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)1,2,3. In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system3,4,5. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts4,6. OPGL expression in T cells is induced by antigen receptor engagement7, 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.

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

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.

Albumin Fusion Proteins

The use of liposomal carriers and the modification of therapeutic molecules through the attachment of poly(ethylene glycol) [PEG] moieties ('pegylation') are the most common approaches for enhancing the delivery of parenteral agents Although 'classical' liposomes (ie phospholipid bilayer vehicles) have been effective in decreasing the clearance of encapsulated agents and in passively targeting specific tissues, they are associated with considerable limitations Pegylation may be an effective method of delivering therapeutic proteins and modifying their pharmacokinetic properties, in turn modifying pharmacodynamics, via a mechanism dependent on altered binding properties of the native protein Pegylation reduces renal clearance and, for some products, results in a more sustained absorption after subcutaneous administration as well as restricted distribution These pharmacokinetic changes may result in more constant and sustained plasma concentrations, which can lead to increases in clinical effectiveness when the desired effects are concentration-dependent Maintaining drug concentrations at or near a target concentration for an extended period of time is often clinically advantageous, and is particularly useful in antiviral therapy, since constant antiviral pressure should prevent replication and may thereby suppress the emergence of resistant variants Additionally, PEG modification may decrease adverse effects caused by the large variations in peak-to-trough plasma drug concentrations associated with frequent administration and by the immunogenicity of unmodified proteins Pegylated proteins may have reduced immunogenicity because PEG-induced steric hindrance can prevent immune recognition Two PEG-modified proteins are currently approved by the US Food and Drug Administration; several others, including cytokines such as interferon-alpha (IFNalpha), growth factors and free radical scavengers, are under development Careful assessment of various pegylated IFNalpha products suggests that pegylated molecules can be differentiated on the basis of their pharmacokinetic properties and related changes in pharmacodynamics Because the size, geometry and attachment site of the PEG moiety play a crucial role in determining these properties, therapeutically optimised agents must be designed on a protein-by-protein basis

Pegylation: a novel process for modifying pharmacokinetics

Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.

There is considerable evidence that osteoclasts are involved in the pathogenesis of focal bone erosion in rheumatoid arthritis Tumor necrosis factor-related activation-induced cytokine, also known as receptor activator of nuclear factor-κB ligand (TRANCE/RANKL) is an essential factor for osteoclast differentiation In addition to its role in osteoclast differentiation and activation, TRANCE/RANKL also functions to augment T-cell dendritic cell cooperative interactions To further evaluate the role of osteoclasts in focal bone erosion in arthritis, we generated inflammatory arthritis in the TRANCE/RANKL knockout mouse using a serum transfer model that bypasses the requirement for T-cell activation These animals exhibit an osteopetrotic phenotype characterized by the absence of osteoclasts Inflammation, measured by clinical signs of arthritis and histopathological scoring, was comparable in wild-type and TRANCE/RANKL knockout mice Microcomputed tomography and histopathological analysis demonstrated that the degree of bone erosion in TRANCE/RANKL knockout mice was dramatically reduced compared to that seen in control littermate mice In contrast, cartilage erosion was present in both control littermate and TRANCE/RANKL knockout mice These results confirm the central role of osteoclasts in the pathogenesis of bone erosion in arthritis and demonstrate distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis

https://cbdm.hms.harvard.edu/assets/Publications/2001pub/PettitAJP.pdf

TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis.

Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional

Pegylated Megakaryocyte Growth and Development Factor Abrogates the Lethal Thrombocytopenia Associated With Carboplatin and Irradiation in Mice

Recent studies show that human osteoclasts develop in vitro from hematopoietic cells; however, special cultures conditions and/or cytokine mobilized peripheral blood are apparently required Here, we report that cells expressing osteoclast markers differentiate from precursors present in nonmobilized peripheral blood mononuclear cells (PBMC), without the addition of stromal cells, growth factors, cytokines or steroids; and characterize their phenotype Three days after establishing high-density PBMC cultures (15 x 10(6) cells/cm2), in serum-containing medium, small adherent colonies of tartrate resistant acid phosphatase positive (TRAP+) cells emerge, amidst massive monocyte cell death These adherent cells have an eccentrically placed, round nucleus, and express low levels of TRAP and sodium fluoride-resistant- alpha-naphthyl-acetate-esterase (NaF-R-NSE) Over the next week, this cell population accumulates phenotypic markers of osteoclasts (vitronectin receptor [VR], calcitonin receptor, TRAP, cathepsin K protein, and mRNA) with increased nuclearity, covering the entire surface by 15 days When cultured on bone, VR+, TRAP+ cells of low multinuclearity appear and cover up to 50% of the surface Resorption lacunae can be observed by day 22 Although these pits are not nearly as numerous as the cells of preosteoclast phenotype, they do represent the activity of a subset of osteoclast-like cells that has achieved osteoclastic maturity under these culture conditions Transcripts for osteoprotegerin ligand (OPGL), an osteoclast differentiation factor (also known as RANKL and TRANCE) are expressed, likely by adherent cells Thus, an adherent population of cells, with preosteoclast/osteoclast phenotypic properties, arises selectively under simple culture conditions from normal PBMC Further characterization of these cells should identify factors involved in the growth, terminal differentiation and activation of osteoclasts

Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors.

It is known that osteoblast precursor cells are found in the low-density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34− cells were cultured under conditions that promote growth and differentiation of mineral-secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34− cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal-specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell-derived osteoblasts displayed osteoblast-specific features. Functionally, these CD34+ cell-derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.

Osteoblast precursor cells are found in CD34+ cells from human bone marrow.

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 micrograms/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 micrograms/kg/d resulted in platelet counts 5-fold above normal. However, doses > 60 micrograms/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.

Pamela Hunt

Papers

Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional

Pegylated Megakaryocyte Growth and Development Factor Abrogates the Lethal Thrombocytopenia Associated With Carboplatin and Irradiation in Mice

Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors.

Osteoblast precursor cells are found in CD34+ cells from human bone marrow.

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis.