Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN-224).

Tetrathiomolybdate (choline salt; ATN-224), a specific, high-affinity copper binder, is currently being evaluated in several phase II cancer trials. ATN-224 inhibits CuZn superoxide dismutase 1 (SOD1) leading to antiangiogenic and antitumour effects. The pharmacodynamics of tetrathiomolybdate has been followed by tracking ceruloplasmin (Cp), a biomarker for systemic copper. However, at least in mice, the inhibition of angiogenesis occurs before a measurable decrease in systemic copper is observed. Thus, the identification and characterisation of other biomarkers to follow the activity of ATN-224 in the clinic is of great interest. Here, we present the preclinical evaluation of two potential biomarkers for the activity of ATN-224: (i) SOD activity measurements in blood cells in mice and (ii) levels of endothelial progenitor cells (EPCs) in bonnet macaques treated with ATN-224. The superoxide dismutase activity in blood cells in mice is rapidly inhibited by ATN-224 treatment at doses at which angiogenesis is maximally inhibited. Furthermore, ATN-224 dosing in bonnet macaques causes a profound and reversible decrease in EPCs without significant toxicity. Thus, both SOD activity measurements and levels of EPCs may be useful biomarkers of the antiangiogenic activity of ATN-224 to be used in its clinical development.

Response to hypoxia involves transforming growth factor-beta2 and Smad proteins in human endothelial cells.

Oxygen deprivation (hypoxia) is a consistent component of ischemia that induces an inflammatory and prothrombotic response in the endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O(2)) increases messenger RNA and protein levels of transforming growth factor-beta2 (TGF-beta2), a cytokine with potent regulatory effects on vascular inflammatory responses. Messenger RNA levels of the TGF-beta2 type II membrane receptor, which is a serine threonine kinase, also increased. The stimulatory effect of hypoxia was found to occur at the level of transcription of the TGF-beta2 gene and involves Smad proteins, a class of intracellular signaling proteins that mediates the downstream effects of TGF-beta receptors. Transient transfection studies showed that the region spanning -77 and -40 base pairs within the TGF-beta2 promoter (harboring a Smad-binding "CAGA box") is activated in hypoxic cells compared with nonhypoxic controls (P <.01). Hypoxia also stimulated transcription from another promoter, 3TP-Lux, a reporter construct responsive to Smads and TGF-beta. In addition, specific binding to a Smad-binding oligonucleotide was observed with nuclear extracts from hypoxic endothelial cells but not from nonhypoxic cells. It is concluded that Smad proteins, which can regulate endothelial responses to mechanical and inflammatory stress, also may play an important role in vascular responses to hypoxia and ischemia.

Stress, peer affiliation, and transforming growth factor-beta1 in differentially reared primates.

A bidirectional regulatory interaction between the central nervous system and the immune system is largely provided by cytokines and their specific receptors, which are expressed by cells of both systems. Transforming growth factor-beta1 (TGF-beta1), produced by glial cells and lymphocytes and regulated by steroid hormones, is one such cytokine. In the current study, we examined the relationship between TGF-beta1 and peer affiliation in bonnet macaques (Macaca radiata) either reared normally or exposed as infants to conditions in which their mothers faced fluctuating requirements for food procurement (variable foraging demand [VFD]). Rearing under VFD conditions has been previously shown to produce dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in these animals. Serum levels of TGF-beta1 after exposure to a moderate stressor had no correlation with peer affiliation under baseline conditions (r=.07), but were highly correlated with affiliation after subsequent challenge with a fear stimulus (r=.62). Affiliation after the fear stimulus also was inversely correlated with baseline levels of affiliation (r=-.71). These data suggest that changes in peripheral TGF-beta1 may be reflective of latent behavioral and biochemical propensities possibly related to affect. Further examination of the effects of early adversity will improve our understanding of the relationship between the HPA axis and immune function.

Successful medical management of orbital abscess in a patient with sickle cell anemia.

The authors describe a patient with sickle cell anemia who had an orbital abscess at the site of a bone infarct during hospitalization for a painful crisis. Because the patient was in close medical observation, the orbital abscess was diagnosed within 48 hours of the onset of symptoms. The patient was treated with a 2-week course of intravenous antibiotics. This resulted in complete resolution of the abscess, as evidenced by clinical improvement and findings on computerized axial tomography scanning. The authors conclude that a heightened suspicion of orbital abscess in sickle cell patients with ocular symptoms will allow the diagnosis of an orbital abscess that can then be cured with antibiotic treatment but without orbital surgery.

