Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.

Recombinant human platelet factor-4 (rhPF4), purified from Escherichia coli, inhibited blood vessel proliferation in the chicken chorioallantoic membrane in a dose-dependent manner. Treatment of several cell types with rhPF4 in vitro suggested that the angiostatic effect was due to specific inhibition of growth factor-stimulated endothelial cell proliferation. The inhibitory activities were associated with the carboxyl-terminal, heparin-binding region of the molecule and could be abrogated by including heparin in the test samples, an indication that sulfated polysaccharides might modulate the angiostatic activity of platelet factor-4 in vivo. Understanding of the mechanisms of control of angiogenesis by endogenous proteins should facilitate the development of effective treatments for diseases of pathogenic neovascularization such as Kaposi's sarcoma, diabetic retinopathy, and malignant tumor growth.

Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4.

Although it is well established that angiogenesis is essential to tumor development, no human protein with high specificity and efficacy for prevention of angiogenesis has been characterized. In a previous study, we demonstrated that recombinant platelet factor 4 (rPF 4) inhibited angiogenesis in the chicken chorioallantoic membrane. In the present study, we have extended that finding to the use of recombinant human platelet factor 4 (rHuPF 4) to inhibit solid tumor growth in the mouse. rHuPF 4 effectively suppressed the growth of the B16-F10 murine melanoma in syngeneic C57BL/6J hosts and prevented the growth of primary tumors of both B16-F10 murine melanoma and HCT 116 human colon carcinoma in semisyngeneic CByB6F1/J female athymic nude mice. These two transformed cell lines were completely insensitive to rHuPF 4 in vitro at levels (50 micrograms/mL) that extensively inhibit normal endothelial cell proliferation. The migration of human endothelial cells was also inhibited at these concentrations of rHuPF 4, suggesting a second mechanism by which rHuPF 4 may modulate capillary development. The observed antitumor effects of rHuPF 4 might be due to the inhibition of angiogenesis. This finding could have implications for the development of novel therapeutic approaches to angiogenic diseases. Alternative, and possibly concurrent, mechanisms of the rHuPF 4 antitumor effect include lymphokine-activated killer cell activation and the induction of other cytokines.

Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity.

An analogue of human platelet factor 4 (PF4) lacking affinity for heparin was specifically designed to evaluate the importance of this property in the antitumor effects of recombinant PF4. The purified protein, recombinant PF4-241 (rPF4-241), failed to bind heparin but retained the ability to suppress the growth of tumors in mice. Daily intralesional injections of rPF4-241 significantly inhibited the growth of the B-16 melanoma in syngeneic mice without direct inhibitory effects on B-16 cell growth in vitro. Similar antitumor effects were observed with the human colon carcinoma, HCT-116, grown in nude mice, indicating that the inhibitory activity was neither tumor-type specific nor T-cell dependent. rPF4-241 inhibited endothelial cell proliferation in vitro with dose dependence similar to the native sequence rPF4. Both rPF4 and rPF4-241 inhibited angiogenesis in the chicken chorioallantoic membrane. The analogue, however, was inhibitory at lower concentrations than rPF4 in the chorioallantoic membrane system and its inhibitory effects were not abrogated by the presence of heparin. The present findings support the conclusion that both rPF4 and rPF4-241 inhibit tumor growth by suppression of tumor-induced neovascularization. The finding that this activity is independent of heparin binding may allow the development of PF4-based angiostatic agents with reduced toxicity and improved bioavailability. These results also suggest that PF4 may play a more specific role in modulation of blood vessel development than previously recognized.

Development of angiogenesis inhibitors for clinical applications.

Angiogenesis, the development of new blood vessels, is associated with many life-threatening pathologies. The neovascularization of tumors for example, allows a blood supply to deliver the required nutrients for tumor development. Inappropriate blood vessel growth also contributes to the pathology of other diseases such as atherosclerosis and arthritis. The process of angiogenesis is beginning to be better understood, and as Ted Maione and Richard Sharpe explain, this understanding has led to the identification of several lead compounds that inhibit this process. At present all of these candidate drugs exhibit severe host toxicity, but more selective angiogenesis inhibitors might be expected to be extremely useful therapeutic agents.

