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.

Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis.

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice.

TALL-1/Blys/BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupus-like changes in transgenic mice expressing TALL-1 under the control of a beta-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220+ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cell-associated autoimmune diseases.

Synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein.

BACKGROUND & AIMS: Genetic diseases reflecting abnormal hepatocyte function are potentially curable through gene therapy. Retroviral vectors offer the potential for permanent correction of such conditions. These vectors generally require cell division to occur to allow provirus entry into the nucleus, initiated in many experimental protocols by partial hepatectomy. We have explored methods to improve the efficiency of retroviral gene transfer that avoid the need for liver damage. METHODS: Triiodothyronine (T3) and keratinocyte growth factor (KGF) were used to induce hepatic proliferation in rats. The effects of intraportal and peripheral administration of a modified retrovirus that encoded the Lac Z gene during growth factor-induced liver hyperplasia were analyzed. RESULTS: T3 initiated hepatocyte proliferation midzonally; after KGF, proliferation was more diffuse. Optimal concentrations of T3 and KGF acted synergistically to induce proliferation in 61% of hepatocytes in the intact liver. This enabled in vivo hepatocyte transduction, leading to gene expression by up to 7.3% of hepatocytes after intraportal retroviral vector administration and 7. 1% after peripheral venous administration. CONCLUSIONS: T3 and KGF act synergistically to induce hepatocyte proliferation in undamaged liver. The liver can be simply transduced with integrating vectors via the peripheral venous system during a wave of growth factor-induced proliferation.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism.

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

Brca1 required for T cell lineage development but not TCR loci rearrangement.

Brca1 (breast cancerl, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell-specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1-/- mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53-/- backgrounds, completely restored survival and development of Brca1-/- thymocytes; peripheral T cell numbers were not totally restored in Brcal-/- p53-/- mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1-/- thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand.

Bone remodelling and bone loss are controlled by a balance between the tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its decoy receptor osteoprotegerin (OPG). In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts. OPGL expression in T cells is induced by antigen receptor engagement, which suggests that activated T cells may influence bone metabolism through OPGL and RANK. Here we report that activated T cells can directly trigger osteoclastogenesis through OPGL. Systemic activation of T cells in vivo leads to an OPGL-mediated increase in osteoclastogenesis and bone loss. In a T-cell-dependent model of rat adjuvant arthritis characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of OPGL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation. These results show that both systemic and local T-cell activation can lead to OPGL production and subsequent bone loss, and they provide a novel paradigm for T cells as regulators of bone physiology.

[Local fibrinolytic treatment in subtotal pulmonary embolism]. / Lokális fibrinolyticus kezelés subtotalis tüdóembóliában.

Authors present an interventional radiological technique used for a successful treatment of 12 patients with massive pulmonary embolism. The method for rapid statement of diagnosis and the range of indications are discussed. In massive pulmonary embolism, after a diagnostic angiography, in 12 patient's mechanical thrombus drilling, in 3 cases thrombus aspiration, in 8 cases thrombus explosion and in other 1 balloon recanalisation was performed combined with a low dose (20,000-60,000 IU/h Streptokinase or the double dose of Urokinase) local fibrinolytic infusion. No major complication was observed and all the patients were successfully treated.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

Keratinocyte growth factor ameliorates dextran sodium sulfate colitis in mice.

Keratinocyte growth factor (KGF) is emerging as an important mediator of mucosal defense and repair in the colon. The aim of the present study was to evaluate and further characterize the effects of exogenous KGF administration utilizing the dextran sodium sulfate (DSS) model of colitis in mice. Colitis was induced via oral administration of DSS (5 g/100 ml) to Balb/c mice for eight days. Intraperitoneal administration of KGF (5 mg/kg, once daily) or vehicle (VEH) was initiated 1 hr prior to the induction of the colitis (N = 10, each group). Mucosal injury of the entire colon was histologically assessed and graded. An approximately fourfold reduction in the crypt damage score was noted in the KGF group when compared to controls (VEH) (2.8 +/- 1.03 and 11.4 +/- 0.78, respectively). The significant reduction of mucosal injury in KGF treated mice confirms that KGF is a key mediator maintaining the integrity of the colonic mucosa.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Intravenous keratinocyte growth factor protects against experimental pulmonary injury.

