Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline-regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61(+) differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.

Platelet activation in preeclampsia is reflected by elevated levels of platelets exposing P-selectin. In plasma, a non-cell bound (soluble) form of P-selectin is present. Elevated levels of this soluble form have been reported in preeclampsia. Plasma P-selectin may consist of two fractions: microparticle (MP)--associated P-selectin and non-MP--associated P-selectin. In the present cross-sectional study, we investigated to which extent plasma P-selectin is MP--associated and whether such MP are elevated in preeclamptic patients. Preeclamptic patients (n=10) were matched with normotensive pregnant women (n=10) and non-pregnant controls (n=10). Plasma P-selectin was measured by ELISA. MP were isolated, double labelled with anti-CD61 (GPIIIa) and anti-CD62P (P-selectin) and subsequently analyzed with flowcytometry. Plasma P-selectin concentration was elevated in preeclamptic patients compared to non-pregnant controls (p=0.007), but not compared to normotensive pregnant women (p=0.210). Plasma P-selectin is partially MP--associated (3-5%). In pregnancy, the fraction of P-selectin exposing platelet-derived MP (PMP) (10.9%) was increased compared to non-pregnant controls (8%). This fraction further increased in preeclamptic patients (15.4%), and significantly differed from normotensive pregnant women (p=0.02). A minor fraction of plasma P-selectin is associated with PMP. The fraction of PMP exposing P-selectin is increased in preeclamptic patients and to a lesser extent in normotensive pregnancy. Because MP associated P-selectin exclusively originates from platelets, this fraction indicates platelet activation. Platelet activation is prominent in preeclampsia and this study proves that at least a part of the plasma P-selectin originates from platelets.

A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity>> 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

BACKGROUND

Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry.

METHODS

Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM+)] or platelet EVs from human plasma [integrin β3 positive (CD61+)]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated.

RESULTS

Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM+ MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61+ EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection.

CONCLUSIONS

None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.

Mast cells (MC) are important cellular components of the immune network in diverse organs. The skin MC has likewise been implicated in IgE- and complement-mediated cutaneous reactions. Such reactions supposedly involve specific cell surface membrane receptors. In this study, the cell surface marker profile of human skin MC was established using monoclonal antibodies (MoAb) against defined CD antigens. MC were isolated from juvenile foreskin (n = 55) and adult mammary skin (n = 5). The reactivity of MC with MoAb was assessed by a combined toluidine blue/immunofluorescence staining technique. Confirming our previous analyses on lung MC, foreskin MC reacted with MoAb against CD9, CD29, CD33, CD43, CD44, CD45, CD46, CD51, CD54, CD55, CD58, CD59, CD61, and CD117 (c-kit). Foreskin MC were also recognized by MoAb to CD47, CD48, CD49d, CD53, CD60, CD63, CD81, CD82, CD84, CD87, CD92, CD97, CD98, and CD99. Recently clustered CD antigens detectable on foreskin MC were CD147 (neurothelin), CD149 (MEM133), CD151 (PETA-3), and CD157 (BST-1). In contrast to lung MC and MC from adult skin, foreskin MC were found to express CD88 (C5aR). Also, cutaneous MC (from both juvenile foreskin and adult mammary skin), but not lung MC, were found to bind the CD32 MoAb IV.3, 2E1, and FLI8.26 (Fc gammaRII). The CD50 antigen (ICAM-3) was detectable on lung MC, but not on foreskin MC or MC of adult mammary skin. In summary, our data show that cutaneous MC and lung MC express an almost identical phenotype; however, in contrast to lung MC, cutaneous MC appear to express substantial amounts of CD32 and to lack CD50. In addition, foreskin MC, unlike MC from adult skin or lung, express CD88.