Differential regulation of transforming growth factor beta-1 gene expression by glucocorticoids in human T and glial cells.

The inflammatory process in the brain requires bidirectional interaction of the immune and nervous systems. Evidently, astrocytic glial cells play an important role in facilitating this communication by releasing immunomodulators and cytokines. Expression of transforming growth factor beta-1 (TGF beta-1), a potent inhibitor of T cell function and glial cell proliferation, is highly regulated in T cells and is believed to be an important component in the molecular interaction between the immune and nervous systems. Comparative analysis of TGF beta-1 gene expression in human T and glial cells by Northern hybridization and S1 nuclease protection assay showed that dexamethasone (DM) caused a significant decrease in the basal and PMA-induced levels of TGF beta-1 mRNA in glial cells but not in T cells. This reduction correlated with a lower level of TGF beta-1 protein production. Transient transfection assay using deletion constructs of the 5' TGF beta-1 gene promoter-containing sequences between -453 and +11 bp identified a region spanning -160 to -60 bp as a potential sequence responsive to regulation by DM in T cells, whereas in glial cells, the overall transcriptional activity of the 5' TGF beta-1 promoter was reduced after DM treatment, but promoter activity within each construct remained constant in response to DM. Thus, a DM-responsive region could not be identified within the TGF beta-1 promoter in glial cells. These findings suggest that TGF beta-1 gene expression is differentially regulated by distinct regulatory elements in T and glial cells, and that extracellular stimulators, including glucocorticoids, can utilize the TGF beta-1-regulatory pathway to affect the functions of neural and immune cells.

Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells.

To determine the mechanism of glucocorticoid (GC)-mediated inhibition of T cell functions, the effect of dexamethasone (DM) on T cell proliferation and interleukin-2 receptor (IL-2R) generation were studied. Dexamethasone inhibited IL-2-induced T cell proliferation by 30%-88%, relative to its concentration within the cultures. The effect of DM on expression of IL-2R alpha (Tac, p55, CD25) and beta (p75) genes in activated T cells was examined next. In T cells stimulated with purified phytohemagglutinin (PHA-p) and 4 beta-phorbol 12-myristate 13-acetate (PMA) addition of DM to the cultures resulted in a 60% reduction in IL-2R alpha and a 30% reduction in IL-2R beta membrane expression compared to T cells cultured in the absence of DM (p < 0.01). Inhibition of membrane IL-2R alpha and IL-2R beta expression by 10(-6) M DM was partially reversible by recombinant human IL-2 (rhIL-2). By Northern blot analysis, DM caused a comparable decrease in IL-2R alpha and in IL-2R beta mRNA levels to membrane receptor expression in mitogen-stimulated T cells. By in vitro transcription assays, DM regulated IL-2R alpha gene expression at a transcriptional level while transcription of IL-2R beta gene was unaffected by DM. The mechanism of action of DM on IL-2R alpha transcription was examined by determining the mRNA levels of the p50 subunit of nuclear factor kappa B (NF-kappa B), a transcription factor that stimulates IL-2R alpha gene expression. The data indicate that 10(-6) M DM increased T cell p50 NF-kappa B mRNA levels by four-fold compared to the levels obtained in the absence of DM. Further, the level of nuclear proteins capable of binding to the NF-kappa B sites in activated T cells increased in response to DM. In sum, DM regulates T cell membrane expression of IL-2R by more than one molecular mechanism.

Effects of repeated stress on T cell numbers and function in rats.

