William P. Hammond

Treatment of Cyclic Neutropenia with Granulocyte Colony-Stimulating Factor

Six patients with cyclic neutropenia were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) for 3 to 15 months. All had a history of recurrent aphthous stomatitis, pharyngitis, lymphadenopathy, fever, and numerous infections during periods of neutropenia. Serial blood-cell counts, findings on bone marrow examination, and signs and symptoms were evaluated before and during the daily administration of G-CSF (3 to 10 micrograms per kilogram of body weight per day), either intravenously or subcutaneously. The kinetics of labeled autologous blood neutrophils and the migration of neutrophils to skin chambers were also measured. Recombinant human G-CSF increased the mean (+/- SEM) neutrophil counts from 717 +/- 171 per microliter to 9814 +/- 2198 per microliter (P = 0.009). In five of the six patients, the cycling of blood-cell counts continued, but the length of the period decreased from 21 to 14 days. The number of days of severe neutropenia was reduced (P = 0.002). Neutrophil turnover increased almost four-fold (P = 0.005), whereas neutrophil migration to a skin chamber was normal. G-CSF therapy reduced the frequency of oropharyngeal inflammation, fever, and infections in these patients. During the first 40 months of treatment, no typical mouth ulcers or bacterial infections occurred; recurrent gingivitis improved. We conclude that G-CSF is effective for the treatment of cyclic neutropenia in humans.

Novel Vascular Endothelial Growth Factor Binding Domains of Fibronectin Enhance Vascular Endothelial Growth Factor Biological Activity

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, &agr;5&bgr;1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to &agr;5&bgr;1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

Long‐term safety of treatment with recombinant human granulocyte colony‐stimulating factor (r‐metHuG‐CSF) in patients with severe congenital neutropenias

Summary . Congenital neutropenias include a heterogenous group of diseases characterized by a decrease in circulating neutrophils. In phase I/II/III studies in patients with severe congenital and cyclic neutropenia, treatment with recombinant human granulocyte colony‐stimulating factor (r‐metHuG‐CSF) resulted in a rise in the absolute neutrophil counts (ANC) and a reduction in infections. We report the effects of long‐term safety of subcutaneous r‐metHuG‐CSF administration in 54 patients (congenital n= 44, cyclic n= 10) treated for 4–6 years. A sustained ANC response was seen in 40/44 severe congenital neutropenia patients and 10/10 cyclic neutropenia patients. Two patients required an increase of > 25% in dose to maintain a clinical response; one patient became refractory to therapy. A significant decrease in the incidence of severe infections and the need for intravenous antibiotics was noted. Significant adverse events noted which may or may not be related to therapy included: osteopenia (n= 15), splenomegaly (n= 12), hypersplenism (n= 1), vasculitis (n= 2), glomerulonephritis (n= 1), BM fibrosis (n= 2), MDS/leukaemia (n= 3), and transient inverted chromosome 5q with excess blasts (n= 1). R‐metHuG‐CSF has been well tolerated in the majority of patients and resulted in a longterm improvement in their clinical status.

Low density lipoprotein receptor activity in freshly isolated human blood monocytes and lymphocytes

Circulating human monocytes and lymphocytes were isolated by counterflow and density gradient centrifugation. Binding and degradation of low density lipoprotein (LDL) occurred predominantly in monocytes and to a much lesser extent in lymphocytes. The findings were consistent with greater LDL receptor activity in freshly isolated monocytes than lymphocytes, in keeping with differences in other cell surface receptors between these two cell types. Therefore, when freshly isolated mixed mononuclear cells are used to study LDL receptor activity in vivo in humans, careful attention needs to be given to the proportions of monocytes and lymphocytes, or alternatively, relatively pure preparations of monocytes should be used.