Induction of dermal and subcutaneous inflammation by recombinant cachectin/tumor necrosis factor (TNF alpha) in the mouse.

The ability of cachectin/tumor necrosis factor (TNF alpha) to induce acute dermal and subcutaneous inflammation was examined in a murine model. A number of other proteins, and diluent alone were examined as controls. After subcutaneous injection into the mouse footpad, recombinant human TNF alpha (rHuTNF alpha) induced acute inflammation with an initial marked dermal and subcutaneous neutrophil infiltrate by approximately 3 h, with a peak between 4 and 24 h and resolution by 79 h. Recombinant interleukin-2, cytochrome c, and heat-inactivated rHuTNF alpha induced negligible inflammation. Recombinant human lymphotoxin (TNF beta), another control protein, also induced acute inflammation in our system. Because TNF alpha and TFN beta are partially homologous, they may be acting through a similar mechanism. This pro-inflammatory effect of TNF alpha may result from chemotactic activity as well as by induction of secondary mediators. Inflammation induced by TNF alpha was partially suppressed by indomethacin treatment, suggesting that products of the cyclo-oxyganase pathway may mediate a portion of the inflammation involved. Five daily injections of rHuTNF alpha into the mouse footpad resulted in a predominantly mononuclear infiltrate and focal fibrosis. These results suggest that TNF alpha may be an important mediator of acute inflammation in vivo and might provide a signal for the production of collagen.

Inhibition of cutaneous contact hypersensitivity in the mouse with systemic or topical spiperone: topical application of spiperone produces local immunosuppression without inducing systemic neuroleptic effects.

We tested the ability of the neuroleptic agent spiperone (8-[3-(p-fluorobenzoyl)propyl]-1-phenyl-1,3,8-triazaspiro-[4.5] decan-4- one) to influence the tissue swelling and leukocyte infiltration associated with T-cell--dependent immune responses, i.e., contact hypersensitivity reactions, in mice. Contact hypersensitivity reactions were elicited by applying the haptens oxazolone or dinitrofluorobenzene topically to one or both ears 5-8 d after epicutaneous sensitization. When spiperone was given subcutaneously at a dose of 30 or 150 mg/kg, 1 h after challenge with oxazolone, cutaneous contact hypersensitivity to this hapten was significantly diminished. When applied topically in concentrations as low as 0.08% (w/w), preparations of spiperone significantly suppressed both the tissue swelling and the leukocyte infiltration associated with the elicitation phase of contact hypersensitivity. Topical treatment with spiperone also suppressed the sensitization phase of contact sensitivity. However, mice treated topically with spiperone, unlike those treated systemically, exhibited no drowsiness or other evidence of central nervous system effects. Spiperone expresses both serotonin and dopamine receptor antagonist activity. However, unlike spiperone, the chemically unrelated serotonin antagonists, trazadone and mianserin, and the dopamine receptor antagonist, haloperidol, were not effective in suppressing contact hypersensitivity. Our results indicate that spiperone can have immunosuppressive effects on contact hypersensitivity reactions in the mouse, even when applied topically in doses that lack neuroleptic effects, and that the mechanism of action of spiperone on the immune response may be independent of its serotonin or dopamine receptor blocking properties.

Cyclosporine inhibits basic fibroblast growth factor-driven proliferation of human endothelial cells and keratinocytes.

Keratinocytes and endothelial cells produce basic fibroblast growth factor (b-FGF), and this cytokine is mitogenic for both cell types. Additionally, b-FGF is stored in the vicinity of keratinocytes and endothelial cells in basement membrane and extracellular matrix, and can be displaced from these "buffers" by various stimuli. Displacement of b-FGF by physical stimuli, such as scratching or rubbing, could explain koebnerization in diseases such as psoriasis. It has been shown that the fungal metabolite cyclosporine A will inhibit the proliferation of keratinocytes in vitro when their proliferation is driven by epidermal growth factor (EGF) and/or bovine pituitary extract. Since b-FGF may be both a positive autocrine and paracrine signal involved in the proliferation of both keratinocytes and endothelial cells, we evaluated the effects of cyclosporine on the b-FGF-driven proliferation of these cell types in vitro. We have shown that normal human keratinocyte and endothelial cell proliferation driven by b-FGF alone can be inhibited by cyclosporine A. Concentrations of cyclosporine A achievable in skin after oral administration can significantly inhibit the b-FGF-driven proliferation of both of these cell types. Basic fibroblast growth factor may be an important signal driving both keratinocyte proliferation and angiogenesis in certain disease states, such as psoriasis, as well as aberrant endothelial cell proliferation as is seen in Kaposi's sarcoma. The efficacy of cyclosporine A in treating these disease states may be due, at least in part, to the ability of cyclosporine A to interrupt b-FGF-mediated autocrine and paracrine feedback loops acting on and between endothelial cells and keratinocytes.

