Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl- phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC).     

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c- derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid- specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

Cytogenetic and histologic correlations in malignant lymphoma

Although a number of studies have indicated correlations between histologic subtypes of tumors and certain nonrandom chromosome changes, cytogenetic studies of lymphoma are in an early stage compared to those of leukemia. No comprehensive analysis of available data has so far been attempted in the literature either. Here we present an analysis of chromosome changes and their correlation with subtypes of lymphoma studied by conventional histology and cell surface markers, as observed in two sets of data: a group of 65 karyotypically abnormal tumors sequentially ascertained and studied by us during the period January 1, 1984 to April 30, 1985, and a larger data set derived by combining our data with those from two published series from the University of Minnesota that are comparable to our data. These combined data, which comprise the largest data set on the cytogenetics of lymphomas assembled so far, enabled a comprehensive analysis of correlation between chromosome change and tumor histology and the patterns of chromosome instability in these tumors. We found several significant associations, some previously described and others now recognized, between nonrandom chromosome gains, breaks, translocations, and deletions and histologic subtypes of tumors that characterize lymphomas. The data indicate that finding of chromosome breaks at certain sites (eg, 8q24, 14q32, 18q21) is of diagnostic value in dealing with cases of unusual lymphoma. Furthermore, nonrandom chromosome breakage exhibited three distinct patterns that reflected three levels of etiologically relevant genetic change.     

Effects of interleukin-11 on the proliferation and cell cycle status of myeloid leukemic cells

Interleukin-11 (IL-11) is a pleiotropic cytokine with effects on many different targets. Within the hematopoietic system, the effects of IL- 11 are largely manifest only through combination with other cytokines, including IL-3 and Steel factor (SF). In the present study, we addressed the question of IL-11 responsiveness within the different types of human leukemic cells, as well as the mechanism of action of IL- 11 at the cellular level. Analysis of a panel of samples from different patients with acute myeloblastic leukemia (AML) and myeloid leukemic cell lines indicated that IL-11 alone was ineffective in supporting myeloid leukemic cell growth but frequently enhanced growth supported by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or SF. In contrast, three acute pre-B lymphocytic leukemia (pre-B-ALL) and two acute T lymphocytic leukemia (T-ALL) lines failed to respond to IL- 11 alone or when combined with other cytokines. The growth enhancement of IL-11 among the AML patient samples was dose dependent and remarkably constant with half-efficient concentrations in the range of 0.3 to 0.4 ng/mL. The thymidine suicide studies with the patient samples revealed that 40% to 50% of the blast cells were in S-phase when exposed for 16 hours to IL-3 and this level was increased to 70% to 90% in response to either IL-11 or IL-6. Our data suggest that the latter two interleukins act synergistically with the direct mitogenic factor, IL-3, in triggering AML blast-cell proliferation. Detailed analysis with several patient samples further revealed that SF and IL- 11 both enhance IL-3-supported clonogenic growth of AML blasts and the combination of all three growth factors yields optimal growth. In contrast, IL-6 does not further enhance the effect of IL-11. These results indicate that SF and IL-11 enhance IL-3-dependent clonogenic growth through two distinct pathways, whereas IL-6 and IL-11 may trigger the same pathway.     

Efficacy of Human Chorion Membrane Allograft for Recession Coverage: A Case Series

Background : Membranes of human placentas have been used in the field of medicine for skin grafts, treatment of burns, and ulcerated skin conditions with great success. The use of placenta allografts in dentistry is a more recent development, with the first commercial product being made available in 2008. The unique inherent biologic properties in placenta allografts enhance wound healing and may propagate regeneration. Methods: Ten healthy adult patients presenting with 21 Miller Class I gingival recession (GR) defects (isolated or adjacent multiple) were surgically treated with a modified coronally advanced flap and chorion membrane for root coverage. Clinical parameters measured at baseline, 3 months, and 6 months were probing depth, clinical attachment level, GR height, width of keratinized gingiva, and assessment of gingival biotype. Statistical analysis was performed to compare the treatment outcomes at the follow‐up intervals. Results: The results showed statistically significant (P <0.001) improvements in all clinical parameters at the 3‐ and 6‐month follow‐ups. The mean percentage of root coverage at the end of 6 months was 89.92% ± 15.59%, and 14 of 21 treated GR defects showed 100% root coverage. The gingival biotype also showed a thick biotype in nine sites that had an initial thin biotype. Conclusions: Fetal membranes possess distinctive properties that can be harnessed to promote periodontal healing. The chorion membrane covered by a modified coronally advanced flap is a new approach that has shown promising results in terms of root coverage, increased width of keratinized tissue, and thickness of the gingival biotype.     

An electronic oral health record to document,plan and educate

The University of British Columbia (UBC) in collaboration with a software developer (Planmeca Oy, Finland) created an electronic oral health record based on the principles of cognitive ergonomics rooted in the European research and development project ‘ORQUEST’ to guide students through medical, dental, social histories, examinations, treatment planning and progress notes. Clinicians in each dental specialty of the Faculty of Dentistry and software engineers cooperated to define the clinical content and workflow of clinical procedures in three phases: (i) development of a radiographic module, (ii) development of medical, dental, social and family histories, intra‐ and extra‐oral examinations, progress notes and treatment planning and (iii) development of the orthodontic section accompanied by an optimisation phase to correct technical problems and clinical content issues. From a practical perspective, this EOHR enhances the clinical performance of students and the quality assurance capacity of the institution. It facilitates audits of clinical productivity and research, and it can be modified with relative ease to suit similar educational and clinical environments in either public or private healthcare settings.