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ABSTRACT: Kaposi sarcoma (KS) is a complex multicellular neoplasm that is commonly associated with AIDS. The pathogenesis of KS is not well understood. KS tumor cells grow poorly in vitro and require medium conditioned by retrovirus-infected T lymphocytes. We observed that conditioned medium (CM) from type II human T-cell leukemia virus (HTLV-II)-infected T cells (HTLV-II CM) induces conversion of endothelial cells (ECs) to a KS tumor cell-like phenotype. ECs grown in HTLV-II CM acquired a spindle-shaped morphology, the ability to express factor XIIIa and other KS cell markers, and a cytokine production profile similar to that of KS cells. We found that HTLV-II CM contains large quantities of scatter factor (SF), an angiogenic cytokine that stimulates cell motility. SF induced ECs to become spindle-shaped and express factor XIIIa. Moreover, SF was found to be a mitogen for KS cells in vitro and was identified within KS lesions in vivo. SF mRNA was present in KS cells in vitro, and antibodies against SF inhibited the growth of KS cells. The receptor for SF, the c-met protein, was expressed by ECs, dermal dendrocytes, and KS tumor cells in vitro and in vivo. HTLV-II CM was highly angiogenic in vivo, which was blocked by antibodies against SF. Based on these findings, we suggest that SF plays a role in the initiation and maintenance of KS lesions.
Proceedings of the National Academy of Sciences 07/1994; 91(12):5281-5. · 9.68 Impact Factor
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ABSTRACT: We investigated the role of interleukin 8 (IL-8) in mediating angiogenesis in human bronchogenic carcinoma. Increased quantities of IL-8 were detected in tumor tissue as compared with normal lung tissue. Immunohistochemical staining of tumors revealed primary localization of IL-8 to individual tumor cells and demonstrated the capacity of tumor to elaborate IL-8. Functional studies that used tissue homogenates of tumors demonstrated the induction of both in vitro endothelial cell chemotaxis and in vivo corneal neovascularization. It is important to note that the addition of neutralizing antisera to IL-8 to these assays resulted in the marked and specific attenuation of these responses. Our observations definitively establish IL-8 as a primary mediator of angiogenesis in bronchogenic carcinoma and offer a potential target for immunotherapies against solid malignancies.
Journal of Experimental Medicine 06/1994; 179(5):1409-15. · 13.85 Impact Factor
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ABSTRACT: Human monocytes (M phi) require stimulation with substances such as bacterial endotoxin [LPS (lipopolysaccharide)] to produce angiogenic activity. In this study, we report that stimulation of M phi with LPS (5 micrograms/ml) in the absence of L-arginine greatly reduced their production of angiogenic activity, as assessed in vivo in rat corneas and in vitro by chemotaxis of human umbilical vein endothelial cells (HU-VECs). D-Arginine did not substitute for L-arginine in the production of angiogenic activity. The nitric oxide synthase (NO synthase, EC 1.14-13.39) inhibitors NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) both inhibited the production of angiogenic activity by LPS-stimulated M phi in the presence of L-arginine, suggesting the involvement of this enzyme in the pathway that generates angiogenic activity. Neither of these substances directly inhibited the M phi-derived angiogenic activity. LPS-induced production of the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) was not significantly reduced when M phi were incubated in the absence of L-arginine. Similarly, L-NMMA and L-NAME did not significantly reduce the LPS-induced production of these cytokines by M phi in the presence of L-arginine. These results suggest that the LPS-stimulation-dependent generation of angiogenic activity by M phi requires an L-arginine-dependent NO-synthase effector mechanism that may be independent of the generation of TNF-alpha and IL-8.
