BACKGROUND

The mouse mammary gland provides a powerful model system for studying processes involved in epithelial tissue development. Although markers that enrich for mammary stem cells and progenitors have been identified, our understanding of the mammary developmental hierarchy remains incomplete.

METHODS

We used the MMTV promoter linked to the reverse tetracycline transactivator to induce H2BGFP expression in the mouse mammary gland. Mammary epithelial cells (MECs) from virgin mice were sorted by flow cytometry for expression of the mammary stem cell/progenitor markers CD24 and CD29, and H2BGFP. Sorted populations were analyzed for in vivo repopulation ability, expression of mammary lineage markers, and differential gene expression.

RESULTS

The reconstituting activity of CD24⁺/CD29⁺ cells in cleared fat pad transplantation assays was not distinguished in GFP⁺ compared to GFP⁻ subpopulations. However, within the CD24⁺/CD29(lo) luminal progenitor-enriched population, H2BGFP⁺, but not H2BGFP⁻, MECs formed mammary structures in transplantation assays; moreover, this activity was dramatically enhanced in pregnant recipients. These outgrowths contained luminal and myoepithelial mammary lineages and produced milk, but lacked the capacity for serial transplantation. Transcriptional microarray analysis revealed that H2BGFP⁺/CD24⁺/CD29(lo) MECs are distinct from H2BGFP⁻/CD24⁺/CD29(lo) MECs and enriched for gene expression signatures with both the stem cell (CD24⁺/CD29⁺) and luminal progenitor (CD24⁺/CD29(lo)/CD61⁺) compartments.

CONCLUSIONS

We have identified a population of MECs containing pregnancy-activated multipotent progenitors that are present in the virgin mammary gland and contribute to the expansion of the mammary gland during pregnancy.

Nitric oxide (NO) can reduce bone loss in chronic bone diseases. NO inhibits or kills osteoclasts, but the mechanism of action of NO in human bone turnover is not clear. To address this, we studied effects of NO on attachment and motility of human osteoclasts on mineralized and tissue culture substrates under defined conditions. Osteoclasts were differentiated in vitro from CD14 selected monocytes in RANKL and CSF-1, and characterized by cathepsin K expression, tartrate-resistant acid phosphatase (TRAP) activity, acid secretion, and lacunar resorption. Cell attachment was labeled with monoclonal antibody 23C6, specific for a binding domain of a key osteoclast attachment protein, the CD51/CD61 integrin dimer (alpha(v)beta(3)), with or without cell permeabilization. A ring of integrin attachment during bone degradation delimits an extracellular acid compartment, while alpha(v)beta(3) forms focal attachments on non-resorbable substrates. On resorbable substrate but not non-resorbable substrate, alpha(v)beta(3) labeling required cell permeabilization, in keeping with the membrane-matrix apposition that excludes large molecules and allows extracellular acidification. Acid secretion was labeled with the fluorescent weak base indicator lysotracker. NO donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), downmodulated acid secretion simultaneously with cytoskeletal rearrangement, with alpha(v)beta(3) redistributed to a discontinuous pattern that labeled, on bone substrate, without membrane permeabilization. These effects were reversible, and an inhibitor of NO synthesis, N(G)-monomethyl-L-arginine (l-NMMA), increased acid secretion and decreased heterogeneity of attachment structures, showing that NO is an autocrine regulator of attachment. A hydrolysis-resistant activating cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate replicated effects of NO donors, while an inhibiting analog, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer, opposed them. On tissue culture or mineralized substrates, NO or cGMP analogs directly regulated motility; after washout cells reattached and survived for days. We conclude that NO is produced by human osteoclasts and regulates acid secretion and cellular motility, in keeping with autocrine and paracrine NO regulation of the resorption cycle.

