The effects of bevacizumab on postoperative complications in patients undergoing colorectal and pancreatic cancer resection.

Bevacizumab (Avastin™; rhuMab VEGF), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), has seen increased use in the perioperative treatment of colorectal and pancreatic cancer. Little is known, however, regarding its impact on surgical outcomes in patients undergoing resection. The objective of this review was to examine if the addition of bevacizumab to existing neoadjuvant regimens increases morbidity after cancer resection.

Inhibition of human erythroid colony-forming units by tumor necrosis factor requires beta interferon.

We have previously reported that inhibition of human CFU-erythroid (E) colony formation by tumor necrosis factor (TNF) is an indirect effect mediated by a soluble factor released from a fraction of marrow accessory cells which are predominantly stromal elements (Means, R. T., Jr., E. N. Dessypris, and S. B. Krantz. 1990. J. Clin. Invest. 86:538-541). Further studies reported here identify a mediator of this effect. The inhibitory effect of recombinant TNF on marrow CFU-E is ablated by neutralizing antibodies to human beta IFN, but not by antibodies to gamma IFN or IL-1. Anti-beta IFN also neutralizes the inhibitory effect of conditioned medium prepared from marrow cells exposed to TNF. Human beta IFN inhibits colony formation by unpurified marrow CFU-E as well as highly purified CFU-E generated from peripheral blood progenitors, and limiting dilution analysis shows that this is a direct inhibitory effect. TNF has been implicated in the pathogenesis of the anemia of chronic diseases since blood TNF levels are elevated in many patients with this syndrome, and since exposure to TNF produces a similar anemia in either humans or mice. The present study demonstrates that beta IFN is a required mediator of this inhibitory effect on erythropoiesis.

Inhibition of human colony-forming-unit erythroid by tumor necrosis factor requires accessory cells.

Recombinant tumor necrosis factor (rTNF) inhibits erythropoiesis in vivo and in vitro. To study the mechanism of this inhibition, the effect of rTNF on highly purified human CFU-erythroid (E) (mean purity 63.5%), which were generated from peripheral blood burst-forming units-erythroid (BFU-E), was compared to its effect on unpurified human marrow CFU-E (mean purity 0.21%). Although growth of colonies from marrow CFU-E was inhibited by rTNF, no significant effect on purified BFU-E-derived CFU-E colony growth was found. Removal of accessory marrow cells by soy bean agglutinin (SBA) ablated the inhibition of marrow CFU-E colonies by rTNF. Inhibition of colony growth was then restored by adding back SBA+ cells, but not by adding T lymphocytes or adherent cells. Conditioned medium prepared from bone marrow mononuclear cells stimulated by rTNF inhibited the growth of colonies from highly purified BFU-E derived CFU-E resistant to direct inhibition by rTNF. These findings indicate that rTNF does not directly inhibit CFU-E, but requires accessory cells to decrease erythropoiesis. These accessory cells reside in the SBA+ cell fraction, but are neither T cells nor adherent cells. Therefore, in order to produce anemia, TNF must induce release or production of a factor that directly inhibits erythroid colony growth.

Studies on red cell aplasia. V. Presence of erythroblast cytotoxicity in G-globulin fraction of plasma.

The marrow cells of a patient with pure red cell aplasia markedly increased their rate of heme synthesis when they were freed from the host environment and were incubated in vitro. When the red cell aplasia was treated with cyclophosphamide and prednisone, marrow cell incorporation of (59)Fe into heme in vitro increased several weeks before a reticulocytosis was apparent, and was the earliest effect noted. The plasma gammaG-globulins of this patient inhibited heme synthesis by normal marrow cells or the patient's own marrow cells obtained after remission of the disease. Since the inhibition of heme synthesis could be the result of damage to erythroblasts, the patient's posttreatment marrow cells or normal marrow cells were labeled with (59)Fe and were then incubated with the patient's pretreatment, treatment, and posttreatment gammaG-globulins as well as normal gammaG-globulins. At the end of this incubation the supernatant and cells were separated and counted. Heme was extracted and also was counted. Treatment of the cells with the patient's pretreatment gammaG-globulins resulted in a release of 40% of the radioactive heme from the cells. This represented the loss of radioactive hemoglobin and was an index of erythroblast cytotoxicity. A progressive disappearance of the cytotoxic factor in the gammaG-globulins occurred in the 3 wk period preceding the onset of reticulocytes in the patient's blood. Posttreatment and normal gammaG-globulins did not produce this effect and increased injury of red cells and lymphocytes was not produced by the patient's pretreatment gammaG-globulins. These studies demonstrate a method for measuring erythroblast cytoxicity and show that red cell aplasia is associated with gammaG-globulins that specifically damage erythroblasts. Whether interference with new erythroblast development also occurs and contributes to the inhibition of heme synthesis has not yet been ascertained.