Mice with combined deficiencies of the low-density lipoprotein receptor (LDLR(-/-)) and the catalytic component of an apolipoprotein B-edisome complex (APOBEC1(-/-)) that converts apoB-100 to apoB-48 have been characterized, and this model of LDL cholesterol-driven atherosclerosis was applied to an investigation of the role of fibrinogen (Fg) in the genesis and progression of the plaque. LDLR(-/-)/APOBEC1(-/-)/FG(-/-) (L(-/-)/A(-/-)/FG(-/-)) triple-deficient mice presented more advanced plaque in their aortic trees and aortic sinuses at 24, 36, and 48 weeks of age compared to L(-/-)/A(-/-) mice, a feature that may result from enhanced platelet activation in these former mice. This is supported by the presence of hypercoagulability, increased CD61 and CD62P on resting platelets, and higher plasma soluble P-selectin in L(-/-)/A(-/-)/FG(-/-) mice as compared to L(-/-)/A(-/-), FG(-/-), or wild-type mice. The elevated higher molecular weight forms of von Willebrand factor (VWF) in L(-/-)/A(-/-)/FG(-/-) mice, revealed by increased VWF collagen binding activity, perhaps resulting from down-regulation of its cleaving metalloproteinase, ADAMTS13, further indicates enhanced platelet activation. Thus, the earlier arterial plaque deposition in L(-/-)/A(-/-)/FG(-/-) mice appears to contain a contribution from enhanced levels of thrombin and activated platelets, a synergistic consequence of an Fg deficiency combined with a high LDL cholesterol concentration.

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19+ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (beta1- and beta2-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19+ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19-CD38++CD45-/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19-CD38++CD45-/dim CD138++ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19+ B cells and a very minor proportion of clonal CD138++ PC that escape from the BM.

OBJECTIVE

To test the hypothesis that fragments of platelet thrombi and vascular endothelium are incorporated into the walls of small cerebral vessels in systemic lupus erythematosus (SLE).

METHODS

Six varied necropsy cases of central nervous system (CNS) SLE and 15 controls were studied. The controls were selected to represent a wide range of diseases in which the cerebral circulation is compromised. Tissue sections were stained by standard histochemical and immunocytochemical methods, the latter using antibodies to platelet membrane glycoprotein IIIa (CD61), and vascular endothelium (CD31).

RESULTS

In four of six cases of CNS SLE characterised by small vessel hyalinization and thickening, fragments of platelet membrane were found in the walls of small cortical and meningeal vessels. Similar findings were not evident in two other SLE cases that were characterised by relatively short clinical histories and an acute vasculitis. One control case of severe polyarteritis nodosa showed platelet fragment deposition in arteries larger than the vessels so affected in SLE.

CONCLUSIONS

Previous studies have suggested that neuropsychiatric symptoms in SLE may be related to repeated episodes of vasculitis in small cerebral vessels that are triggered by antiphospholipid antibodies. Concurrent thrombus formation might facilitate the incorporation of platelet fragments into small vessel walls. This process contributes to the thickening and irregularity of small vessels, a major feature of longstanding cases of CNS SLE.

The polymer poly-N-acetylglucosamine (pGlcNAc) containing fiber material is becoming increasingly important as a topical agent for hemostasis at wound sites. The pGlcNAc polymeric fiber provides hemostasis through redundant mechanisms that include platelet activation for fibrin network formation. The research presented here better defines the mechanism for the effect of pGlcNAc containing fibers on platelet-mediated processes. Adsorption experiments demonstrated that pGlcNAc fibers tightly bind most major plasma proteins and a specific sub-set of platelet surface proteins, including the integrin beta(3) subunit (CD61) and the von Willebrand receptor GP1b (CD42b). The result of this interaction is a platelet-dependent acceleration of fibrin gel formation. Accelerated fibrin polymerization is sensitive to factor XII inhibition by corn trypsin inhibitor and integrin inactivation with integrilin. Confocal microscopy studies show that when platelet integrins contact plasma protein-saturated pGlcNAc fibers, an increase in intracellular free calcium for platelet activation occurs to drive surface expression of phosphatidyl serine (PS). Thus, a catalytic surface for thrombin generation and accelerated fibrin clot formation results from the interaction of platelets with pGlcNAc. These findings, when considered with the observation that pGlcNAc fibers also induce red blood cell agglutination and vasoconstriction, provides an explanation for the ability of the pGlcNAc material to provide hemostasis in a wide variety of clinical applications.

