Zebrafish blastomere screen identifies retinoic acid suppression of MYB in adenoid cystic carcinoma.

Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target MYB-driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of c-myb expression. Retinoic acid treatment also decreased c-myb gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient-derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.

Preimplantation High-Resolution HLA Sequencing Using Next Generation Sequencing.

Hematopoietic stem cell transplantation (SCT) is the only therapeutic option in a number of heritable hematologic disorders and hematologic cancers. Many parents and families fail to find an HLA-identical donor for their affected family member. In such cases, conceiving for a "savior baby" remains the only option, especially in countries without access to national registries. By means of next generation sequencing (NGS) techniques, in a single experiment on single-cell products of in vitro fertilization, a healthy HLA-identical embryo can be implanted in the uterus of a concerned mother. The patient can therefore benefit from cord blood SCT along with confirming that the fetuses are not suffering from the heritable disorder. This study is an attempt to study the feasibility of preimplantation HLA sequencing on single blastomeres using NGS. Two couples who had previously undergone preimplantation genetic diagnosis of ß-thalassemia and their overall 10 embryos were studied and their 5 HLA loci were typed in high resolution through multiple displacement amplification and NGS of single cells. For 88.9% of the 90 HLA alleles, conclusive HLA typing in 4 digit sets was made. HLA alleles were typed; 1 ambiguity in the allelic group and 4 ambiguities in the protein level were observed that were then unraveled by haplotype analysis. Amplification efficiency was 93.3% with an allele drop-out (ADO) rate of 22.2% (6 alleles dropped from a maximum of 27 possible ADOs). In this study the feasibility of a new method of preimplantation HLA sequencing via combining the state-of-the-art techniques used in single-cell whole genome amplification, preimplantation genetic diagnosis, and high-resolution HLA typing by NGS has been shown. This method can make preimplantation HLA sequencing a practicable technique in families desperate for an HLA-matched donor.

Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

[Cytokines in acute myeloid leukemia]. / Cytokiny w ostrej bialaczce szpikowej.

The cytokines belong to the group of main factors regulating of leukaemia cell proliferation. Most of information comprised in the literature concern the behaviour of single cytokines in the cell culture in vitro. In the organism of leukaemia patient many different cytokines, adhesion molecules, growth factors and other substances probably act in the same time. Therefore the aim of study was the determination of plasma concentrations of interleukin 1B, 3, 4, 6, 8, G-CSF and P-selection in 26 patients with acute myeloblastic leukaemia-aml, among them 14 patient in the course of exacerbation-aml-e and 12 being in the remission-aml-r, classified as type M1 and M2 according to the FAB classification. Control group consisted of 15 healthy volunteers. The cytokine measurements were performed by means of immunoradiometric, immunoenzymatic and radioimmunologic kits. In the patients with aml-e significant increase of plasma concentrations of IL-1B, IL-3, IL-6, G-CSF, P-selectin and essential decrease of IL-4 was found. Significant decrease of IL-4 and P-selecting concentrations in the patients with aml-r and lack of changes in IL-8 concentrations in the both groups of patients was demonstrated. The observed differences of concentrations may additionally confirm the role of studied cytokines and P-selectin in the regulation of leukaemia cell proliferation.

Expression of interleukin-1 system mRNA in single blastomeres from human preimplantation embryos.

Gathering knowledge about the molecular events during preimplantation development is one of the most important challenges in in-vitro fertilization (IVF). The interleukin-1 (IL-1) system has been shown to be intimately involved in embryonic implantation. The aim of our study was to detect the major components of the IL-1 system in single blastomeres from human preimplantation embryos and to relate our findings to the further development of the biopsied embryos in vitro. Single blastomeres were removed from morphologically normal embryos obtained from dipronuclear zygotes and examined by reverse transcription (RT)-nested polymerase chain reaction (PCR). Expression of beta-actin (external standard), IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-1 receptor (IL-1R) type I mRNA were related to embryonic development and IVF outcome. Blastomeres from 12 embryos were examined: beta-actin and IL-1R type I mRNA were detected in all blastomeres (100%) whereas IL-1beta could be detected in only nine of the blastomeres (75%). IL-1ra was expressed in only two (17%) of the blastomeres and those were simultaneously positive for IL-1beta. Both IL-1ra positive embryos were arrested in development before reaching blastocyst stage. Five embryos (three of them IL-1beta mRNA positive and two IL-1beta mRNA negative) were transferred as blastocysts; none of the transfers resulted in a pregnancy. We postulate that embryos expressing IL-1ra mRNA in a detectable amount appear more likely to be arrested in early developmental stages.