Erythropoietin receptors in polycythemia vera.

The role of erythropoietin (EP) in polycythemia vera (PV) is controversial, with some experiments suggesting that erythroid progenitors in PV are exquisitely sensitive to EP and EP dependent, and others suggesting that PV progenitors are EP independent. We have examined the characteristics of the EP receptor (EP-R) on erythroid colony-forming cells (ECFC) from patients with PV. In contrast to normal ECFC, which have two classes of EP-R, with 20% showing high affinity (Kd = 0.13 nM; range, 0.04-0.20 nM) and the remainder lower affinity (Kd = 0.37 nM; range, 0.28-0.57 nM), PV ECFC show a single class of 851 low affinity EP-R with Kd = 0.72 nM (range, 0.36-0.85 nM). ECFC from patients with secondary (EP driven) polycythemia or anemia show two classes of EP-R (Kd = 0.18 and 1.10 nM, respectively). Attempts to remove tightly bound EP from putative high affinity EP-R in PV did not reveal any higher affinity receptors. Determination of molecular size by crosslinking showed two proteins of 90 and 100 kD similar to those seen with normal EP-R. These studies indicate the PV ECFC have EP-R that are structurally similar to normal EP-R but lack the higher binding affinity for EP.

Distinct roles of erythropoietin, insulin-like growth factor I, and stem cell factor in the development of erythroid progenitor cells.

Erythropoietin (EP), insulin-like growth factor I (IGF-I) and stem cell factor (SCF) each reduce apoptosis of human erythroid progenitor cells. To determine if these growth factors have additional roles in stimulating erythropoiesis, the proliferation, maturation, and survival of highly purified human erythroid colony-forming cells (ECFCs) were studied during the application of different combinations of these growth factors in a serum-free liquid culture. EP maintained cell viability and supported heme synthesis during erythroid maturation, with little increase in viable cell number or stimulation of DNA synthesis. The addition of SCF with EP resulted in a substantial increase in DNA synthesis, which was greater than that seen with the addition of EP and was associated with a large expansion in the number of ECFCs. Thus EP, by itself, produces little increase in cell proliferation, and expansion of the number of erythroid cells depends upon the presence of SCF with EP. The addition of IGF-I with EP led to enhanced heme synthesis and moderate cellular proliferation, but also greatly enhanced nuclear condensation and enucleation in the late erythroblasts. Thus EP, by itself, is not sufficient for complete end-terminal nuclear condensation/enucleation and the presence of IGF-I is necessary for this complete process. While EP greatly reduced apoptosis during 16 h of incubation at 37 degrees C, the addition of SCF and IGF-I with EP had little additional effect, but these additions enhanced DNA synthesis > 3.4-fold. Thus SCF may have an additional role in directly stimulating proliferation through a process that is distinct from apoptosis. Our observations indicate that EP prevents apoptosis and maintains erythroid cell viability and development. IGF-I enhances erythroid maturation and proliferation, but the proliferation of erythroid progenitors is mainly controlled by the addition of SCF with EP, independent of an effect on apoptosis.

Polycythemia vera blood burst-forming units-erythroid are hypersensitive to interleukin-3.