Proceedings of the National Academy of Sciences 06/1994; 91(10):4190-4. · 9.68 Impact Factor
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ABSTRACT: Psoriasis is a common inherited skin disease that is characterized by hyperproliferation of epidermal keratinocytes and excessive dermal angiogenesis. A growing body of evidence supports a key pathogenetic role for activated keratinocytes in the angiogenic response that accompanies psoriasis. We investigated the role of psoriatic epidermis in the aberrant expression of angiogenesis by examining the ability of pure populations of multipassaged keratinocytes obtained from the skin of normal individuals and psoriatic patients to induce angiogenesis in vivo in the rat corneal bioassay and endothelial cell chemotaxis in vitro. Media conditioned by keratinocytes from psoriatic patients, including both symptomless skin and psoriatic plaques, induced vigorous angiogenic responses in over 90% of corneas tested and potently stimulated directional migration of capillary endothelial cells in vitro. In contrast, conditioned medium from normal keratinocyte cultures was weakly positive in less than 10% of corneas assayed and failed to stimulate endothelial cell chemotaxis. Furthermore, keratinocytes from psoriatic skin exhibited a 10- to 20-fold increase in interleukin-8 production and a seven-fold reduction in thrombospondin-1 production. The angiogenic activity present in keratinocyte-conditioned media from psoriatic patients was suppressed by adding either highly purified thrombospondin-1 (125 ng) or following the addition of either normal keratinocyte-conditioned media or neutralizing interleukin-8 antibody. We conclude that psoriatic keratinocytes are phenotypically different from normal keratinocytes with respect to their angiogenic capacity and that this aberrant phenotype is attributable to a defect in the overproduction of interleukin-8 and a deficiency in the production of the angiogenesis inhibitor thrombospondin-1.
American Journal Of Pathology 05/1994; 144(4):820-8. · 4.89 Impact Factor
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ABSTRACT: Previous investigations have shown that macrophages play a pivotal role in the induction of angiogenesis in both physiological and pathological settings. This investigation examines the relative production of the angiogenic modulator thrombospondin-1 (TSP1) by activated and nonactivated monocytes and macrophages. TSP1, a multifunctional extracellular matrix molecule, has been reported recently to inhibit angiogenesis both in vitro and in vivo. To examine the relationship between the level of TSP1 production by macrophages and expression of the angiogenic phenotype, murine monocytelike cells (WEHI-3) and human peripheral blood monocytes were each activated in vitro and examined for TSP1 production and angiogenic activity in rat corneal bioassay. Nonangiogenic monocytes produced low levels of TSP1 messenger RNA. Surprisingly, activated, potently angiogenic monocytes and macrophages exhibited as much as a sixfold increase in steady state TSP1 messenger RNA over unstimulated levels. Biosynthetic labeling studies demonstrated that TSP1 protein secretion increased in conjunction with increased TSP1 messenger RNA levels in angiogenic macrophages. The results demonstrate that activated monocytes and macrophages actively produce the angiogenic modulator TSP1 and suggest that TSP1 production may be a component of the angiogenic phenotype. In addition, the data suggest that the ability of macrophages to mediate angiogenesis results from a complex interplay of positive and negative regulators.
American Journal Of Pathology 10/1993; 143(3):678-84. · 4.89 Impact Factor
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Behring Institute Mitteilungen 09/1993;
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ABSTRACT: Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2-terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19-residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.
The Journal of Cell Biology 08/1993; 122(2):497-511. · 10.26 Impact Factor
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ABSTRACT: Angiogenic factors produced by monocytes-macrophages are involved in the pathogenesis of chronic inflammatory disorders characterized by persistent angiogenesis. The possibility was tested that interleukin-8 (IL-8), which is a cytokine that is chemotactic for lymphocytes and neutrophils, is also angiogenic. Human recombinant IL-8 was potently angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells. Angiogenic activity present in the conditioned media of inflamed human rheumatoid synovial tissue macrophages or lipopolysaccharide-stimulated blood monocytes was equally blocked by antibodies to either IL-8 or tumor necrosis factor-alpha. An IL-8 antisense oligonucleotide specifically blocked the production of monocyte-induced angiogenic activity. These data suggest a function for macrophage-derived IL-8 in angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.
Science 01/1993; 258(5089):1798-801. · 31.20 Impact Factor
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ABSTRACT: A rabbit corneal pocket model was used to demonstrate that physiologic concentrations of human recombinant (r) IL-8 may induce corneal neovascularization. Computer-assisted analysis of sequential fluorescein angiograms showed that rIL-8 doses ranging from 2 to 40 ng/cornea (P = 0.01), but not high dose rIL-8 (400 ng/cornea), results in neovascularization within 14 days. Repeat fluorescein angiograms 6 weeks after placing angiogenic doses of rIL-8 demonstrated significant regression (P = 0.01) of the vascularity present at 2 weeks, suggesting that IL-8 angiogenesis undergoes dynamic modulation similar to that normally seen in wound healing. To our knowledge, this is the first study showing an angiogenic role for IL-8, a finding that emphasizes the interplay between inflammation and wound healing. Our results imply that corneal-derived IL-8 may be important in corneal neovascularization, in particular, and that IL-8 may modulate wound healing in general. Finally, these results raise the possibility that corneal-derived cytokines, such as IL-8, may obfuscate the effects of agents tested in experimental corneal pocket models.