Management of rhesus isoimmunization by preimplantation genetic diagnosis.

A genetic assay by single blastomere analysis was developed for rhesus (RhD) blood group typing of early cleavage stage embryos. The method, which is based on the simultaneous amplification of an RhD-specific sequence and an internal control in single cells, was applied for the selective transfer of RhD-negative embryos in a family of an RhD sensitized woman and a heterozygote partner. The RhD status of two out of three biopsied embryos was determined. According to their amplified products, both were typed as RhD-negative and transferred to the uterus. Pregnancy was not achieved.

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

[Post mortem HLA phenotyping of donors of eyes. Evaluation of a serological method of microlymphotoxicity on lymphocytes and PHA lymphoblasts]. / Phénotypage HLA post mortem des donneurs d'yeux. Evaluation d'une méthode sérologique de microlymphocytotoxicité sur lymphocytes et lymphoblastes PHA.

To increase the number of HLA typed corneas a microlymphocytotoxicity assay on lymphocytes and PHA lymphoblasts was investigated. A double immunofluorescence technique using magnetic beads coated with anti-T8 (for class I) and anti-DR (for class II) monomorphic antibodies was applied. Blood samples from 50 non selected donors were obtained. HLA class I and class II typing was possible in 74% of the cases. Using PBL's (Peripheral Blood Lymphocytes) HLA class I could be defined in 29 out of 50 Cases and class II in 15 out of 50 cases. On PHA blasts HLA class I and class II antigens could be defined in 32 and 33 out of 50 cases respectively. Mean time of culture was 10 days (6-20). No influence of donor age and post partum time could be observed. By combination of both methods a significant proportion of eye donors could be reliably typed within a short time.

The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres.

A detailed investigation into the activity of the homotypic, Ca2+-dependent cell-cell adhesion system (CDS) in the early mouse embryo has revealed its involvement in the synchronizing of the time of polarization of 8-cell blastomeres, and the orienting of the axis of polarization. Since polarization marks an important and early event in the process of cell diversification in the mouse embryo, it is concluded that the CDS provides an important component of the system by which the temporal and spatial elements of normal development are integrated.

Changes in the distribution of membranous organelles during mouse early development.

The unfertilized oocyte, fertilized egg and early embryo (2-cell to 16-cell) of the mouse have been examined immunocytochemically for the distribution of antigens associated with the endoplasmic reticulum, the lysosomal and acidic vesicle fraction (100 kD antigen), Golgi apparatus (135 kD antigen) and coated vesicles (clathrin). The distribution of these antigens has also been examined in isolated 8-cell and 16-cell-stage blastomeres of various ages and phenotypes. Endoplasmic reticulum is detected only weakly in the oocyte and egg, but is seen abundantly at later stages both in association with the nuclear membrane and evenly distributed throughout the cytoplasm, except in regions of cell:cell apposition from which it is excluded. Intracellular clathrin is associated with the spindle in mitotic and meiotic cells. During interphase, clathrin is distributed throughout the cell until the mid-8-cell stage when it is concentrated into the apical region of the cell under the region of membrane at which a surface pole of microvilli will form subsequently. Thus, the cytoplasmic polarization of clathrin precedes overt polarization at the surface. At mitosis, the clathrin relocates to the spindle and is distributed to both daughter cells. It resumes an apical location beneath the surface pole of microvilli in polar daughter 1/16 cells, but remains dispersed in apolar daughter 1/16 cells. Both the lysosomal and Golgi antigens are distributed throughout the cytoplasm until the early 16-cell stage. In pairs of 16-cell blastomeres both antigens aggregate in a single cluster and do so whether the surface phenotype of the blastomeres is polar or apolar. The position of this cluster is not consistently related to the point of contact with the other cell in the pair but there is a suggestion that in cells with a polar surface phenotype the polar foci of Golgi/lysosomal antigens are located between the nucleus and the surface pole at earlier time points, but shift to a position between the basolateral membrane and the nucleus at the later time point. In intact 16-cell embryos also, the aggregated Golgi/lysosomal antigens of polar cells appear to localize to the basal region. The distributions of these various organelles in embryonic cells reported here show a number of differences from those reported previously for mature, differentiated cells.

