Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.     

Human platelets exert cytotoxic effects on tumor cells

Monocytes are thought to play a role in host resistance to tumor cell growth in animals and humans. In addition, platelets are known to be involved in tumor metastases. To investigate the interaction of these two cell types and their effect on tumor cells, human monocytes and platelets were examined using an in vitro monocyte-tumor cell cytotoxicity assay. Monocytes alone resulted in 32% +/- 1.5 (mean +/- SEM) tumor cell kill. When platelets were added to monocytes in a 1:1 ratio, an increase in cytotoxicity to 61% +/- 3.2 was observed. The cytotoxicity noted when platelets were added to a fixed number of monocytes and tumor cells was dependent on the number of platelets added. A decrease in cytotoxicity from 32% +/- 1.5 to 12% +/- 2.3 was observed when contaminating platelets were removed from monocyte preparations. Platelets added to tumor cells in the absence of any monocytes were also toxic, resulting in a maximum kill of 95% at a 4:1 platelet/tumor cell ratio. Secreted products of freshly isolated platelets may be responsible for much of the observed cytotoxicity, since supernatants from the platelets were toxic for tumor cells. Platelets pretreated with a cyclooxygenase inhibitor (ASA) or a lipoxygenase inhibitor had decreased cytotoxicity compared with untreated platelets. Our results indicate that products of platelet arachidonate metabolism are toxic for tumor cell lines. They also suggest that the role of the platelet must be considered when studying monocyte-tumor cell cytotoxicity.     

Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells

The cell surface molecule Ly-6A/E provides a convenient marker for primitive stem cells in the hematopoietic tissues of both fetal and adult mice. However, previous studies have shown that Ly-6A/E expression by lymphocytes is variable depending on the haplotype of the Ly-6 locus. Therefore, strain-specific variation in Ly-6A/E expression by bone marrow (BM) cells was investigated. The results show that Ly-6a mice have, on average, 50% of the number of BM cells expressing Ly-6A/E relative to that for Ly-6b mice. Furthermore, among the 5% of BM cells that do not express antigens characteristic of mature T, B, myeloid, or erythroid lineages, which include the primitive hematopoietic stem cell compartment, Ly-6a mice have, on average, more than fivefold fewer Ly- 6A/E+ cells relative to that for Ly-6b mice. Isolation of Ly-6A/E- and Ly-6A/E+ cells from mice of both haplotypes showed that, whereas 99% of the marrow repopulating activity (MRA) of C57BL/Ka (Ly-6b) mice could be recovered in the Ly-6A/E+ fraction, only about 25% of the MRA of BALB/c (Ly-6a) was recoverable in the same population. On a per-cell basis, the Ly-6A/E+ cells that were isolated from BALB/c mice were essentially equivalent in MRA to those isolated from C57BL/Ka mice. Thus, whereas a large percentage of the hematopoietic stem cells of Ly- 6a mice do not express the Ly-6A/E molecule, the antigen may be used to isolate a subset of stem cells from these mice. These results show that hematopoietic stem cell phenotype can vary between mouse strains and imply that caution should be exercised in the identification of human stem cell antigens such as CD34, because a similar variability may occur between individual humans. To further explore the influence of Ly- 6 haplotype on Ly-6A/E expression by specific cell subsets, lymph-node lymphocytes from a panel of mouse strains were analyzed by multiparameter flow cytometry for correlated expression of Ly-6A/E, CD4, and CD8. All Ly-6a strains examined had less than 20% Ly-6A/E+ cells, and those cells were predominantly CD8+ T lymphocytes. In contrast, the Ly-6b strains had greater than 30% Ly-6A/E+ cells, and those cells included CD4+, CD8+, and B lymphocytes.     

Transient leukemoid proliferation of the cytogenetically unbalanced +21 cell line of a constitutional mosaic boy

A newborn without any signs of Down's syndrome was found to have an acute proliferation that remitted without drug therapy. Chromosomal analysis of blood, bone marrow, and skin cells revealed that the child was a constitutional mosaic with normal cells and a low number of cells in which one no. 21 chromosome was replaced by a probably isochromosome for the no. 21 long arm: 46,XY/46,XY,i(21q). The abnormal cell line of the mosaic appeared to be selectively involved in this proliferation.     

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.     

Utilization of supplemental iron by premature infants fed fortified human milk

We measured red blood cell iron incorporation (RBC-inc) in 13 human milk-fed premature infants (birthweight 1037 +/- 289 g, gestational age 27 +/- 2 wk, weight at start of study 1571 +/- 426 g) who were receiving full tube-feedings of human milk fortified with a commercial human milk fortifier (FortHM). The relative RBC-inc of supplemental iron (2 mg/kg/d of ferrous sulfate) was assessed using 57Fe sulfate mixed directly into a 24-h volume of FortHM, and 54Fe sulfate given as a bolus between two FortHM feedings the next day. RBC-inc was similar between the two methods of supplemental iron administration (4.7 +/- 2.5% vs 4.6 +/- 1.5%, respectively). Although these values are lower than RBC-inc expected from iron native to human milk, the relatively large amount of iron in the supplements contributed most of the iron incorporated into RBC by the infants. There was a significant positive correlation between the reticulocyte count and RBC-inc. As the high nutrient (especially calcium) content of the FortHM did not interfere with iron utilization, adding iron directly to FortHM, or incorporating it into commercial fortifiers, may be a practical method to provide iron to premature infants.     

Basic isoferritin and hypercalcaemia in renal cell carcinoma

A 63-year-old man with iron loss anaemia and hypercalcaemia was found to have a renal cell carcinoma. Despite the iron-deficient blood and bone marrow picture, the serum ferritin concentration was markedly raised. This was mainly due to a “basic isoferritin”. The serum parathormone concentration was normal. The serum ferritin and calcium concentrations returned to normal after the tumour was removed. We propose that the renal cell carcinoma cells in this patient secreted the basic isoferritin as well as humoral factor(s) responsible for hypercalcaemia.     

Mortality and failure among tuberculosis patients who did not complete treatment in Vietnam: a cohort study

Background  

Tuberculosis treatment failure and death rates are low in the Western Pacific Region, including Vietnam. However, failure or death may also occur among patients who did not complete treatment, i.e. reported as default or transfer-out. We aimed to assess the proportion failures and deaths among new smear-positive pulmonary tuberculosis patients with reported default or transfer-out.