American Journal Of Pathology 01/1993; 141(6):1279-84. · 4.89 Impact Factor
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ABSTRACT: The anticarcinogenic action of the garlic constituent diallyl sulfide (DAS), was examined in the hamster buccal pouch and forestomach. Groups of hamsters were topically treated, for up to 14 weeks, with a 0.5% solution of the buccal pouch and forestomach carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Prior to, during and after DMBA treatment, groups of hamsters were also treated, on alternate days, with a 1% solution of DAS. In addition to tumor formation, the induction of gamma-glutamyl transpeptidase (gamma GT) buccal pouch epithelial lesions served as an additional presumptive index of in vivo carcinogenesis/anticarcinogenesis. DAS resulted in a significant reduction in buccal pouch tumor frequency, buccal pouch tumor burden, buccal pouch gamma GT lesion frequency and forestomach tumor frequency. In a separate experiment, DAS also reduced the level of autoradiographically quantified unscheduled DNA repair synthesis (UDS) in pieces of hamster buccal pouch concurrently exposed in vitro to the potent buccal pouch carcinogen N-methyl-N-benzylnitrosamine (MBN). This study demonstrates that DAS is an effective anticarcinogenic agent in squamous mucosa of the hamster and suggests novel cost-effective strategies for the rapid identification of tissue-specific anticarcinogens and a quantitative assessment of their efficacy.
Cancer Letters 11/1992; 66(3):207-16. · 4.24 Impact Factor
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ABSTRACT: Scleroderma is a disease characterized by proliferative vascular lesions in which monocytes/macrophages may play a key role. Monocytes were isolated from 14 scleroderma patients and 11 normal controls and cultured with or without lipopolysaccharide (LPS) (5 micrograms/ml). Monocyte-conditioned medium was assayed in the rat corneal bioassay for angiogenesis. Conditioned medium from normal monocytes was nonangiogenic, as was conditioned medium from scleroderma monocytes. While conditioned medium from LPS-activated normal monocytes was potently angiogenic in 11/13 corneas, conditioned medium from LPS-activated scleroderma monocytes was angiogenic in only 3/14 corneas. Levels of the angiogenic cytokine tumor necrosis factor-alpha (TNF-alpha) were measured in conditioned medium from scleroderma and normal monocytes. TNF-alpha levels were not significantly different in patient and control groups and thus do not account for the decreased angiogenic activity exhibited by scleroderma monocytes. As monocytes require activation to produce angiogenic activity, we determined the cell surface binding of monoclonal antibodies to activation-related (HLA-DR, 3D8, and 8D7) and other (Leu-M5) markers on monocytes by radioimmunoassay. Monocytes were cultured alone, with LPS (5 micrograms/ml), or with interferon-gamma (IFN) (200 units/ml). The usual increase in binding of anti-HLA-DR on stimulation of scleroderma monocytes with IFN was slightly less than that of controls. IFN-stimulated monocytes bound less anti-8D7 than controls. Anti-3D8 and anti-Leu-M5 binding was comparable in both groups. These results suggest that scleroderma monocytes do not produce normal levels of angiogenic activity with LPS stimulation, have some altered markers of activation on their cell surfaces, and may thus contribute to the aberrant vascular proliferation found in this disease.
Clinical Immunology and Immunopathology 09/1992; 64(2):153-60.