Homeobox genes encode for regulatory proteins central to hematopoietic differentiation and proliferation. Previously, we identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-DeltaH3). Here, we demonstrate that this mutation results in a significantly truncated protein with impaired DNA-binding efficiency. Electrophoretic mobility shift assays (EMSA) confirm that wild-type HOXA11 (HOXA11-WT) interacts in vitro with the DNA-binding consensus sequence for HOXA11, and that this interaction is most efficient when the TALE transcription factor, Meis1b, is also present. However, the binding between HOXA11-DeltaH3 and DNA is abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA-binding capacity. We investigated whether the point mutation also affected the physical protein-protein interaction between HoxA11 and Meis1b. Using GST pulldown assays, we find Meis1b interactions with both HOXA11-WT and HOXA11-DeltaH3 in the presence of DNA. DNAse treatment decreased these interactions, suggesting that the interaction is a protein-protein association, and DNA may serve to stabilize this interaction. Stable expression of FLAG-HOXA11-WT or -DeltaH3 in K562 cells significantly impacts megakaryocytic differentiation. Staurosporine (STSP) induced K562 cells to differentiate into a megakaryocytic phenotype. Treatment leads to an increase in surface expression of the megakaryocytic/platelet-specific antigen, CD61, and causes morphological changes consistent with megakaryocytic differentiation. CD61 surface expression on STSP treated HOXA11-WT and -DeltaH3 expressing cells was significantly reduced as compared to untransfected K562 cells. Interestingly, we found only a slight difference in CD61 expression between wild-type and mutant HOXA11 K562. These data suggest that HoxA11 inhibition of differentiation may involve nonhomeodomain sequences. Finally, our laboratory has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood and murine ES cell culture cocultured on OP9 for 6 days in the absence of leukemia inhibitory factor (LIF). This finding suggests HoxA11 may be endogenously expressed in very early hematopoietic precursor cells. Taken together, these data begin to give us insight into the molecular mechanisms by which HoxA11 may be involved in regulating megakaryocytic differentiation.

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss.

OBJECTIVE

To test prospectively whether the antiplatelet effect of a 600 mg loading dose of clopidogrel is attenuated in patients receiving atorvastatin and simvastatin for at least 4 weeks prior to coronary artery stenting.

RESULTS

Blood samples were obtained at least 2 h after receiving 100 mg aspirin and 600 mg clopidogrel and prior to coronary stenting from 90 patients without statin therapy and 90 patients with statin (atorvastatin and simvastatin) therapy for at least 4 weeks. Maximal and residual platelet aggregation was evaluated with optical aggregometry in response to ADP (5 and 20 micromol/l). Surface expression of IIb/IIIa (CD61) and P-selectin (CD62) was assessed with whole blood flow-cytometry at baseline and following stimulation (5 and 20 micromol/l ADP). Inhibition of ADP-induced platelet aggregation was not impaired in the presence of concomitant statin therapy. Moreover, patients with and without statin therapy did not differ in respect to all flow-cytometric parameters obtained.

CONCLUSIONS

The antiplatelet effect of a high, 600 mg loading dose of clopidogrel is not diminished in patients receiving atorvastatin and simvastatin for at least 4 weeks prior to coronary stenting.

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 μg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 μmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347-2946 vs. 3061, 446-4901 PMPs/μL; median ad range, n = 9, P < 0.001), collagen 10 μg/mL (5317, 2027-15935 vs. 10252, 4187-46316 PMPs/μL; n = 13, P < 0.001), collagen 1.5 μg/mL (1078, 528-2820 vs. 1465, 582-5948 PMPs/μL; n = 21, P < 0.001) and TRAP-6 (2008, 1621-2495 vs. 2840, 2404-3031 PMPs/μL; n = 3, P < 0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395-2908 vs. 3045, 1119-5494 PMPs/μL, n = 6, P < 0.05), collagen 1.5 μg/mL (1006, 780-1309 vs. 2422, 1839-3494 PMPs/μL, n = 3, P < 0.01) and TRAP-6 (904, 761-1224 vs. 2840, 2404-3031 PMPs/μL, n = 3, P < 0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMPs release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

ADAM15, a member of the ADAM (a disintegrin and metalloprotease) family, is a membrane protein containing both protease and adhesion domains and may, thus, be involved in tumor invasion and metastasis. The aim of this study was to analyze the expression of ADAM15 and its potential ligand, integrin alpha(v)beta3 (CD51/CD61), in lung carcinoma cell lines and tissues. Most small cell lung carcinomas (SCLCs) and non-SCLC cell lines were ADAM15, alpha(v) and beta3 integrin mRNA positive. Half of the cell lines expressed ADAM15, and three expressed the alpha(v)beta3 heterodimer at the cell surface as shown using flow cytometry. Paraffin sections of pulmonary epithelial tumors, including SCLCs (n=26), squamous cell cancer (SCCs, n=27) and adenocarcinomas (ACs, n=17) were stained with antibodies to the ectosolic and cytosolic domain of ADAM15 and alpha(v)beta3 integrin complex. The results were scored (0-12, according to Remmele's score). Normal epithelial cells of the lung were negative or slightly positive for ADAM15 (score<2). The score was always significantly higher for tumor cells. ACs showed the strongest staining (tumor center; ADAM15ecto; mean+/-SEM; 5.47+/-1.04), whereas SCLCs only showed weak ADAM15 expression (2.67+/-0.42; SCCs: 3.62+/-0.62). Frequently, significantly stronger ADAM15 expression has been shown in tumor cells located at the front of invasion compared with those within solid formations. Overall analysis of all tumor specimens and each tumor type revealed no significant correlation between tumor stage or degree of differentiation and ADAM15 ectosolic or cytosolic domain expression in tumor cells. Both molecules are often co-localized in the same tumor cells in ADAM15- and alpha(v)beta3 integrin-positive carcinomas. In summary, lung carcinoma cell lines and tissues were frequently ADAM15 positive.