Mice were exposed to pure oxygen for various times to explore the pulmonary platelet trapping associated with alveolar damage, its mechanism, and its role in the lesions. Platelet sequestration, evaluated by electron microscopy and by injection of radiolabeled platelets, was detectable after 72 h and reached a maximum after 96 h of exposure (i.e., shortly before death). Circulating platelets (analyzed by Facscan) showed some increase in the expression of CD11a and CD62, but little change in CD31 and CD61. Both platelet activation and lung sequestration were dependent on TNF-alpha, since antibody against TNF-alpha reduced the expression of CD11a on circulating platelets and their sequestration in the lung. Lung platelet sequestration was also decreased by anti-CD11a MoAb. Northern blot analysis of lung mRNA isolated at 96 h of oxygen exposure revealed a 7-fold increase in CD54 (intercellular adhesion molecule-1 [ICAM-1]) and a 2.5-fold increase in TNF-alpha mRNAs respectively. These results demonstrate that the platelet pulmonary trapping induced by hyperoxia is dependent upon TNF-alpha and the CD11a-CD54 adhesion molecules. However, platelet trapping does not appear to play an important pathogenic role in acute oxygen injury, since treatments that decrease trapping (anti-TNF-alpha, anti-CD11a, or antibody-induced thrombocytopenia) did not markedly attenuate the alveolar damage.

Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34(+) cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1 beta (IL-1 beta), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and Flt-3L. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61(+) population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N--32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1 beta, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34(+) cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications.

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

In this study, we investigated the bone cell activity in patients with osteogenesis imperfecta (OI) treated and untreated with neridronate. We demonstrated the key role of Dickkopf-1 (DKK1), receptor activator of nuclear factor-κB ligand (RANKL), and tumor necrosis factor alpha (TNF-α) in regulating bone cell of untreated and treated OI subjects. These cytokines could represent new pharmacological targets for OI.

Bisphosphonates are widely used in the treatment of children with osteogenesis imperfecta (OI) with the objective of reducing the risk of fractures. Although bisphosphonates increase bone mineral density in OI subjects, the effects on fracture incidence are conflicting. The aim of this study was to investigate the mechanisms underlying bone cell activity in subjects with mild untreated forms of OI and in a group of subjects with severe OI treated with cycles of intravenous neridronate.

Sclerostin, DKK1, TNF-α, RANKL, osteoprotegerin (OPG), and bone turnover markers were quantified in serum of 18 OI patients (12 females, mean age 8.86 ± 3.90), 8 of which were receiving cyclic intravenous neridronate, and 21 sex- and age-matched controls. The effects on osteoblastogenesis and OPG expression of media conditioned by the serum of OI patients and anti-DKK1 neutralizing antibody were evaluated. Osteoclastogenesis was assessed in cultures from patients and controls.

DKK1 and RANKL levels were significantly increased both in untreated and in treated OI subjects with respect to controls. The serum from patients with high DKK1 levels inhibited both osteoblast differentiation and OPG expression in vitro. High RANKL and low OPG messenger RNA (mRNA) levels were found in lymphomonocytes from patients. High amounts of TNF-α were expressed by monocytes, and an elevated percentage of circulating CD11b-CD51/CD61+ osteoclast precursors was observed in patients.

Our study demonstrated the key role of DKK1, RANKL, and TNF-α in regulating bone cell activity of subjects with OI untreated and treated with bisphosphonates. These cytokines could represent new pharmacological targets for OI patients.

Epidemiological data indicate that there is a strong association between intake of trans-18 : 2 fatty acids (TFA) and sudden cardiac death. There is little known about the mechanisms by which TFA exert harmful effects on the cardiovascular system. The present in vitro study is the first to demonstrate the effects of membrane-incorporated C18 : 2 TFA on human aortic endothelial cell (HAEC) function. Trans-18 : 2 fatty acids were incorporated to a greater extent (2-fold) in the phospholipid fraction of endothelial cells than that of cis-18 : 2; furthermore, these fatty acids were enriched to a similar extent in the TAG fraction. Flow cytometric analysis indicated that TFA treatment of HAEC significantly increased the expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 (CD54) and vitronectin receptor (CD51/CD61). Incorporation of TFA into membranes increased HAEC adhesion to fibronectin- or vitronectin-coated plates by 1.5- to 2-fold, respectively. Neutrophil and monocyte adhesion to HAEC monolayers was nearly proportional to adhesion molecule expression. TFA treatment also induced the release of monocyte chemoattractant protein-1 by nearly 3-fold in non-stimulated HAEC. Furthermore, we examined the role of TFA on in vitro angiogenic assays. Chemotactic migration of TFA-treated HAEC toward sphingosine-1-phosphate (SPP) was significantly increased compared with controls. Conversely, capillary morphogenesis of TFA-treated HAEC was significantly inhibited in response to SPP, suggesting that TFA incorporation suppresses endothelial cell differentiation. In conclusion, these in vitro studies demonstrated that TFA play a role in the induction of pro-inflammatory responses and endothelial cell dysfunction.

