Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

Interleukin-1beta and tumor necrosis factor-alpha produce distinct, time-dependent patterns of acute arthritis in the rat knee.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synergistically induce and sustain arthritis. Two competing hypotheses of arthritis induction are 1) that TNF preferentially mediates inflammation, whereas IL-1 impels bone destruction, or 2) that either cytokine controls the entire process. In this study, these propositions were tested in two experiments by instilling IL-1beta or TNF-alpha into one knee of Lewis rats (n = 6/group) to incite arthritis, after which semiquantitative scores for inflammation, bone resorption, osteoclasts, and cartilage integrity were acquired. In the induction study, IL-1beta or TNF-alpha (3, 10, or 30 micro g) was given once to incite arthritis. After 2 days, IL-1beta induced significant, dose-dependent increases in inflammation (mild to marked), bone resorption (minimal to moderate), and osteoclasts (minimal to moderate). In contrast, TNF-alpha induced minimal to mild inflammation but had little impact on resorption or osteoclasts. Both IL-1 and TNF (>/=10 micro g) yielded mild cartilage degeneration. Most lesion scores in TNF-treated rats were significantly lower than those in animals given the same dose of IL-1beta. In the persistence study, rats were injected once with IL-1 or TNF (10 micro g) and maintained for 2, 3, or 7 days. IL-1beta significantly enhanced inflammation (all 3 days), bone resorption (days 2 and 3), osteoclasts (days 2 and 3), and cartilage matrix loss (days 2 and 3), whereas TNF-alpha augmented inflammation (days 2 and 3) and cartilage degeneration (day 2) but not bone resorption or osteoclasts. Thus, both IL-1beta and TNF-alpha can launch inflammation, but IL-1beta drives skeletal destruction.

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.

Evidence that the protein tyrosine phosphatase (PC12,Br7,Sl) gamma (-) isoform modulates chondrogenic patterning and growth.

One of the earliest events in bone morphogenesis is the condensation of embryonic mesenchymal cells into chondroblasts and their subsequent proliferation and differentiation into chondrocytes. During this time, certain signaling cascades operate to establish proper patterning and differentiation of the cartilaginous skeleton. Characterization of the signaling pathways involved in these processes remains to be accomplished. We have identified a novel murine cytosolic tyrosine phosphatase termed PTPPBS gamma (+/-) which is a member of the PTP PC12,Br7,Sl (PTPPBS) family. Spatio-temporal expression analysis of the members of this tyrosine phosphatase family demonstrates significant expression of the gamma (-) splice variant in the cartilaginous skeleton. Using an embryonic mandibular explant culture system to serve as a model for cartilage formation, we examined the potential roles of the PTPPBS gamma phosphatase by loss-of-function studies achieved with antisense oligodeoxynucleotides. These studies demonstrated that loss of expression of the PTPPBS gamma (-) isoform resulted in abnormal patterning of Meckel's cartilage and an increase in the size of the chondrogenic regions. In gamma antisense-treated explants, bromodeoxyuridine-pulse labeling studies revealed increased proliferation of chondroblasts bordering along precartilaginous condensations and bordering populations of maturing chondrocytes. These studies provide evidence that in early skeletal development, PTPPBS gamma may regulate the rate of chondroblast proliferation in the cartilaginous skeleton.

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

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.