Decreased Granulocyte-Macrophage Colony-Stimulating Factor Production by Human Neonatal Blood Mononuclear Cells and T Cells

ABSTRACT: Impaired production and delivery of neutrophils to the site of infection have been implicated in the increased susceptibility of the neonate to infection. Because granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) play critical roles in the production of neutrophils from marrow precursors, we assessed the ability of leukocytes from neonates and adults to produce GM-CSF, G-CSF, and, for comparison, macrophage colony-stimulating factor (M-CSF) after stimulation with concanavalin A ± phorbol myristate acetate [blood mononuclear cells (MC) and T lymphocytes] or lipopolysaccharide (monocytes). MC and monocytes from adult and neonatal subjects produced mRNA for GM-CSF, G-CSF, and M-CSF, whereas T cells produced only GM-CSF mRNA. Neonatal MC and T cells accumulated only −30% as much GM-CSF mRNA as did adult MC and T cells. In contrast, the accumulation of GM-CSF mRNA by neonatal and adult monocytes was similar. Neonatal MC also accumulated similar amounts of G-CSF mRNA and somewhat more M-CSF mRNA than did adult MC; results with monocytes were similar to those with MC. Results of colony-stimulating activity bioassays on supernatants from neonatal and adult MC stimulated with concanavalin A paralleled the mRNA results. Although the overall number of colonies generated using neonatal and adult supernatants was similar, neonatal MC supernatants generated significantly more (p < 0.05) monocyte-containing colonies (72 ±19 versus 46 ± 11), significantly fewer (p < 0.05) eosinophil-containing colonies (7 ± 6 versus 23 ± 13), and similar numbers of granulocyte-containing colonies (59 ± 23 versus 63 ± 11) compared with adult MC supernatants. Because GM-CSF is a major determinant of eosinophil production in these assays, these data suggested diminished amounts of GM- CSF in the neonatal culture supernatants. Similarly, GM- CSF concentrations in neonatal MC and T cell culture supernatants averaged 40 to 50% of the concentrations in adult culture supernatants as determined by ELISA (p < 0.01). Whether the modestly diminished GM-CSF production by neonatal T cells contributes t the observed deficiency of granulocyte production in neonates, which occurs when demand is increased in response to infection, remains to be determined.

Thrombopoietin is synergistic with other hematopoietic growth factors and physiologic platelet agonists for platelet activation in vitro

Thrombopoietin (TPO) is the primary physiologic regulator of platelet production. The effect of TPO on platelet function, both alone and in combination with other hematopoietic growth factors, adenosine diphosphate (ADP), and epinephrine, was investigated using fluorescent‐labeled antibodies to the activation‐dependent antigen CD62 (P‐selectin) and flow cytometry. TPO stimulated CD62 expression on normal human platelets, and this expression was completely inhibited by the soluble extracellular domain of the TPO receptor, MPL. The growth factors granulocyte colony‐stimulating factor (G‐CSF) and erythropoietin (EPO), but not interleukin‐3 (IL‐3) or stem‐cell factor (SCF), also stimulated platelet activation. The combination of EPO, SCF, ADP, and epinephrine with TPO were synergistic for platelet CD62 expression. These data further support a role for TPO in modulating platelet function. Am. J. Hematol. 54:225–232, 1997

Genetic Tracing of Arterial Graft Flow Surface Endothelialization in Allogeneic Marrow Transplanted Dogs

In order to trace genetically the source of fallout endothelialization on arterial grafts, six beagle dogs with successful autologous bone marrow transplantation received composite tandem aortic grafts with an isolated, totally impervious Dacron graft and a porous Dacron graft for 12 weeks. For impervious segments, five of 12 fresh tissue samples were Factor VIII/von Willebrand factor + (FVIII/vWF) and seven had faint or negative signals; three of the FVIII/vWF + samples had alpha-actin + smooth muscle cells. Polymerase chain reaction (PCR) study showed eight had a pure donor DNA genotype and four had donor/host mixed, with the donor predominant. Of 12 AgNO3-stained samples, 11 showed pure donor type and one had donor/host mixed, with the donor predominant. For porous segments, all 12 fresh samples had positive flow surface FVIII/vWF and alpha-actin cells. PCR showed all these samples and all 12 AgNO3-stained samples had donor/host mixed type, but the host pattern was predominant. Porous graft healing appears to involve both cellular fallout and tissue ingrowth, and bone-marrow-derived cells may be a source for fallout.