Because polycythemia vera (PV) is a clonal hematopoietic stem cell disease with a trilineage hyperplasia, and interleukin-3 (IL-3) stimulates trilineage hematopoiesis, we have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-E) to recombinant human IL-3 (rIL-3). Whereas the growth of normal blood BFU-E in vitro rapidly declined by 40 and 60% after 24 and 48 h of incubation without 50 U/ml of rIL-3, the growth of PV BFU-E declined by only 10 and 30% under the same conditions, demonstrating a reduced dependence on rIL-3. A reduced dependence of PV BFU-E on recombinant human erythropoietin (rEP) was also present. Dose-response experiments showed a 117-fold increase in PV BFU-E sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEP, compared to normal BFU-E, whereas blood BFU-E from patients with secondary polycythemia responded like normal BFU-E. Endogenous erythroid colony (EEC) formation, which is independent of the addition of rEP, was reduced by 50% after erythroid colony-forming cells were generated from PV BFU-E in vitro without rIL-3 for 3 d, whereas rEP-stimulated erythroid colonies were unaffected. These studies demonstrate a striking hypersensitivity of PV blood BFU-E to rIL-3, which may be the major factor in the pathogenesis of increased erythropoiesis without increased EP concentrations.

Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.

Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.

Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development.

The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.

Splenic erythroid response to friend polycythemia virus: time course in vitro after infection in vivo.

When spleen cells removed from plethoric BALB/c mice shortly after infection with Friend polycythemia virus were cultured, they subsequently increased their rate of hemoglobin synthesis in vitro without the addition of the hormone erythropoietin. The increased 59Fe incorporation into hemoglobin in vitro was part of a well-defined single wave unlike the progressive increase in hemoglobin synthesis that occurs in vivo. The peak occurred within the same total time (85-105 hr after infection), irrespective of either the virus dose or time after infection when the cells were removed from the animal and cultured. However, the magnitude of the peak increased with an increase in either of these two variables. Medium change experiments indicated that the time during which the peak occurred was not artificially determined by depletion of some medium component or the accumulation of an inhibitor. This system may be useful in separating the early events of Friend virus infection from the late effects on erythroid differentiation.

Pure red cell aplasia associated with administration of sustained-release procainamide.

Pure red cell aplasia developed in a patient with small-cell lung cancer who was also taking sustained-release procainamide. Shortly after discontinuation of the procainamide preparation, a reticulocytosis and increasing hemoglobin level were observed.

Upper airway obstruction as a complication of oral anticoagulation therapy. Report of three cases.

While taking orally administered anticoagulants, three patients had hemorrhages into their retropharyngeal and submandibular spaces, suffering eventual acute airway obstruction. One of the patients died. Despite the life-threatening nature of this complication of anticoagulant therapy, the diagnosis was obscure and initially veiled in complaints of sore throat or hoarseness, suggesting infection. Thorough investigation of such complaints is necessary in patients receiving anticoagulation therapy. If a hematoma is discovered, the patient should be admitted to the hospital for close observation and prompt reversal of anticoagulation with plasma. Intubation or tracheostomy also may be required.

Bone-marrow imaging in pure red blood cell aplasia.

Bone-marrow scintigraphy with indium chloride in 111 was performed on a patient with pure red blood cell aplasia before and after successful treatment with immunosuppressive drugs. The return of erythroid precursors to the bone marrow was accompanied by a substantial increase in the marrow uptake of 111In. The distribution of 111In in the posttreatment scan was indistinguishable from that of 52Fe. These results indicate that indium chloride in 111 is a useful agent for the delineation of erythroid cellularity within bone marrow.

Cimetidine-induced neutropenia: a possible dose-related phenomenon.

Neutropenia associated with high-dose cimetidine therapy developed in a patient in whom earlier therapy, as well as rechallenge with low-dose cimetidine, did not result in neutropenia. As other factors cannot be implicated, a dose-related toxicity of cimetidine appears likely. Although the mechanism of cimetidine-induced neutropenia is unknown, it may involve the previously demonstrated ability of histamine H2 receptor antagonists to block the histamine-induced initiation of DNA synthesis in bone marrow stem cells.

Beneficial effect of hepatitis in leukemic reticuloendotheliosis.

A patient with leukemic reticuloendotheliosis had splenomegaly, neutropenia, and a severe underproduction anemia. During a three-year period, the hematocrit was never in the normal range, and periodic transfusions were required. However, after an episode of hepatitis that was positive for B surface antigen, the spleen became smaller, the number of neutrophils increased, the transfusion requirement disappeared, and the hematocrit rose to normal. Several mechanisms for this observation are proposed.