A tumor immunotherapy technique based on modulation of the idiotype anti-idiotype network.

In this communication I propose a technique for manipulating the idiotype anti-idiotype network with the aim of enhancing the immune response to tumors. The theoretical basis for this technique follows from Jerne's network theory of the immune system. By tolerizing the host to immunoglobulin bearing idiotypes directed against tumor associated antigens it should be possible to dampen the expected anti-idiotype response to these idiotypes. A reduced anti-idiotype response should result in an increased idiotype response and thus a greater anti-tumor immune response.

Endogenous gamma interferon production may protect against hepatic cirrhosis and administration of exogenous gamma interferon may protect individuals prone to cirrhosis.

Hepatic cirrhosis is characterized by the replacement of normal liver parenchyma by collagenous fibrous tissue. Although hepatocytes in the adult retain the ability to divide, under certain circumstances hepatocyte death leads to replacement with fibroblasts and collagen. Whether a particular form of hepatocyte injury leads to cirrhosis is dependent upon the stimulus for the injury and is also highly variable between individuals. It has recently been shown that gamma interferon inhibits collagen synthesis in vitro and fibrosis in vivo. I suggest that individuals who are prone to hepatic cirrhosis from a given stimulus are low producers of gamma interferon while high gamma interferon producers are relatively protected from cirrhosis. I also hypothesize that exogenous gamma interferon administration may halt or slow the progression of cirrhosis in patients with early progressive cirrhosis. Alternatively, endogenous gamma interferon production could be stimulated in these patients with progressive cirrhosis. One agent which may be useful for inducing endogenous gamma interferon is GE-132, an organogermanium.

The low incidence of multiple sclerosis in areas near the equator may be due to ultraviolet light induced suppressor cells to melanocyte antigens.

It has been shown by Elmets et al. (1) that ultraviolet irradiation of the skin followed by the application of a normally sensitizing antigen results in tolerance induction to the antigen. This tolerance is due to the induction of suppressor T-cells specific for the antigen. Multiple sclerosis is more common in certain climates distant from the equator where there is less ultraviolet light (sunlight). I postulate that people near the equator are exposed to more sunlight and that the ultraviolet light from the sunlight aids in the induction of suppressor cells specific for melanocyte associated antigens. Since melanocytes are of neural crest origin and share antigenic determinants with neural tissues these suppressor cells may act to protect against multiple sclerosis which is probably an autoimmune disease directed against neural tissues.

The LAV/HTLV-III virus may evade elimination by the immune system by inducing low zone tolerance to itself.

The acquired immunodeficiency syndrome (AIDS) is caused by the LAV/HTLV-III virus. The incubation period for AIDS is prolonged and on the order of years. We hypothesize that during this prolonged incubation period the LAV/HTLV-III virus is replicating very slowly and is present in extremely low concentrations. The concentrations of the virus may be low enough that the virus induces a low zone tolerance to itself in the T-cell arm of the immune system. B-cells which are resistant to direct low dose tolerance induction may remain responsive to the LAV/HTLV-III virus in a direct fashion without specific helper T-cells. Thus, anti-LAV/HTLV-III antibody may be produced even though the more important cellular immune response has been crippled by the virus. We also outline two hypothetical approaches for breaking this tolerance and restoring the cellular immune response to the LAV/HTLV-III virus.

Neonatal tolerance increases efficacy of antisera production.