Epithelial cells of the intestine seem to act as antigen-presenting cells to surrounding lymphoid tissue and may be crucial to maintain the pool of peripheral T lymphocytes. The scope of this study was to carry out an immunophenotypic and ultramicroscopic analysis of purified human enterocytes to elucidate their role as antigen-presenting cells, in the immune responses in the gut-associated lymphoid tissue. A method has been developed to obtain purified and viable human enterocyte populations, later labeled with relevant monoclonal antibodies directed to leukocyte antigens and subjected to cytofluorometric analysis. Phenotypic analysis revealed the presence of markers common to "classical" antigen-presenting cells (CD14, CD35, CD39, CD43, CD63 and CD64), reinforcing the idea that enterocytes may act as such. Moreover, several integrins (CD11b, CD11c, CD18, CD41a, CD61 and CD29) were also found. CD25 (IL-2 receptor alpha chain) and CD28, characteristic of T cells, were detected on the surface of these cells; this latter finding rises the possibility that enterocytes could be activated by IL-2 and/or via CD28 through binding to its ligands CD80 or CD86. Finally, the presence of CD21, CD32, CD35 and CD64 that may bind immune complexes via Fc or C3, suggests their participation in the metabolism of immune complexes. Furthermore, the finding of a Birbeck's-like granule in the cytoplasm of the cells, shows that enterocytes contain an ultramicroscopic feature previously thought to be characteristic of Langerhans' cells, an antigen-presenting cell. The phenotype detected on the surface of enterocytes, along with their ultramicroscopic characteristics, suggests that they may play an important role in the immune responses elicited in the gut, presenting antigens to surrounding lymphoid cells, and establishing cognate interactions with them.

BACKGROUND

Prostate stromal cells can be readily cultured in vitro. Are these proliferating cells representative of stromal cells in situ? Since the expression of cell surface molecules, like the cluster of differentiation (CD) antigens, can be affected by changes in physiological conditions cultured stromal cells may differ from uncultured stromal cells in their complement of CD molecules.

METHODS

Prostate stromal cells were prepared from tissue specimens and cultured. Flow cytometry was used to analyze the expression of 107 CD molecules in the resultant cells. Expression of the CD molecules by prostate cells in situ was done by immunohistochemistry.

RESULTS

The expression of a number of cell surface molecules such as CD10, CD13, CD26, and CD44 is elevated in prostatic stromal cells cultured in vitro. These are markers of epithelial cells in tissue. Other molecules expressed by the cultured stromal cells include CD29, CD49a, CD49b, CD49d, CD49f, CD51/61, CD54, CD55, CD56, CD58, CD59, CD61, CD71, CD79b, CD81, CD82, CD90, CD95, CD107a, CD130, and CD147. Among these are stromal, epithelial, and nonstromal/nonepithelial markers as defined by tissue immunohistochemistry.

CONCLUSIONS

Cultured stromal cells express a number of CD molecules normally found in other cell types of the prostate. Cells can express different CD molecules under different conditions.

