than in control subjects (29% vs. 8%; P <.0002). Furthermore, patients with CVI formed significantly more of these aggregates in response to all platelet agonists than did control subjects. There were no significant differences between baseline numbers of aggregates or response to agonists in patients who had CVI with (n = 10) or without (n = 14) ulceration. Patients with CVI had more circulating platelet-neutrophil aggregates than control subjects (7.2% vs. 3.6%; P =.05). The addition of platelet agonists to the blood of patients with CVI resulted in more platelet-neutrophil aggregates than in control subjects. Monocyte CD11b expression was higher in patients with CVI than in control subjects (7.5 vs. 3.7; P <.01), with no differences noted in CD11b expression between patients with or without ulceration. Neutrophil CD11b expression was low and similar in control subjects and patients with CVI.

CONCLUSIONS

All classes of CVI are associated with significantly increased percentages of platelet-monocyte aggregates and increased percentages of platelet-neutrophil aggregates throughout the circulation. The presence of more of these aggregates and the increased propensity to form aggregates in the presence of platelet agonists in all classes of CVI suggests an underlying state of platelet activation and increased reactivity that is independent of the presence of ulceration. The increased expression of monocyte CD11b throughout the circulation in all classes of CVI suggests that although systemic monocyte activation occurs in CVI, its presence is independent of VSU as well.

It has been shown that the mammary luminal lineage could be maintained by luminal stem cells or long-lived progenitors, but their identity and role in breast cancer remain largely elusive. By lineage analysis using Wap-Cre mice, we found that, in nulliparous females, mammary epithelial cells (MECs) genetically marked by Wap-Cre represented a subpopulation of CD61+ luminal progenitors independent of ovarian hormones for their maintenance. Using a pulse-chase lineage-tracing approach based on Wap-Cre adenovirus (Ad-Wap-Cre), we found that Ad-Wap-Cre-marked nulliparous MECs were enriched with CD61+ alveolar progenitors (APs) that gave rise to CD61- alveolar luminal cells during pregnancy/lactation and could maintain themselves long term. When transformed by different oncogenes, they could serve as cells of origin of heterogeneous mammary tumors. Thus, our study revealed a type of long-lived AP within the luminal lineage that may serve as the cellular origin of multiple breast cancer subtypes.

The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be induced. Signaling focal contacts via ITGβ3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.

The aim of the paper was to perform a chronological assessment of the phenomenon of delayed rupture of the spleen, to assess the phenomenological order about the sub-capsular hematoma transformation to determine the causal relationship with trauma as hypothetical cause of death. 80 cases of blunt trauma with splenic capsular hematoma and subsequent rupture of the spleen were evaluated: 38 had an acute rupture of the spleen, 42 presented a break in days or weeks after the traumatic injury. Time between the traumatic event and delayed rupture of the spleen is within a range of time from one day to more than one month. Data recorded included age, sex, type of trauma, injury severity score, grade of splenic injury, associated intra-abdominal injuries, pathologic specimen evaluation. Immunohistochemical investigation of perisplenic hematoma or laceration was performed utilizing polyclonal antibodies anti-fibrinogen, CD61 and CD68, and showed structural chronological differences of sub-capsular hematoma. Expression of modification and organization of erythrocytes, fibrinogen, platelets and macrophages provides an informative picture of the progression of reparative phenomena associated with sub-capsular hematoma and subsequent delayed splenic rupture. Sub-capsular splenic hematoma dating, which we divided into 4 phases, is representing a task in both clinical practice and forensic pathology.

Platelets have been proved to exacerbate influenza infection and its complications. Inhibition of platelet activation may be a feasible method for preventing severe infection and secondary acute lung injury (ALI). Isofraxidin (IFD) is a natural coumarin isolated from the plants Sarcandra glabra and Siberian ginseng, and exerts anticancer, antioxidant and antiinflammatory effects. In the present study, we examined the therapeutic effects of IFD in ADP- or arachidonic acid (AA)-induced platelet aggregation model and in influenza A virus (IAV)-induced ALI mouse model. The results showed that IFD significantly inhibited platelet aggregation induced by ADP and AA in vitro in a concentration-dependent manner as well as the release of soluble P-selectin and platelet factor 4. Moreover, IFD significantly relieved IAV-induced lung inflammation, reduced the expressions of platelet activation biomarkers (P-selectin and CD61), decreased the serum levels of TNF-α, IL-1β, IL-6 and MIP-2, suppressed peripheral platelet aggregation and prolonged the survival time of infected mice. The western blotting results also demonstrated that IFD reduced the phosphorylation levels of PI3K, AKT and p38 in the activated platelets stimulated by ADP and IAV infection. But IFD did not have any effects on IAV replication. It indicated that IFD ameliorated IAV-induced severe lung damage and lethal infection by suppressing platelet aggregation via regulating PI3K/AKT and MAPK pathways.

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.