Keratinocyte growth factor (KGF) administered by intratracheal instillation is well documented to stimulate the proliferation of alveolar and bronchial cells. In the present study, intravenous KGF was also shown to stimulate the proliferation of alveolar and bronchial cells in mice and rats, although to a lesser degree than intratracheal KGF. Despite the decreased potency of intravenous KGF on pulmonary cell 5-bromo-2'-deoxyuridine incorporation compared with intratracheal KGF, intravenous KGF was very effective in preventing experimental bleomycin-induced pulmonary dysfunction, weight loss, and mortality in either mice or rats and experimental hyperoxia-induced mortality in mice. The effectiveness of intravenous administration of KGF in preventing lung injury suggests that the mechanisms of the protective effect of KGF may involve more than pulmonary cell proliferation and also suggests the potential use of systemic KGF for clinical trials in settings of pulmonary injury.

Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice.

Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G alpha q class genes, G alpha q and G alpha 11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [G alpha q(-/+);G alpha 11(-/-) or G alpha q(-/-);G alpha 11(-/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, G alpha q(-/-);G alpha 11(-/+) newborns suffered from craniofacial defects. Mice lacking both G alpha q and G alpha 11 [G alpha q(-/-);G alpha 11(-/-)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in G alpha q and G alpha 11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development.

Keratinocyte growth factor promotes healing of left-sided colon anastomoses.

BACKGROUND: Inadequate healing and consequent leakage from bowel anastomoses are a significant cause of postoperative morbidity and mortality. Systemic application of keratinocyte growth factor (KGF) has been shown to promote mucosal healing in models of colitis in rats and mice. The aim of the present study was to evaluate the effect of systemic KGF administration on healing of colonic anastomoses in rats. METHODS: Rats underwent laparotomy, division of the left colon, and sigmoido-sigmoidostomy. KGF (5 mg/kg) or vehicle were administered intraperitoneally in two groups (n = 30 per group) 12 hours prior to surgery, and then once daily until sacrifice (6 animals per group; 2, 4, 7, 12, and 21 days after surgery). Bursting pressure measurements, histologic evaluation, morphometric analysis, mucin and collagen staining, and hydroxyproline measurements of the anastomotic site were performed. RESULTS: Administration of KGF significantly increased anastomotic bursting pressure on postoperative days 2, 4, and 7 by 34%, 49%, and 19%, respectively. Histology, mucin staining, and measurements of the colonic crypt depth showed markedly less extended inflammation with an increased acidic mucin content and a significantly thickened mucosal layer in the KGF treated group when compared with vehicle-treated animals. CONCLUSIONS: KGF promotes healing of colonic anastomoses in rats during a 1-week postoperative period following large bowel surgery. KGF may be acting to accelerate host reparative processes as well as to enhance protection of the anastomotic wound bed by increased colonic epithelium proliferation, increased mucus production, and reduction of the inflammatory activity at the anastomotic site.

osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation. In this study the physiological role of OPG is investigated by generating OPG-deficient mice. Adolescent and adult OPG-/- mice exhibit a decrease in total bone density characterized by severe trabecular and cortical bone porosity, marked thinning of the parietal bones of the skull, and a high incidence of fractures. These findings demonstrate that OPG is a critical regulator of postnatal bone mass. Unexpectedly, OPG-deficient mice also exhibit medial calcification of the aorta and renal arteries, suggesting that regulation of OPG, its signaling pathway, or its ligand(s) may play a role in the long observed association between osteoporosis and vascular calcification.

Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.