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ABSTRACT: We showed previously that thiol-containing compounds inhibited the production of macrophage-mediated angiogenic activity. Since thiol-containing compounds may act on macrophages by affecting activation and inhibiting the production of oxygen free-radicals, we studied the effects of oxygen free-radical scavengers on production of angiogenic activity by elicited mouse peritoneal macrophages and lipopolysaccharide stimulated normal human monocytes. Monocyte/macrophage conditioned media were potently angiogenic when assayed in rat corneas, while conditioned media, from oxygen free-radical scavenger-treated cells were not. The inhibitory effect of oxygen free-radical scavengers was due to a direct effect on monocyte/macrophage production of angiogenic activity but was not due solely to a decrease in the production of the macrophage-derived angiogenic cytokine tumor necrosis factor-alpha. We conclude that oxygen free-radical scavengers are potent inhibitors of the production of macrophage-mediated angiogenic activity.
Cell Biology International Reports 06/1992; 16(5):415-25.
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ABSTRACT: Macrophage (M phi)-mediated angiogenesis is believed to play an important role in the pathogenesis of rheumatoid arthritis. Gold sodium thiomalate, which is used in the treatment of rheumatoid arthritis, is a potent inhibitor of the production of m phi-derived angiogenic activity. To determine the mechanism of this inhibition, we studied the effects of thiol containing compounds (TCCs) on elicited mouse peritoneal m phi and lipopolysaccharide stimulated normal human monocytes. Monocyte/m phi conditioned media were potently angiogenic when assayed in rat corneas, while conditioned media from viable monocyte/m phi s treated with TCCs (at concentrations of 8.3-16.6 x 10(-5) M) were not. TCCs inhibited production of angiogenic activity by the m phi s rather than affecting other components of the angiogenic response such as the angiogenic factors or the target microvasculature of the rat cornea. Levels of the angiogenic mediator tumor necrosis factor-alpha (TNF-alpha) were not decreased in conditioned media of monocyte/m phi s treated with TCCs. We conclude that TCCs are potent inhibitors of the production of m phi-mediated angiogenic activity. This action of TCCs on m phi s may be in part responsible for the mechanism of action of therapeutic gold compounds in rheumatoid arthritis.
Agents and Actions 12/1991; 34(3-4):350-7.
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ABSTRACT: In order to test the hypothesis that the property of resistance to cytotoxicity is an acquired trait of premalignant oral mucosal epithelium, cell dissociates were prepared from in vivo initiated hamster buccal pouch epithelium (HBPE), non-initiated HBPE and malignant HBPE cell lines. These cell types were evaluated for resistance to the cytotoxic effects of the inducing carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). A mitoinhibition assay and a clonogenicity assay were used to assess the ability of these cells to replicate or form colonies in the presence of 40 microM DMBA. Replication of primary plated HBPE cells was inhibited by 100% in both assays. PO II, a cell line derived from non-initiated, paraffin-oil-exposed HBPE, was inhibited by 97 and 100% in the mitoinhibition and colony-forming assays respectively. This same cell line, like primary plated HBPE, lacked the transformation-linked traits of angiogenesis and anchorage-independent growth. By contrast, three malignant HBPE cell lines, two derived during long-term culture of DMBA-initiated HBPE, and one from a DMBA-induced HBPE carcinoma, were inhibited by only 34% or less in the assays for resistance to cytotoxicity. Primary cell cultures derived from HBPE initiated in vivo with twice-weekly topical applications of a 0.5% solution of DMBA in paraffin oil, for 3 or 5 weeks, were inhibited to an intermediate degree, indicating the presence of DMBA-resistant cells. In addition, DMBA-resistant cell colonies were observed in cell cultures prepared at 2, 6 and 10 weeks after completing the 5 week initiation regimen. Progenitors of the resistant cells, persisting in vivo for several weeks after initiation, may represent early preneoplastic cell populations in this experimental model.