Effects of ethanol on cultured human megakaryocytic progenitors.

Ethanol intoxication may result in the suppression of platelet production in human beings through direct toxicity to the bone marrow. To determine whether this suppression is due in part to a toxic effect on megakaryocytic progenitors, human bone marrow was cultured for 15 days in plasma clots, and megakaryocytic colonies were enumerated. Addition of ethanol to cultures at the beginning of incubation resulted in a reduction in megakaryocytic colonies only when the initial ethanol concentration exceeded 1 g/dl, well above physiologically tolerated levels. However, when cultures were treated with lower concentrations of ethanol (0.5 g/dl) after seven days of incubation, a reduction of megakaryocytic colony formation also occurred. These results suggest that development of a human cell more differentiated than the megakaryocytic progenitor is sensitive to ethanol toxicity at or near concentrations observed in vivo and is in accord with clinical features of the reversible alcohol-induced thrombocytopenic syndrome.

Inhibition of human erythroid colony-forming units by interferons alpha and beta: differing mechanisms despite shared receptor.

Previous investigations have demonstrated that interferons alpha, beta, and gamma (alpha-, beta-, and gamma-IFN) are potent inhibitors of erythropoiesis in vitro. By utilizing a cell population enriched for human erythroid colony-forming units (CFU-E), we have previously demonstrated that the inhibitory effects of beta- and gamma-IFNs are direct effects, not requiring the presence of accessory cells, and that the inhibitory effect of recombinant human (rh) gamma-IFN could be corrected by high concentrations of rh erythropoietin (Epo). In this study, we compared the effects of rh(alpha)-IFN on cells enriched for CFU-E to its effects on unpurified marrow cells and found that although h(beta)-IFN (which shares a common receptor with alpha-IFN) directly inhibits CFU-E colony formation, the effect of rh(alpha)-IFN is indirect and is mediated by a soluble factor released from T lymphocytes in response to rh(alpha)-IFN. However, rh(alpha)-IFN enhanced the direct inhibitory effect of rh(gamma)-IFN on CFU-E not inhibited by rh(alpha)-IFN. The inhibitory effects of neither alpha- nor beta-IFN could be overcome by high levels of rhEpo. These findings imply that alpha- and beta-IFN exert different cellular effects despite binding to the same receptor. Failure of rhEpo to correct CFU-E colony inhibition by alpha- and beta-IFNs but not by gamma-IFN also suggests a mechanism for the differing degrees of response to different doses of rhEpo in patients with the anemia of chronic disease.

Specific binding of interferon-gamma to high affinity receptors on human erythroid colony-forming cells.