Two monoclonal antibodies, F4 and F11, were raised to newborn rat bone cell suspensions. These antibodies are shown by immunocytochemistry on tissue sections to recognize an antigen shared between osteoclasts, megakaryocytes, and platelets. Immunoprecipitation analysis of the antigen from C6 rat glial cells followed by SDS-PAGE showed a heterodimeric molecule with a characteristic integrin-like shift in apparent molecular mass upon reduction (137/78 kD nonreduced; 118/100 kD reduced); the low-molecular-mass band comigrates with the beta 3 subunit precipitated with polyclonal antihuman vitronectin receptor antiserum, and the high-molecular-mass band comigrates with the alpha v subunit precipitated with a polyclonal antiserum to a C-terminal amino acid sequence of human alpha v. Antibody F4 strongly cross-reacts with human cells and is shown in cross-blocking experiments and immunoprecipitation analysis with a human melanoma cell line DX3 to recognize a seemingly identical molecule as identified by anti-alpha v beta 3 monoclonal antibody 23C6. Expression of F4 and F11 is reduced in platelets from a patient heterozygous for Glanzmann's thrombasthenia. Taken together, these results indicate that F4 and F11 recognize rat CD61, the integrin beta 3 chain, which, as was confirmed with polyclonal anti CD61 antisera, is highly expressed in rat osteoclasts. These antibodies may be useful tools in investigating the biochemical nature and biologic function of beta 3 integrins in rat osteoclasts. Additionally, because high expression of beta 3 in vivo is restricted to osteoclasts, megakaryocytes, and platelets, these antibodies may be used to help identify osteoclasts in tissue sections and bone cell suspensions.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated T cells acquire endothelial cell (EC) plasma membrane constituents during transendothelial migration. This was assessed using an in vitro model system in which human peripheral blood CD4+ T cells migrated through confluent monolayers of HUVEC. Flow cytometry of migrated CD4+ T cells demonstrated that activated, but not resting, T cells acquired a variety of endothelial surface determinants, including CD31, CD49d, CD54, CD61, and CD62E. The extracellular domains of these molecules were detected on migrated T cells with mAbs, including those directed to the ligand-binding regions. A number of approaches were employed to document that the acquisition of these molecules was uniquely accomplished by activated T cells and clearly involved transfer from both resting and TNF-alpha-activated EC. Acquisition of endothelial markers by activated T cells occurred as part of the transfer of membrane components, as migrating T cells acquired EC membranes prelabeled with the lipophilic dye, 3,3'-dihexadecyloxacarbocyanine perchlorate (DiOC-16), along with EC surface proteins. Thus, during transendothelial migration, activated T cells acquire endothelial membrane components, and as a result may deliver them to perivascular sites.

Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.

OBJECTIVE

To comparatively study the expressive conditions of platelet activation related factors (GP I b, GP II b- III a and GMP-140) in healthy subjects and patients with coronary heart disease (CHD) of blood-stasis (BS) or non-blood-stasis (non-BS) syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes.

METHODS

With case control design adopted, patients with the CHD (40 of BS, 37 of non-BS) and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity (MFI) of GP I b, GP II b- III a, and GMP-140 (CD42b, CD61, CD62p) in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing.

RESULTS

MFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome (P<0.05); MFI of CD42b was lower in the CHD patients than in the healthy control (P<0.05), but showing insignificant difference between BS and non-BS syndrome (P>0.05); at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GP II b HPA-3 and GP I b HPA-2 polymorphism loci (P>0.05).

CONCLUSIONS

(1) The activities of GP II b- III a and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. (2) The level of GP I b was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. (3) Levels of GP II b- III a, GP I b and GMP-140 were not related with the number of affected coronary branches in CHD patients. (4) The changes in amino-acids expression induced by the two loci brought no significant influence on GP I b and GP II b- III a activities.

Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS-145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/IIIa blockers.

Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24(+)CD61(+)CD29(hi), CD24(+)CD61(+)CD29(lo), and CD24(+)CD61(-)CD29(lo) cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFβ signaling as key regulators of CD24(+)CD61(+)CD29(lo) cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells.

To determine whether cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes, we evaluated the expression of a cell surface marker panel in breast cancer cell cultures derived from various subtypes of human breast carcinoma. We used a four-color flow cytometry strategy to immunophenotype seven human breast cancer cell cultures and four reference breast cancer cell lines. We analyzed 28 surface markers selected based on their potential to distinguish epithelial or mesenchymal lineage, to identify stem cell populations, and to mediate cell adhesion and migration. We determined their ability to form mammospheres and analyzed luminal cytokeratins CK18, CK19, and myoepithelial/basal CK5, SMA (alpha-smooth muscle actin), and vimentin expression by western blot. All cell surface markers showed a unimodal profile. Ten/28 markers were homogenously expressed. Four (CD66b, CD66c, CD165, CD324) displayed negative/low expression. Six (CD29, CD55, CD59, CD81, CD151, CD166) displayed homogenous high expression. Eighteen (CD9, CD10, CD24, CD26, CD44, CD47, CD49b, CD49f, CD54, CD61, CD90, CD105, CD133, CD164, CD184, CD200, CD227, CD326) were heterogeneously expressed. Spearman's rank test demonstrated a significant correlation (p< 0.001) between mesenchymal phenotype and breast cancer cell cultures. Breast cancer cell cultures, all CD44+, displayed concomitant high expression of only three antigens (CD10, CD54, CD90), and low expression of CD326; cell cultures formed mammospheres and expressed CK5, SMA and vimentin, and were weakly CK19-positive. We demonstrate that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry. Therefore, cell cultures can be used for in vitro modeling of breast cancer subtypes; immunophenotyping may mirror breast cancer heterogeneity and reveal molecular characteristics of individual tumors useful for testing target therapy.

beta(3) Integrin has been identified as a cellular receptor for Hantaan virus, which causes hemorrhagic fever with renal syndrome (HFRS). To investigate the relationship between intensity of the platelet membrane beta(3) integrin (CD61) and disease severity, the percentage of CD61-positive platelets and the mean fluorescence intensities (MFI) of platelet CD61 were determined in patients with HFRS by flow cytometry. The intensity levels of CD61 in patients with HFRS were significantly higher than those in the controls and correlated with the clinical phases of the disease. The CD61 intensity at the oliguric phase was inversely correlated with platelet count and serum albumin, and positively correlated with white blood cell count, blood urea nitrogen, serum creatinine, and alanine aminotransferase levels. The results suggest that the intensity levels of platelet CD61 were elevated and associated with clinical phases and disease severity in patients with HFRS, and the intensity of platelet beta(3) integrin in patients with HFRS may be indicative of disease severity.

OBJECTIVE

The aims of our study were to determine the basal platelet activation state in women with preeclampsia compared with normotensive pregnant women and nonpregnant women and to investigate the platelet reactivity on in vitro stimulation with adenosine diphosphate or thrombin receptor activation peptide.

METHODS

Platelet expression of CD61 (fibrinogen receptor), CD42a (von Willebrand factor receptor), CD62P (P-selectin), CD63 (Glycoprotein 53), and PAC-1 binding (activated fibrinogen receptor) were determined in 20 pairs of women with preeclampsia/normotensive pregnant women and in 12 nonpregnant women, with the use of flow cytometry.

RESULTS

Basal platelet expression of CD61, CD42a and CD62P, and adenosine diphosphate-stimulated CD62P expression were increased in women with preeclampsia compared with normotensive pregnant women. Platelets from women with preeclampsia and normotensive pregnant women differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation, which was demonstrated by increased expression of CD61, CD62P, and CD63.

CONCLUSIONS

This study supports the notion that platelets are important in the pathophysiologic condition of preeclampsia.

OBJECTIVE

A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations.

METHODS

Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice.

RESULTS

The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice.

