John Tarpley

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

The candidate neuroprotective agent artemin induces autonomic neural dysplasia without preventing peripheral nerve dysfunction.

Artemin (ART) signals through the GFRα—3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfrα—3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.

Role of hypoxia in growth factor responses: differential effects of basic fibroblast growth factor and platelet-derived growth factor in an ischemic wound model.

Both recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer are potent inducers of new tissue generation in models of normal dermal repair. However, their therapeutic targets include chronic wounds, which are frequently characterized by local tissue hypoxia. To explore the potential of recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer to stimulate more clinically relevant repair, we created an ischemic dermal wound on the rabbit ear by ligating two of the arteries which feed the ear. Both recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer stimulated marked neovascularization of the wound (p < 0.0001), but only recombinant platelet‐derived growth factor‐BB homodimer accelerated and augmented granulation tissue formation (p = 0.01) and reepithelialization. This study is the first demonstration of a direct angiogenic effect of recombinant platelet‐derived growth factor‐BB homodimer in vivo. India ink perfusion coupled with endothelial cell‐specific histochemistry showed that nearly all the neovessels in all wounds were functional, indicating rapid capillary morphogenesis. In the nonischemic (normal) rabbit ear, recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer accelerated healing comparably, as expected. Higher doses of recombinant basic fibroblast growth factor also failed to elicit stimulatory effects in ischemic wounds. These results indicate that differential responsiveness to growth factors is related to local tissue hypoxia, angiogenesis alone is an insufficient stimulus for repair. These data also suggest new therapeutic approaches for the treatment of chronic wounds.

Novel erythropoiesis stimulating protein (darbepoetin alfa) alleviates anemia associated with chronic inflammatory disease in a rodent model

OBJECTIVE We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy. METHODS Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly. RESULTS Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing. CONCLUSION ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Hematologic effects of stem cell factor (SCF) and leukemia inhibitory factor (LIF) in vivo: LIF-induced thrombocytosis in SCF-primed mice

Abstract: Stem cell factor (SCF) administered as daily bolus injections in dose‐response experiments in mice causes a progressive and dramatic dose‐dependent panleukocytosis characterized by neutrophilia, eosinophilia, monocytosis, and lymphocytosis. SCF causes circulating platelet numbers to be dose‐dependently increased after 2 weeks of daily injections. Leukemia inhibitory factor (LIF) administered as daily bolus injections in mice causes a peripheral leukopenia that is largely due to peripheral lymphopenia. LIF causes thrombocytosis peaking after approximately 1 w. Coinjection of SCF and LIF for 1 to 2 wk in mice does not cause a much greater thrombocytosis than the maximum thrombocytosis achievable with SCF or LIF alone. On the other hand, daily injection of SCF for 5 days followed by daily injection of LIF for 5 to 6 d in mice causes a very substantial increase in platelets that was lineage‐specific in terms of not being accompanied by a generalized leukocytosis. In contrast, only a very modest thrombocytosis was noted in SCF‐primed LIF‐treated rats. LIF causes a large increase in the cytoplasmic volume of splenic megakaryocytes in mice, but not in rats. In conclusion, SCF‐induced priming followed by LIF‐induced maturation of megakaryocytes causes a substantial selective increase in the numbers of circulating platelets in mice.

Prolonged neutropenia in a novel mouse granulocyte colony-stimulating factor neutralizing auto-immunoglobulin G mouse model

Therapeutic use of recombinant human cytokines in humans can result in the generation of drug-specific antibodies. To predetermine the maximum potential effects of a granulocyte colony-stimulating factor (G-CSF) neutralizing auto-immunoglobulin G (auto-IgG) response during recombinant human G-CSF therapy, we developed a mouse model of mouse G-CSF (mG-CSF) neutralizing auto-IgG response. Mice were immunized and boosted with mG-CSF chemically conjugated to either keyhole limpet hemocyanin or ovalbumin on an alternating schedule. Sera were analyzed for mG-CSF-specific titers and full blood counts were performed on a Technicon H-1E. On day 252, tissues were collected for histology. IgG was protein A affinity purified from pooled mG-CSF autoimmune sera. Mice immunized with mG-CSF conjugates produced mG-CSF-specific auto-IgG responses that lasted for the length of the study. Significant neutropenia (p(max) < 0.004) was concurrent with the rise in mG-CSF-specific IgG titers. However, neutrophil counts remained at approximately 20% of preimmunization levels through day 252. Endogenous mG-CSF neutralizing auto-IgG had no significant effect on hemoglobin, erythrocyte, lymphocyte, eosinophil, basophil, and platelet counts, and had minor, transient, or no effects on monocyte counts. Bone marrow colony assays from mG-CSF autoimmune mice demonstrated no significant effect of G-CSF neutralization on the numbers or proliferative capacity of preneutrophil lineage progenitors. Purified IgG from mG-CSF autoimmune mice neutralized mG-CSF in vitro. High-titer G-CSF neutralizing auto-IgG in adult mice partially inhibited steady-state granulopoiesis and had little or no effect on steady-state levels of other hematopoietic cells.

Adult rodent double skeletal stain

Rodent embryo double skeletal staining has long played a role in toxicological studies and is now an important part of selected genetic studies involving knockout or transgenic animals. However, phenotypic changes are sometimes not seen until animals reach adulthood. This study expands a previously developed embryonic staining method for use with adult mice.