Identification of the regulatory inputs that direct megakaryocytopoiesis and platelet production is essential for the development of novel therapeutic strategies for the treatment of thrombosis and related hematologic disorders. We have previously shown that primary human megakaryocytes express the N-methyl-d-aspartate acid (NMDA) receptor 1 (NR1) subunit of NMDA-type glutamate receptors, which appear to be pharmacologically similar to those identified at neuronal synapses, responsible for mediating excitatory neurotransmission in the central nervous system. However, the functional role of NMDA receptor signaling in megakaryocytopoiesis remains unclear. Here we provide evidence that demonstrates the fundamental importance of this signaling pathway during human megakaryocyte maturation in vitro. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA extracted from CD34+-derived megakaryocytes identified expression of NR2A and NR2D receptor subunits in these cells, as well as the NMDA receptor accessory proteins, Yotiao and postsynaptic density protein 95 (PSD-95). In functional studies, addition of a selective NMDA receptor antagonist, MK-801 inhibited proplatelet formation, without affecting proliferation or apoptosis. Exposure of CD34+ cells to MK-801 cultured for 14 days in the presence of thrombopoietin induced a decrease in expression of the megakaryocyte cell surface markers CD61, CD41a, and CD42a compared with controls. At an ultrastructural level, MK-801-treated cells lacked alpha-granules, demarcated membranes, and multilobed nuclei, which were prominent in untreated mature megakaryocyte controls. Using immunohistochemistry on sections of whole tibiae from c-Mpl knockout mice we demonstrated that megakaryocytic NMDA receptor expression was maintained following c-Mpl ablation. These data support a fundamental role for glutamate signaling in megakaryocytopoiesis and platelet production, which is likely to be independent of thrombopoietin-mediated effects.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((alphabeta)3gammadeltaepsilon) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10gammaepsilon relative to (alphabeta)3deltaab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but "horizontal" repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of "vertical" repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.

BACKGROUND

Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross-linking nucleic acids and thus affects all cells containing DNA or RNA.

METHODS

Fourteen double-dose single-donor PCs were divided into two study arms. The double-dose PCs were split in two identical units, PCT and conventional control PCs. Study Arm A consisted of seven PCT PCs with corresponding untreated controls, whereas Study Arm B consisted of seven PCT PCs with corresponding gamma-irradiated control. Metabolic changes and agonist-induced platelet (PLT) response were evaluated during storage for up to 12 days.

RESULTS

Higher rate of PLT destruction, illustrated by reduced PLT content, increased lactate dehydrogenase levels, and higher CD61+ microparticle formation rate, were observed after PCT. Generally PCT accelerated metabolic changes in PCs and reduced agonist-induced (collagen or thrombin receptor activator peptide [TRAP]) aggregation responses. Flow cytometric analysis of CD62P and CD42b (GPIbalpha) expression showed higher spontaneous PLT activation in PCT PCs from 5 days of storage. Correspondingly, a reduced capacity for up regulation of CD62P expression and down regulation of CD42b was observed in PCT PLTs after stimulation by the agonists ADP or TRAP.

CONCLUSIONS

Generally reduced in vitro PLT quality was observed after PCT during storage for up to 12 days, with marked reduction from 5 days of storage. Compared to conventional PCs, reduced agonist-induced aggregation and glycoprotein expression were observed after PCT during storage, corresponding to significantly higher level of spontaneous PLT activation in PCT PCs. Clinical studies of efficacy and safety of PCT PCs stored for more than 5 days are recommended.

Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The αIIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. αIIb pairs exclusively with β3 to form the αIIbβ3 integrin. β3 (CD61) also pairs with αv (CD51) to form the αvβ3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express αIIbβ3 and αvβ3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express αvβ3, and most fetal liver HSCs do not express either integrin. By using αIIb deficient embryos, we show that αIIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta.

Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.

To evaluate the impact of ex vivo expanded megakaryocyte (MK) progenitors on high-dose chemotherapy-induced thrombocytopenia, we conducted a phase II study in 10 patients with relapsed lymphoma. Two fractions of peripheral blood progenitor cells (PBPC) were cryopreserved, one with enough cells for at least 2 x 10(6) CD34+ cells/kg and a second obtained after CD34+ selection. Ten days before autologous stem cell transplantation, the CD34+ fraction was cultured with MGDF+SCF for 10 days. After BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy, patients were reinfused with standard PBPC and ex vivo expanded cells. No toxicity was observed after reinfusion. The mean fold expansion was 9.27 for nucleated cells, 2 for CD34+ cells, 676 for CD41+ cells, and 627 for CD61+ cells. The median date of platelet transfusion independence was day 8 (range: 7-12). All patients received at least one platelet transfusion. In conclusion, ex vivo expansion of MK progenitors was feasible and safe, but this procedure did not prevent BEAM-induced thrombocytopenia. Future studies will determine if expansion of higher numbers of CD34+ cells towards the MK-differentiation pathway will translate into a functional effect in terms of shortening of BEAM-induced thrombocytopenia.

As microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS-PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.