CONCLUSIONS

In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Most patients who became critically ill following infection with COVID-19 develop severe acute respiratory syndrome (SARS) attributed to a maladaptive or inadequate immune response. The complement system is an important component of the innate immune system that is involved in the opsonization of viruses but also in triggering further immune cell responses. Complement activation was seen in plasma adsorber material that clogged during the treatment of critically ill patients with COVID-19. Apart from the lung, the kidney is the second most common organ affected by COVID-19. Using immunohistochemistry for complement factors C1q, MASP-2, C3c, C3d, C4d, and C5b-9 we investigated the involvement of the complement system in six kidney biopsies with acute kidney failure in different clinical settings and three kidneys from autopsy material of patients with COVID-19. Renal tissue was analyzed for signs of renal injury by detection of thrombus formation using CD61, endothelial cell rarefaction using the marker E-26 transformation specific-related gene (ERG-) and proliferation using proliferating cell nuclear antigen (PCNA)-staining. SARS-CoV-2 was detected by in situ hybridization and immunohistochemistry. Biopsies from patients with hemolytic uremic syndrome (HUS, n = 5), severe acute tubular injury (ATI, n = 7), zero biopsies with disseminated intravascular coagulation (DIC, n = 7) and 1 year protocol biopsies from renal transplants (Ctrl, n = 7) served as controls. In the material clogging plasma adsorbers used for extracorporeal therapy of patients with COVID-19 C3 was the dominant protein but collectin 11 and MASP-2 were also identified. SARS-CoV-2 was sporadically present in varying numbers in some biopsies from patients with COVID-19. The highest frequency of CD61-positive platelets was found in peritubular capillaries and arteries of COVID-19 infected renal specimens as compared to all controls. Apart from COVID-19 specimens, MASP-2 was detected in glomeruli with DIC and ATI. In contrast, the classical pathway (i.e. C1q) was hardly seen in COVID-19 biopsies. Both C3 cleavage products C3c and C3d were strongly detected in renal arteries but also occurs in glomerular capillaries of COVID-19 biopsies, while tubular C3d was stronger than C3c in biopsies from COVID-19 patients. The membrane attack complex C5b-9, demonstrating terminal pathway activation, was predominantly deposited in COVID-19 biopsies in peritubular capillaries, renal arterioles, and tubular basement membrane with similar or even higher frequency compared to controls. In conclusion, various complement pathways were activated in COVID-19 kidneys, the lectin pathway mainly in peritubular capillaries and in part the classical pathway in renal arteries whereas the alternative pathway seem to be crucial for tubular complement activation. Therefore, activation of the complement system might be involved in the worsening of renal injury. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury.

Keywords: COVID-19; complement—immunological term; endothelial injury; kidney; lectin pathway activation.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. In this study, the MDSC population was evaluated in adult patients with primary immune thrombocytopenia (ITP), where cell-mediated immune mechanisms are involved in platelet destruction. Our data demonstrated that both the numbers and suppressive functions of MDSCs were impaired in the peripheral blood and spleens of patients with ITP compared with healthy control patients. High-dose dexamethasone (HD-DXM) treatment rescued MDSC numbers in patients with ITP. And DXM modulation promoted the suppressive function of MDSCs induced in vitro. Moreover, the expression of interleukin 10 and transforming growth factor β was significantly upregulated in DXM-modulated MDSCs compared with the unmodulated cultures. DXM-modulated MDSCs inhibited autologous CD4(+)T-cell proliferation and significantly attenuated cytotoxic T lymphocyte-mediated platelet lysis, further indicating enhanced control over T-cell responses. Elevated expression of the transcription factor Ets1 was identified in DXM-modulated MDSCs. Transfection of Ets-1 small interfering RNA efficiently blocked regulatory effects of MDSCs, which almost offset the augmentation of MDSC function by DXM. Meanwhile, splenocytes from CD61 knockout mice immunized with CD61(+)platelets were transferred into severe combined immunodeficient (SCID) mouse recipients (C57/B6 background) to induce a murine model of severe ITP. We passively transferred the DXM-modulated MDSCs induced from bone marrow of wild-type C57/B6 mice into the SCID mouse recipients, which significantly increased platelet counts in vivo compared with those receiving splenocyte engraftment alone. These findings suggested that impaired MDSCs are involved in the pathogenesis of ITP, and that HD-DXM corrected MDSC functions via a mechanism underlying glucocorticoid action and Ets1.

OBJECTIVE

To elucidate the mechanisms of thrombocytopenia in alcoholic liver diseases, we investigated activation status of platelets in patients with alcoholic fatty liver (Al-FL), alcoholic liver cirrhosis (Al-LC) or hepatitis-C liver cirrhosis C (C-LC).

METHODS

Platelet activation was evaluated by flow cytometry using monoclonal antibodies against P-selectin (CD62P) and the fibrinogen receptor (PAC-1), both specific for platelet activation, and anti-CD61 antibody for the presence of microparticles (PMP) in seven patients with Al-FL, thirteen patients with Al-LC and, as a non-alcoholic liver disease control, nine patients with C-LC. As a normal control, seventeen healthy subjects without liver dysfunction were also evaluated.