In this investigation we demonstrate that neonatal tolerance can be used to increase the specificity of antisera. We have shown that if mice are immunized with a human B cell line then the antisera they produce also reacts strongly with human T cells. However, if the mice are first neonatally tolerized to a human T cell line and then immunized with the B cell line the antisera becomes much more specific for the B cell line. In theory this approach, of initially tolerizing the animal to uninteresting antigens, may also make it possible to produce monoclonal antibodies to rare cellular determinants more efficiently.

Induction of local inflammation by recombinant human platelet factor 4 in the mouse.

Platelet factor 4 (PF-4) has been shown to be chemotactic for neutrophils and monocytes in vitro. To assess whether these observations have in vivo relevance, we tested the ability of recombinant human PF-4 (rPF-4) to induce acute and chronic dermal inflammation in the mouse. When injected as a single dose intradermally, rPF-4 induced an acute inflammatory response that peaked at 6 to 12 hr and which resolved by 36 hr. Injection of an equivalent amount of cytochrome c, buffer alone, or an amino-terminal PF-4 peptide failed to elicit a significant inflammatory response; however, the carboxy-terminal PF-4 peptide retained proinflammatory properties. The inflammatory infiltrate induced by a single injection of either rPF-4 or the 41 amino acid carboxy-terminal peptide was composed of neutrophils and smaller numbers of mononuclear cells. Repeated injection of rPF-4 resulted in nearly equal numbers of neutrophils and mononuclear cells. Moreover, marked dermal fibrosis developed after only 5 days of daily injection of rPF-4. Although relatively high concentrations of rPF-4 were required to elicit an inflammatory response, these concentrations may be locally attainable during platelet aggregation. Our findings thus support the hypothesis that PF-4 may contribute to the development of inflammatory responses at sites of platelet aggregation.

Buspirone inhibits contact hypersensitivity in the mouse.

We have observed that 8-4-[4-2-pyrimidyl)-1-piperazinyl]butyl]-8-azaspiro [4.5]decane-7.9-dione, an agent commonly known as buspirone HCl, possesses immunosuppressive activity when administered either topically or systemically, as assessed in a mouse model of contact hypersensitivity. Topical or systemic administration of buspirone significantly reduced the tissue swelling and leukocyte infiltration associated with the elicitation phase of contact hypersensitivity. Buspirone is a safe, widely used drug which has a history of use in humans throughout the world. These data demonstrate a previously unknown pharmacologic activity of buspirone.

Improvement of in vitro fertilisation after treatment with buserelin, an agonist of luteinising hormone releasing hormone.

Treatment with buserelin, an agonist of luteinising hormone releasing hormone, and human menopausal gonadotrophin was compared with the conventional treatment of clomiphene citrate and human menopausal gonadotrophin in the outcome of in vitro fertilisation. Seventy seven infertile women had 83 cycles of treatment with buserelin and human menopausal gonadotrophin, and concurrently another 328 infertile women were treated with clomiphene citrate and human menopausal gonadotrophin. Seven (8%) cycles were cancelled owing to inadequate super-ovulation or ovarian hyperstimulation in the women receiving buserelin and 103 (31%) were cancelled because of poor follicular development in those receiving clomiphene citrate. The mean number of oocytes recovered was significantly higher with buserelin (9.5 (SD 4.5) v 5.5 (2.2)) as was the mean number of embryos obtained (4.3 (2.4) v 2.9 (1.7)). Significantly more women who had an embryo transfer became clinically pregnant after treatment with buserelin (53% (30/57) v 30% (48/159), or 36% v 14% of treatment cycles). Altogether 33% (10) of pregnancies in women treated with buserelin were multiple compared with 23% (11) in those treated conventionally. Of the 17 completed pregnancies in women treated with buserelin, 11 resulted in the birth of live babies (eight singletons, two sets of twins, and one set of triplets) and six failed, five before 12 weeks' gestation and one at 22 weeks. The 13 continuing pregnancies (32 weeks) were eight singletons, two sets of twins, and three sets of triplets. Of the 48 completed pregnancies in women treated with clomiphene citrate, 35 resulted in the birth of live babies (26 singletons, five sets of twins and four sets of triplets) and 13 failed, eleven before 12 weeks' gestation and two by 27 weeks. Buserelin increased the chance of pregnancy after in vitro fertilisation compared with conventional treatment, but the risk of multiple pregnancy may be increased.