Carcinogenesis 05/1991; 12(4):617-22. · 5.70 Impact Factor
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Methods in Enzymology 02/1991; 198:440-50. · 2.04 Impact Factor
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ABSTRACT: Inhibition of DNA synthesis was observed and quantitated in hamster buccal pouch epithelium exposed in vivo and in vitro to indirect acting carcinogens. Topical application of a 0.5% solution of the potent hamster buccal pouch carcinogen 7,12-dimethylbenz[a]-anthracene (DMBA) acutely inhibited epithelial DNA synthesis by 40-65%, as indicated by a decrease in [3H]thymidine incorporation over a period of 24 h. When applied twice at a concentration of 2%, N-methyl-N-benzylnitrosamine (MBN), another potent buccal pouch carcinogen, inhibited epithelial DNA synthesis by 76% within a period of 4 h. A similar acute inhibitory effect on DNA synthesis was observed when explants of buccal pouch mucosa, exhibiting continuous cell replication, were exposed in vitro in the presence of MBN or DMBA for periods up to 12 and 24 h, respectively. The inhibitory effect of DMBA was greater than that of other polycyclic aromatic hydrocarbons of lesser carcinogenic potency in this tissue. This study demonstrates that the metabolic activation of indirect acting carcinogens leading to acute cytotoxicity and inhibition of DNA synthesis occurs rapidly in hamster buccal pouch mucosa exposed to these agents in vitro as well as in vivo. The experimental imposition of an acute inhibitory pressure, applied as demonstrated in this report, may enable the detection of precancerous cells which have acquired the property of resistance to this cytotoxic effect in the course of carcinogenesis. In principle, the in vitro approach, coupled with autoradiography, may be useful in identifying microscopic foci of resistant preneoplastic cells in samples of human oral mucosa. 24R01 34160
Cancer Letters 10/1990; 53(2-3):163-73. · 4.24 Impact Factor
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ABSTRACT: A secreted inhibitor of angiogenesis that is controlled by a tumor suppressor gene in hamster cells has been found to be similar to a fragment of the platelet and matrix protein thrombospondin. The two proteins were biochemically similar and immunologically crossreactive and could substitute for one another in two functional assays. Human thrombospondin inhibited neovascularization in vivo and endothelial cell migration in vitro, as does the hamster protein, gp140. gp140 sensitized smooth muscle cells to stimulation by epidermal growth factor, as does human thrombospondin. The thrombospondin gene has been localized on human chromosome 15. These results demonstrate a function for the ubiquitous adhesive glycoprotein thrombospondin that is likely to be important in the normal physiological down-regulation of neovascularization. In addition, they raise the possibility that thrombospondin may be one of a number of target molecules through which a tumor suppressor gene could act to restrain tumor growth.
Proceedings of the National Academy of Sciences 10/1990; 87(17):6624-8. · 9.68 Impact Factor
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ABSTRACT: As normal cells undergo neoplastic transformation, multiple suppressor gene functions are lost or inactivated. However, the relative contribution that individual suppressor gene defects play in the sequential evolution of solid tumors has not yet been evaluated in a readily analyzable experimental model of carcinogenesis. The present study was undertaken to: (a) document the loss of suppressor genes implicated in the control of angiogenic activity, anchorage and serum growth requirements, and proliferative life span, in populations of hamster buccal pouch (HBP) keratinocytes (Kr) initiated in vivo with the chemical carcinogen 7,12 dimethylbenz(a)anthracene and (b), to determine what combination of defective suppressor genes are necessary for tumorigenesis. Kr were isolated from HBPs at various times after treating the mucosal surfaces in vivo with 7,12 dimethylbenz(a)anthracene. Cells or their conditioned culture media were evaluated for: (a) angiogenic activity in vivo and in vitro, (b) anchorage independent growth, (c) growth in low serum, (d) immortality, and (e) tumorigenicity. Angiogenic activity and immortality were the first two phenotypes detected with anchorage independence and tumorigenesis emerging late in the carcinogenic process. Hybrids generated between Kr which were angiogenic, but otherwise negative for all other phenotypic traits, and a transformed and tumorigenic HBP carcinoma cell line, E1-1, were suppressed for all phenotypes except angiogenic activity and none of the hybrids were tumorigenic. In contrast, Kr positive for angiogenic activity, anchorage independence, immortality, and tumorigenicity and hybrids generated between these cells and E1-1 carcinoma, were tumorigenic. However, hybrids between a nontumorigenic, anchorage independent, immortal but nonangiogenic Kr, and the E1-1 line were not tumorigenic. These results suggest that (a) angiogenic activity is an early phenotypic trait that emerges as a result of the loss of a suppressor gene function and (b) loss of this function is essential but not sufficient for the development of HBP tumors.
Laboratory Investigation 10/1990; 63(3):298-306. · 3.64 Impact Factor
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Northwestern dental research 02/1990; 2(1):15-9.
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Northwestern dental research 02/1990; 2(1):6-8.