Therapy-related leukemia associated with myelofibrosis. Blast cell characterization in six cases.

Six patients exhibiting severe pancytopenia or overt leukemia associated with myelofibrosis after chemotherapy for malignant disease have been investigated by immunologic techniques and ultrastructural cytochemistry. Initially, five patients displayed severe thrombocytopenia contrasting with mild neutropenia and anemia. Bone marrow biopsies showed a clear megakaryocytic proliferation and an excess of immature mononuclear cells. The demonstration of peroxidase activities at the ultrastructural level and immunofluorescence labeling with a panel of monoclonal antibodies, including an antiplatelet glycoprotein Ib and an antiglycoprotein IIb-IIIa complex, on blood or marrow cells, permitted identification of otherwise unidentifiable promegakaryoblastic proliferation. In two patients, the use of an immunoperoxidase technique with an antifactor VIII-R-Ag antibody has allowed direct confirmation of this diagnosis on bone marrow sections. This megakaryoblastic proliferation was not pure and was variably associated with blasts of other cell lines (erythroblasts or myeloblasts). Changes in the population of blasts were observed during evolution in two patients. The sixth patient had a mild thrombocytopenia associated with severe neutropenia and anemia. Bone marrow biopsy displayed a myelofibrosis and immature cells, without megakaryocytic proliferation. Ultrastructural study revealed a pure basophil-mast cell proliferation. In conclusion, in five of six patients with secondary acute leukemia associated with myelofibrosis, a proliferation of promegakaryoblasts was demonstrated using both immunofluorescent and ultrastructural cytochemical techniques.

Decreased macrophage-mediated suppression of lymphocyte activation in chronic renal failure.

Prostaglandin-dependent adherent cell suppressor activity was assessed in patients with end-stage renal insufficiency. Proliferative responses of uremic peripheral blood mononuclear cells to optimal concentrations of phytohemagglutinin and concanavalin A were impaired. Responses to the galactosyl-directed lectins, soybean agglutinin and peanut agglutinin, were, however, normal or supranormal. The addition of 1 microgram/ml of indomethacin, to cell cultures resulted in relatively less potentiation of blastogenic responses to the galactosyl-directed lectins in cells from uremic patients (soybean agglutinin, p less than 0.02; peanut agglutinin, p less than 0.05). Similarly, depletion of adherent cells markedly enhanced blastogenesis induced by the galactosyl-directed lectins in normal cell cultures, whereas the effect was much less pronounced (soybean agglutinin, p less than 0.02; peanut agglutinin, p less than 0.02) in uremic cells. Reduced activity of the adherent cell suppressor system in patients with renal failure might be associated with altered sensitivity of uremic lymphocytes to soluble mediators of suppression. The lymphocytes of uremic patients, depleted of adherent cells, were relatively resistant to the inhibitory action of prostaglandin E1 (0.001 microgram/ml, p less than 0.05, and 0.01 microgram/ml, p less than 0.02) on galactosyl-directed, lectin-induced mitogenesis. In contrast, dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-5) M), and 3-isobutyl-1-methyl xanthine (20 micrograms/ml) inhibited both control subject and patient cultures to the same extent. Prostaglandin E1 in combination with methyl isobutyl xanthine produced, in adherent-cell-depleted control subjects, levels of cyclic AMP that were significantly higher than in cells from uremic patients (p less than 0.05). Thus, depressed adherent cell suppressor activity in patients with renal failure may result in part from impaired generation of cyclic AMP by lymphocytes.

Cosegregation of monoclonal antibody reactivity and cell behaviour in the mouse preimplantation embryo.

Expression of an antigen, recognized by a monoclonal antibody raised against PC13 embryonal carcinoma, is described in mouse preimplantation embryogenesis. The antigen is found in the cytoplasm of ovulated ova and is first noted on the cell surface of the 1-cell embryo 20 h post-ovulation. Surface labelling of blastomeres is uniform until the 8-cell stage when antigen expression becomes polarized along the radial axis of the embryo. Two major populations of blastomeres are distinguishable on division to the 16-cell morula. Dissociation of morulae in calcium-free medium yields large, polar, antigen-positive cells and small apolar cells with reduced levels of detectable antigen. A third, minor population of small, antigen-negative cells is also found in vivo. Large and small blastomeres differ in their ability to relocate within the embryo when aggregated with intact 16-cell-stage embryos. The small blastomeres of the 16-cell morula contribute significantly to the inner cell mass while the large antigen-positive cells are found only in the trophectoderm.

