Dimitry Michael Danilenko

FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.

ABSTRACT The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

Growth factors and cytokines in hair follicle development and cycling: recent insights from animal models and the potentials for clinical therapy

Hair growth disorders, particularly those that lead to hair loss (alopecia), are common and frequently cause significant mental anguish in affected individuals. The mechanisms underlying the majority of these disorders are unknown. However, insights into the specific molecular mechanisms of hair follicle development and cycling have recently been made using animal models, particularly mice that over- or underexpress a specific gene for a growth factor or cytokine. Other animal models have demonstrated that certain growth factors and cytokines can prevent much of the alopecia caused by cancer chemotherapeutic agents. These animal models have confirmed the importance of growth factors and cytokines in hair follicle development and cycling, and have formed the foundation for potential clinical therapy of hair growth disorders, particularly alopecia. Nevertheless, important questions concerning their efficacy, safety and delivery will need to be answered before successful clinical therapy of any hair growth disorder becomes a reality.

Effect of recombinant human keratinocyte growth factor (rHuKGF) on the immunopathogenesis of intestinal graft-vs.-host disease induced without a preconditioning regimen.

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6→(C57BL/6 X DBA/2)F1-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNFα, did not develop endotoxemia, and did not die. LPS augmented serum TNFα release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-α, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-γ mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-γ gene knockout grafts, suggesting that IFN-γ may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.

The role of interferon‐γ, nitric oxide and lipopolysaccharide in intestinal graft‐versus‐host disease developing in F1‐hybrid mice

(C57BL/6 × DBA/2)F1‐hybrid mice injected with lymphoid cells from wild‐type, C57BL/6 donors develop acute, lethal graft‐versus‐host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 μg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor‐α (TNF‐α) into the serum. To explore the role of interferon‐γ (IFN‐γ) in the pathogenesis of intestinal GVHD we used IFN‐γ gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild‐type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 μg LPS into recipients of wild‐type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of Nωnitro l‐arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN‐γ gko grafts. These findings indicate that donor‐derived IFN‐γ is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN‐γ gko grafts develop high levels of LPS‐induced TNF‐α release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent→F1‐hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF‐α, and that this depends on donor‐derived IFN‐γ.

Pharmacokinetics, pharmacodynamics, and safety assessment of palifermin (rHuKGF) in healthy volunteers

Palifermin is a recombinant human keratinocyte growth factor approved to decrease the incidence and duration of severe oral mucositis in patients with hematologic malignancies who received myelotoxic therapy requiring hematopoietic stem cell support.

Noninsulin-dependent diabetes mellitus occurs in mice ectopically expressing the human Axl tyrosine kinase receptor.

The axl tyrosine kinase receptor is aberrantly expressed on myeloid cells of many individuals afflicted with chronic myelogenous leukemia (CML) and other myeloid leukemias. Although previous studies demonstrated this kinase to have oncogenic potential, it is not known whether axl actively participates in the onset and/or progression of CML. We addressed this question by generating transgenic mice possessing constitutive ectopic expression of human axl throughout cells of the myeloid hematopoietic lineage through the use of the granulocyte colony‐stimulating factor (GCSF) receptor promoter. The transgenics did not exhibit hematopoietic malignancies, but did exhibit phenotypic characteristics associated with noninsulin‐dependent diabetes mellitus (NIDDM) including hyperglycemia and hyperinsulinemia, severe insulin resistance, progressive obesity, hepatic lipidosis, and pancreatic islet dysplasia. The obese‐diabetes phenotype was similar to that observed in the agouti and melanocortin‐4−/− mutants, however the axl transgenics were not hyperphagic. Axl transgenic animals expressed elevated serum tumor necrosis factor (TNF)‐alpha levels that were further enhanced upon in vitro lipopolysaccharide (LPS) stimulation of peripheral blood. Administration of the axl ligand, gas6, to peripheral transgenic blood samples eliminated excessive TNF‐alpha production in response to LPS stimulation. As a means to better understand axl‐gas6 biology, transgenic animals were produced which systemically expressed the gas6‐binding axl proteolytic cleavage product. A more severe NIDDM phenotype occurred in these mice. The observed phenotypes may be related to the axl receptor or proteolytic cleavage product competing with related axl family receptors for binding of the gas6 ligand. We conclude that axl expression in myeloid cells in itself does not lead to the onset or progression of leukemia and suggest that ectopic axl expression affects endogenous modulation of TNF‐alpha production indirectly resulting in the NIDDM phenotype. J. Cell. Physiol. 181:433–447, 1999.