Interferon-gamma (IFN-gamma) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, but IFN-gamma receptors are not present on red cells and have never been demonstrated on erythroid progenitor cells. We obtained highly purified day 6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity to measure binding of radioiodinated recombinant human IFN-gamma ([125I]rhIFN-gamma). When [125I]rhIFN-gamma was incubated with day 6 ECFC, 77% of the binding was inhibited by excess unlabeled rhIFN-gamma, but no inhibition occurred with a variety of growth factors and glycoproteins. Specific binding was directly proportional to the cell concentration with a straight line passing through the origin, and equilibrium was reached at 0 degree C by 24-48 hours. Saturation of specific binding occurred at a [125I]rhIFN-gamma concentration of 1.0 nM and internalization was demonstrated with further incubation at 37 degrees C. Scatchard analysis showed a single class of binding sites and at a high ECFC cell purity of 80-89%, 1910-2070 binding sites per ECFC were present with a Kd of 0.01-0.02 nM. As day 5 ECFC developed into more mature day 7-day 12 cells, with incubation at 37 degrees C in vitro, specific binding for [125I]IFN-gamma greatly decreased. These experiments delineate specific binding sites for IFN-gamma on human erythroid progenitor cells and indicate that the enhanced sensitivity to rhIFN-gamma inhibition of mature day 3-day 6 burst-forming units-erythroid may be a result of enhanced specific binding. Human IFN-gamma is a multifunctional lymphokine, secreted by activated T lymphocytes and NK cells, which exerts antiviral, antiproliferative, and immunomodulatory activities on a wide variety of cells [1,2]. With regard to hematopoietic cells, IFN-gamma has been reported to inhibit the growth of granulocyte-macrophage colony-forming units, burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) in vitro [3-7]. Most recently, mature day 3 to day 6 BFU-E have been shown to be most sensitive to the inhibitory effect of recombinant human (rh) IFN-gamma, while primitive day 1 to day 2 cells and later day 7 cells were less affected [7]. Incubation of rhIFN-gamma with mature BFU-E inhibits hemoglobin accumulation and produces apoptosis of the maturing erythroid cells [7]. Moreover, since blood IFN-gamma levels are elevated and vary directly with the degree of the anemia, in patients with hematologic malignancies [8] and HIV-seropositivity [9], IFN-gamma appears to have a prominent role in producing the anemia associated with chronic disease [10,11]. Although characterization of human IFN-gamma receptors has been extensively performed for a variety of human cells including fibroblasts, lymphocytes, monocytes, granulocytes, eosinophiles, platelets, and many tumor cells [12-17], IFN-gamma receptors have not been identified on red cells [12] and the presence plus the extent of IFN-gamma receptors on progenitor cells, including human erythroid progenitor cells, remains unknown. A method has been reported from our laboratory by which human erythroid colony-forming cells (ECFC) can be highly purified, starting with peripheral blood BFU-E, in a sufficient amount for analysis of cytokine binding [18-20]. In this paper, we report the results of [125I]rhIFN-gamma binding to day 6 ECFC in vitro and demonstrate the presence of specific binding that is saturable at 1.0 nM. Scatchard analysis reveals that there are 1910-2070 rhIFN-gamma binding sites per ECFC with a Kd of 0.01-0.02 nM and, as with erythropoietin (EP) and insulin-line growth factor I (IGF-I) receptors, specific binding is highest with the earliest BFU-E studied and declines progressively as the erythroid progenitors mature.

Combined effect of friend polycythemia virus and erythropoietin on erythroid burst formation in vitro.

The infection of mouse bone marrow or spleen cells in vitro with the polycythemia strain of Friend virus (FVP) results in the formation of erythroid bursts after 4-6 days in plasma clot or methylcellulose cultures in the absence of added erythropoietin (Ep). We have now used this cell culture system to study the effect of added Ep on FVP-induced burst formation. The number of bursts that developed following infection of bone marrow cells with equilibrium density gradient purified FVP was increased by the addition of low concentrations of highly purified human urinary Ep (70,000 units/mg protein) to the infected cultures. The addition of as little as 0.001 unit per ml of Ep to infected cultures resulted in a 2-fold increase in burst formation. Increasing the concentration of Ep to 0.05 unit per ml caused a 4-fold increase in the number of bursts that developed. These concentrations of Ep were ineffective in inducing erythroid bursts when added to cultures of uninfected cells. The addition of Ep within 6 hr after FVP infection was necessary to observe increased burst formation. These experiments demonstrate that FVP-burst formation is enhanced by extremely low levels of Ep. Although a precise mechanism for the increase in FVP-induced erythroid bursts with added Ep is not apparent from these experiments, a number of possibilities are discussed.

Friend virus production and heme synthesis in primary mouse spleen cell cultures.

A cell culture method has been developed in which spleen cells from Friend virus (FV) infected mice can be studied for virus production as well as erythroid differentiation. Primary spleen cell cultures from plethoric Balb/c mice were initiated at 24, 48 or 73 h after FV infection. These cells manifested a well-defined wave of heme synthesis at approximately 64, 48, or 23 h, respectively, of cell culture. Assays for spleen focus-forming virus (SFFV) and helper murine leukemia virus (MuLV-F) production in these cultures revealed that the peak rates of production of both viruses occurred at essentially the same time as the peaks of heme synthesis. The time at which the peaks of virus production and heme synthesis occurred in vitro was related to the time interval after infection (80-105 h) rather than the time at which the cells were placed in cell culture or the number of hours of cell culture. Medium change experiments suggested that the temporal relation between heme synthesis and virus production was an intrinsic feature of FVP infected cells in this in vitro system.