[Angioimmunoblastic lymphadenopathy]. / Angioimmunoblastos lymphadenopathia.

Cytological, histological and ultrastructural characteristics of a cose of an extranodal and nodal angioimmunoblastic lymphadenopathy persisting during several years are given. Angiomatous proliferation in the case reported seemed to be identical both in the lymph node and in the cutaneous infiltration. Types of cells seen in the angioimmunablastic lymphadenopathy did not differ from those of other immunoreactive processes. In the extranodal infiltrates in addition to other phenomena thickening of the immunological techniques decrease of the natural cytotoxicity and the number of T-lymphocytes could be observed.

Distribution of antibody- and lectin-binding sites on dissociated blastomeres from mouse morulae: evidence for polarization at compaction.

The distribution of binding sites for rabbit anti-species antiserum, Concanavalin A (Con A) and peanut agglutinin (PNA) on dissociated blastomeres from 2- to 16-cell mouse embryos has been investigated using direct and indirect fluorescence techniques. With each ligand, paraformaldehyde-fixed blastomeres from 2- to 8-cell precompact embryos were uniformly surface labelled; the majority (77%) of late compact 8-cell blastomeres showed quantitative polarization of surface labelling; and 16-cell blastomeres were either polarized (53.3%) or uniformly surface labelled. Binding of fluorescein-conjugated PNA increased at the 16 cell stage. Labelling patterns on unfixed blastomeres were similar to those on fixed blastomeres except that surface label was patched and became internalized, most rapidly from the less heavily labelled areas of 8- and 16-cell blastomeres. Quantitative polarization of binding sites at postcompaction stages was detected after (i) fixation, (ii) pretreatment and labelling in the presence of azide, cytochalasin D and/or colcemid, or (iii) labelling with monovalent Fab1 antibody fragments. It is probably due, therefore, to the presence of microvilli at the heavily labelled pole, which increase surface area and are known to become to the outer surface of the compact morula (Ducibella, Ukena, Karnovsky & Anderson, 1977). The possibility that the cleavage of polarized blastomeres into dissimilar daughter blastomeres could provide a mechanism for the spatial differentiation of the inner cell mass and trophectoderm of the blastocyst is briefly discussed.

Studies in acute leukemia. II. Chemotaxis of leukemic blasts from patients with acute nonlymphoid leukemia: its relationship with IgG Fc receptors and role of surface-bound immunoglobulin.

Leukemic blasts from patients with acute myeloblastic and acute monocytic leukemia were studied for their chemotaxis towards casein- or endotoxin-activated serum as well as for the presence of Fc receptors and surface immunoglobulins. Leukemic blasts from patients with acute myeloblastic leukemia lacked both Fc receptors and surface immunoglobulin and did not migrate towards chemoattractants. Leukemic blasts from patients with acute monocytic leukemia, however, demonstrated heterogeneity with regard to Fc receptors, surface immunoglobulins and chemotaxis, and could be divided into three groups: in group I, leukemic blasts which lacked both Fc receptors and surface immunoglobulin moved poorly; in group II, large proportions of leukemic blasts and Fc receptors but lacked surface immunoglobulins. These blasts migrated well towards the chemoattractants, and in group III, leukemic blasts which had both Fc receptors and surface immunoglobulins lacked chemotactic property. A correlation between the presence or absence of Fc receptors and chemotaxis was observed. In group III, lack of blast cell migration appears to be due to passive binding of IgG via the Fc receptors.

Aberrant blastogenic response to LPS in experimental gingivitis of elderly subjects.

The blastogenic response of peripheral blood leukocytes to lipopolysaccharide (LPS), phytohemagglutinin (PHA) and LPS/PHA mixtures was followed over a short course of experimental gingivitis in elderly subjects (65-81 years) who strictly avoided oral hygiene procedures for periods up to 9 d. The leukocytes responded poorly to LPS, PHA and to LPS/PHA combinations. The concomitant heightened sensitivity of the gingiva to dental plaque among the elderly subjects may relate to the altered leukocyte response in this age group.