Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.     

Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic diseases

Translocations and deletions of the short arm of chromosome 12 [t(12p) and del(12p)] are common recurring abnormalities in a broad spectrum of hematologic malignant diseases. We studied 20 patients and one cell line whose cells contained 12p13 translocations and/or 12p deletions using fluorescence in situ hybridization (FISH) with phage, plasmid, and cosmid probes that we previously mapped and ordered on 12p12-13. FISH analysis showed that the 12p13 translocation breakpoints were clustered between two cosmids, D12S133 and D12S142, in 11 of 12 patients and in one cell line. FISH analysis of 11 patients with deletions demonstrated that the deletions were interstitial rather than terminal and that the distal part of 12p12, including the GDI-D4 gene and D12S54 marker, was deleted in all 11 patients. Moreover, FISH analysis showed that cells from 3 of these patients contained both a del(12p) and a 12p13 translocation and that the affected regions of these rearrangements appeared to overlap. We identified three yeast artificial chromosome (YAC) clones that span all the 12p13 translocation breakpoints mapped between D12S133 and D12S142. They have inserts of human DNA between 1.39 and 1.67 Mb. Because the region between D12S133 and D12S142 also represents the telomeric border of the smallest commonly deleted region of 12p, we also studied patients with a del(12p) using these YACs. The smallest YAC, 964c10, was deleted in 8 of 9 patients studied. In the other patient, the YAC labeled the del(12p) chromosome more weakly than the normal chromosome 12, suggesting that a part of the YAC was deleted. Thus, most 12p13 translocation breakpoints were clustered within the sequences contained in the 1.39 Mb YAC and this YAC appears to include the telomeric border of the smallest commonly deleted region. Whether the same gene is involved in both the translocations and deletions is presently unknown.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Additive effect of erythropoietin and heme on murine hematopoietic recovery after azidothymidine treatment [see comments]

The ability of combination treatment with erythropoietin (Epo) and heme to rescue hematopoietic activity in mice from the suppressive effect of azidothymidine (AZT) was determined. Exposure of mice to AZT for 5 weeks produced marked anemia, thrombocytopenia, neutropenia, and weight loss, whereas mice that received Epo and heme for 3 subsequent weeks showed significant alleviation of AZT cytotoxicity. Treatment with Epo (10 U for 5 times/week) stimulated hematopoietic recovery in the AZT- treated animals and reduced the severe anemia and thrombocytopenia by 3 weeks. Administration of a lower Epo dose (1 U Epo) resulted in only a modest retardation of AZT-induced anemia, although, when combined with heme, there was a great improvement in recovery of erythropoiesis. The combination of heme with Epo (10 U) produced the optimum response, resulting in almost normal recovery of bone marrow cellularity as well as recovery of burst-forming units-erythroid (BFU-E) and splenic hematopoietic progenitor content (colony-forming unit-spleen [CFU-S]) by the end of 3 weeks of post-AZT treatment. Treatment with heme alone markedly enhanced the recovery of BFU-E and CFU-S, as well as body weight post-AZT; however, this recovery was not to the extent seen in combination with Epo (10 U). Long-term bone marrow cultures (LTBMCs) established from mice exposed to AZT for 8 weeks showed a marked reduction in cellularity and this was completely alleviated when mice received heme and Epo (10 U) for 3 weeks after 5 weeks of AZT administration. The additive effect of heme and Epo was seen in BFU-E production, as well as in CFU-S production, in LTBMCs. Thus, heme exerts a significant protective effect on hematopoietic progenitors in vivo and may be of potential clinical use in combination with Epo to promote effective erythropoiesis in the setting of AZT therapy.     

CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.     

Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies

To characterize the nature of the immunoreactive transferrin receptor in human serum, antisera were developed to peptide sequences of the extracellular domain of human transferrin receptor between amino acids 107 and 120 and the intracellular domain between amino acids 40 and 54. Antisera against the extracellular domain exhibited reactivity against both purified intact receptor and immunopurified circulating receptor, whereas antisera against the intracellular domain reacted only with intact receptor. Using competitive binding enzyme-linked immunosorbent assays, transferrin receptor in ultracentrifuged sera from normal subjects and patients with sickle cell anemia could be detected with antisera against the extracellular but not the intracellular domain. When the pellet obtained by ultracentrifugation of these sera was assayed after solubilization in 1% teric (polyoxyethylene-9-lauryl ether), only 0.6% of total serum receptor was detected in normal subjects and 3.8% in subjects with sickle cell disease. Roughly equal amounts of this pelleted immunoactivity were detected with antibodies against the extracellular and intracellular domains. These results indicate that less than 1% of transferrin receptor in normal human sera is intact receptor consistent with an exosomal origin and that virtually all circulating transferrin receptor is in the form of a truncated extracellular domain.