Induction of the urokinase plasminogen activator system by oncostatin M promotes endothelial migration

Oncostatin M (OSM) is an inflammatory cytokine produced by activated macrophages and T‐lymphocytes. We have previously demonstrated that OSM‐induced endothelial cell migration, unlike endothelial cell proliferation and spindle formation, is independent of basic fibroblast growth factor expression (Wijelath et al. [1997] J. Cell. Sci. 110:871–879). To better understand the mechanism of OSM‐induced endothelial cell migration, this study examined the potential role of the plasminogen activator system in promoting OSM mediated endothelial cell migration. OSM stimulated increased mRNA levels of urokinase‐plasminogen activator (uPA) and urokinase‐plasminogen activator receptor (uPAR) in a time and dose‐dependent manner. Transcriptional run‐off and mRNA stability analysis demonstrated that the increase in uPA and uPAR mRNA levels was due to both increased gene transcription and mRNA stability. The increase in mRNA correlated with increased protein levels of both uPA and uPAR. This increase was reflected in elevated levels of membrane‐bound plasmin activity. OSM‐induced endothelial cell migration was only partially dependent on plasmin activity since incubating endothelial cells without plasminogen or, in the presence of aprotinin, resulted in suppression of endothelial cell migration, indicating that OSM promoted endothelial cell migration through both a plasmin‐dependent and ‐independent mechanism. Our results imply a role for OSM in promoting endothelial cell migration via a plasmin‐dependent pathway and a uPAR‐mediated pathway. Together, these and other recent studies support a role for OSM in modulating the different phases of angiogenesis. J. Cell. Biochem. 79:239–248, 2000.

Internal thoracic artery grafts for the entire heart at a mean of 12 years

BACKGROUND There is consensus today that the long-term results of bypassing the left anterior descending artery with an internal thoracic artery (ITA) graft are superior to those of a saphenous vein graft. Our hypothesis for this study was that three-vessel revascularization with only ITA grafts would also give excellent results. METHODS Using our previously described techniques to enhance the length of ITA grafts by skeletonization and high mediastinal mobilization, we were able to perform tension-free, three-vessel revascularization using only ITA grafts in 125 (83%) of a consecutive series of 150 patients with three-vessel occlusive coronary disease. We followed 100% of these 125 exclusive ITA graft patients (average of 3.9 anastomoses per patient) to their time of death (59; 47.2%) or current living status (66; 52.8%). RESULTS Combined intraoperative graft flows averaged 225 mL/min. Of the 125 patients in this study (average age, 63.5 years), 121 (96.8%) lived beyond 40 days. Of these 121 patients, 55 (45%) died at a mean of 7 years postoperatively and 66 (55%) are still living at a mean of 12.1 years. Of these 121 patients, 112 (93%) had angina at baseline. Of these 112, 92 (85%) were angina free at a mean of 9.1 years postoperatively. Freedom from infarction was 100% at 5 years and 97% at 10 years. Freedom from reintervention was 90% at a mean of 9.8 years. CONCLUSIONS Use of ITA grafts for three-vessel coronary revascularization provides excellent results and is both practical and appropriate for many patients.

The role of lymphocytes and monocytes in hematopoietic growth factor production by peripheral blood mononuclear cells

Stimulated peripheral blood mononuclear cells (MNC) are one of the richest described physiologic sources of colony-stimulating activity. To understand the molecular basis for, and the cellular sources of, this MNC activity, we cultured purified human lymphocytes and monocytes for 2 hr to 6 days and examined colony-stimulating factor (CSF) gene activity by Northern blot analysis. We show that MNC are capable of expressing messenger RNA for macrophage (M)-CSF, granulocyte (G)-CSF, GM-CSF, and multi-CSF when stimulated with mitogens. The time courses of induction of these genes differ, with G-CSF induction preceding that of the other CSFs. In addition, the spectra of CSFs produced by cell populations enriched for lymphocytes, monocytes, or macrophages differ. The implications of these findings for the selective activation of hematopoiesis are discussed.