Although stress has been reported to affect various functions of the immune system, the mechanism that mediate these effects remain unclear. Thus we examined the effects that 1, 7, and 14 days of stress could have on various aspects of immune and endocrine function in rats. Rats subjected to repeated stress (7 and 14 days) showed significant decreases in the total number of mononuclear cells, particularly suppressor/cytotoxic (CD8) T cells, in the spleen and blood. The mitogenic responses of T cells to phytohemagglutinin (PHA) and concanavalin-A (Con A) were also significantly diminished at these times, as well as after acute (1 day) stress in the case of PHA stimulation. The mechanisms of this impaired T cell mitogenesis were explored by assessing the effects of stress on T cell interleukin 2 (IL-2) production and T cell responsiveness to IL-2. T cells from repeatedly stressed rat showed a decreased production of IL-2 in response to PHA, although their proliferative response to exogenous IL-2 was normal. Repeated stress also decreased body weight and spleen weight, increased adrenal weight, and decreased plasma levels of triiodothyronine and testosterone. These results suggest that lower levels of IL-2 production during stress could be one reason for the decreased mitogen responsiveness of T cells, often seen with stress. This is important because defective IL-2 production could also lead to significant impairment of immunoregulatory T cell generation and thus a predisposition to malignancy or autoimmune disease that some have associated with stress.

Commitment to erythroid or granulocyte-macrophage differentiation is associated with loss of macromolecular insoluble cold globulin.

In mice a macromolecular insoluble cold globulin (MICG) is present on the surface of resting and stimulated peripheral T lymphocytes, thymocytes, fetal prothymocytes and hemopoietic stem cells forming spleen colonies, colony-forming unit-spleen (CFU-S). Here we demonstrate that removal of MICG positive cells from bone marrow by treatment with antibody and complement does not affect the number of erythroid, burst-forming unit-erythroid (BFU-E) and granulocyte-macrophage, colony-forming unit-granulocyte macrophage (CFU-GM) progenitors developing in vitro. Thus, commitment of stem cells to lineage specific differentiation is associated with the loss of the MICG marker. Furthermore, the removal of MICG positive T lymphocytes from bone marrow does not affect the growth of BFU-E or CFU-GM.

Ontogeny of interleukin 2 responsive cells in the fetal thymus.

The ontogeny of IL 2-responsive cells in the thymus of CBA/J mice was examined in neonatal animals and in fetuses at 14, 16, and 18 to 20 days gestation. The thymocytes were tested for responsiveness to 2 micrograms/ml Con A, TCGF, IL 2, and co-stimulation by Con A plus TCGF or IL 2. These responses were compared with those of thymocytes of 6- to 8-wk-old CBA/J. Thymocytes (1 X 10(5)) were cultured, and the reaction was measured at maximum response (96 hr). Neonatal animals gave an unusually high response to TCGF or partially purified IL 2 alone, approximately five times greater than the adult. A low but significantly enhanced proliferation, stimulated by partially purified IL 2 alone, was observed with 14-day fetal thymocytes, even though cultures of these cells in medium alone had higher background proliferation than any other age tested. In the co-stimulator reaction, proliferation significantly above background was measured at 16 days of gestation with Con A plus TCGF. The magnitude of the co-stimulator reaction increased with age, especially between the 16th and 18th day of gestation and immediately after birth.

Macromolecular insoluble cold globulin (MICG): a marker for pluripotential hemopoietic stem cells.

In embryonic mice pluripotential hemopoietic stem cells (PHSC) originate in the yolk sac and migrate to the fetal liver and from there to the bone marrow. Hemopoietic cells from yolk sac and fetal liver also migrate to the thymic primordium, and within the thymic environment these prothymocytes differentiate into mature T cells. We have recently demonstrated that macromolecular insoluble cold globulin (MICG), a T cell marker, is synthesized and inserted into the plasma membrane of embryonic prothymocytes as soon as these cells appear in the early thymus. In addition, we have shown that MICG+ cells are present within the fetal liver before the thymus has fully formed. In the present study we show that pluripotential hemopoietic stem cells in the fetal liver and bone marrow have MICG on their surface and represent a subpopulation of these MICG+ cells. The implications of these findings in relationship to stem cell differentiation and isolation are discussed.

Macromolecular insoluble cold globulin (MICG): a marker for embryonic prothymocytes in the mouse.

Adult thymocytes and peripheral T cells synthesize and insert a glycoprotein into their plasma membrane that is termed macromolecular insoluble cold globulin (MICG). This protein, in contrast to other T cell markers, was shown to be present in embryonic prothymocytes as soon as they appear in the thymic rudiment. Furthermore, some hematopoietic cells in the fetal liver were shown to contain MICG at a period in gestation before the completion of lymphocyte migration to the thymus. These results are discussed in terms of the requirements for embryonic cell migration.