RESULTS

Compared with the healthy controls, the platelet count was significantly decreased in patients with alcoholic liver diseases or C-LC. Ten days after discontinuation of alcohol intake, the platelet count was significantly higher in both the Al-FL and Al-LC groups than that measured on admission. There was an inverse correlation between the platelet count and PMP, a marker of platelet activation. The Al-FL, Al-LC and C-LC groups showed significantly higher percentages of platelets positive for CD62P than the healthy controls. The PAC-1 positivity was increased only in the C-LC group. PMP were significantly increased in the Al-FL, Al-LC and C-LC groups compared to that in the healthy group. In the Al-LC group, PMP were significantly decreased 10 days after discontinuation of alcohol intake from that measured on admission.

CONCLUSIONS

Patients with alcoholic liver diseases have increased platelet activation, which may contribute to the occurrence of thrombocytopenia. The formation of PMP might be one of the important factors of thrombocytopenia in alcoholic liver diseases.

Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60-966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-β1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-β1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

Immunophenotypic analysis of transient myeloproliferative disorder (TMD) and acute myeloid leukemia (AML) using multiparameter flow cytometry might provide insight into their relationship. We retrospectively analyzed the expression of multiple lymphoid, myelomonocytic, and megakaryocytic antigens on blast proliferations in 18 patients with Down syndrome (DS; AML, 9; TMD, 9). The AMLs and TMDs shared several immunophenotypic characteristics. Blasts in all expressed CD45, CD38, and CD33; most AMLs and all TMDs were CD36+; and the majority expressed CD41 and CD61, suggesting megakaryocytic differentiation. The majority of cases were CD34+, CD14-, and CD64-. There was aberrant expression of the T-cell-associated antigen CD7 in most AMLs and TMDs. CD56 was expressed aberrantly in 5 AMLs and 7 TMDs. The major difference between the disorders was the pattern of expression of myeloid markers CD11b and CD13; each was expressed in 8 AMLs but only 2 TMDs. Blasts were HLA-DR-positive in 3 AMLs vs 7 TMDs. Blasts in TMD and AML in DS have a characteristic immunophenotype distinct from AML in other settings. The immunophenotypic similarities suggest a biologic relationship between the disorders; however, distinct immunophenotypic differences also were observed.

We have previously demonstrated that immune platelet destruction observed in an AIDS-free HIV-infected patient was associated with the presence of a cross-reactive antibody recognizing both HIV-glycoprotein (gp)120 and platelet gpIIIa (CD61). We have now investigated the presence of such antibodies in other HIV-infected patients, together with the molecular structure of the cross-reactive epitope. Platelet gpIIb/IIIa antibodies were characterized in sera from HIV-infected patients with immune thrombocytopenic purpura by means of an ELISA and a radio-immunoprecipitation procedure (RIP). The platelet antibodies were purified and tested for their ability to recognize HIV-gp. We also tried to characterize the antibody target epitope on HIV-gp120 using recombinant gp and synthetic peptides. IgG with anti-gpIIb/IIIa activity were detected, by means of an ELISA with purified gpIIb/IIIa, in 101/138 (73%) sera from HIV-infected patients with immune thrombocytopenic purpura. The platelet antibodies were purified from 23 sera by absorption/elution on purified immobilized platelet gpIIb/IIIa, and recognition of gpIIIa was confirmed in eight cases with a RIP. Furthermore, the presence of a cross-reactive antibody between HIV-gp120 and platelet gpIIIa was demonstrated in 18/18 patients (including the eight with a confirmed gpIIIa antibody) by the ability of the serum HIV-gp160/120 antibodies to bind to purified gpIIb/IIIa. The cross-reactive epitope was shown to be independent of the carbohydrate moieties of gp120, since deglycosylation of two recombinant (r)-gp120s did not abolish antibody binding. However, the antibody did not recognize synthetic gp120 peptides spanning 355 of the 516 amino acids of gp120, particularly the four regions exhibiting sequences of four or five consecutive amino acids that are identical between r-gp120 and gpIIIa. Our results thus support the hypothesis that the cross-reactive antibody recognizes the conformational structure of gp120. These results strongly suggest that molecular mimicry between HIV-gp120 and platelet gpIIIa may be important in the pathogenesis of immune thrombocytopenia in AIDS-free HIV-infected patients.