Tibor T. Glant

Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-α-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.

Osteolysis: basic science.

Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has been gained on the basic science of periprosthetic bone loss. Initially termed cement disease, it now generally is accepted that, in most instances, osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris, possibly facilitated by local hydrodynamic effects. Tissue explant, animal, and cell culture studies have allowed us to compile an appreciation of the complexity of cellular interactions and chemical mediators involved in osteolysis. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular responses and effects on bone include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin 6. However, an increasing number of other proinflammatory and antiinflammatory cytokines, prostenoids, and enzymes have been shown to play important roles in this process. The ultimate goal of basic research is to develop novel strategies for evaluation and treatment of patients with osteolysis. Although initial animal studies are promising for possible pharmacologic treatment and prevention of osteolysis, well-controlled human trials are required before agents such as bisphosphonates can be recommended for general clinical use.

Composition and morphology of wear debris in failed uncemented total hip replacement

Interfacial membranes collected at revision from 11 failed uncemented Ti-alloy total hip replacements were examined. Particles in the membranes were characterised by electron microscopy, microchemical spectroscopy and particle size analysis. Most were polyethylene and had a mean size of 0.53 micron +/- 0.3. They were similar to the particles seen in the base resin used in the manufacture of the acetabular implants. Relatively few titanium particles were seen. Fragments of bone, stainless steel and silicate were found in small amounts. Most of the polyethylene particles were too small to be seen by light microscopy. Electron microscopy and spectroscopic techniques are required to provide an accurate description of this debris.

Anti-CD44 treatment abrogates tissue aedema and leukocyte infiltration in murine arthrtis

A ubiquitous cell adhesion receptor, CD44, preferentially binds hyaluronan, a poly-saccharide macromolecule that is present in most extracellular matrices. Hyaluronan molecules have large hydrodynamic volumes that entrap substantial amounts of water and can therefore control tissue hydration (swelling). CD44 is overexpressed by synovial cells and leukocytes, and hyaluronan is overproduced in the rheumatoid synovium and in other inflammatory sites. Nevertheless, the role of the CD44–hyaluronan interaction during inflammation is unclear. Our evidence shows that the CD44 receptor plays a critical role in governing the migration of inflammatory leukocytes into the extravascular compartment of the synovium in murine arthritis. An anti-CD44 antibody induces a rapid loss of CD44 from both leukocytes and synovial cells and displays an inhibitory effect on cell–extracellular matrix interactions in the synovium. As a result, the administration of such an antibody abrogates tissue swelling and leukocyte infiltration, two major components of inflammation.

Animals lacking link protein have attenuated perineuronal nets and persistent plasticity.

Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.

Wear Debris in Total Joint Replacements.

&NA; In vivo degradation of prosthetic implant materials is increasingly recognized as a major factor limiting the durability of total joint arthroplasty. In vivo degradation occurs primarily by means of wear processes that can generate large quantities of particulate debris. This debris can stimulate an adverse local host response leading to periprosthetic bone loss, which can compromise implant fixation and bone stock. The authors review the basic mechanisms of implant degradation and the host response to particulate degradation products, particularly in the context of the pathogenesis of osteolysis. Submicron polyethylene particles (mean size, 0.5 &mgr;m) are the dominant type of wear particle present in periprosthetic tissues associated with uncemented hip replacements. Polyethylene wear can be minimized by improving the quality of the polyethylene, avoiding use of large‐diameter (greater than 28 mm) femoral heads in total hip arthroplasty, and improving the design and fabrication of modular connections, which can be important sources of threebody wear particles. Advances in the understanding of the basic mechanisms of osteolysis are critical to the development of preventive measures that will minimize the clinical impact of this phenomenon.

The Effects of Particulate Wear Debris, Cytokines, and Growth Factors on the Functions of MG-63 Osteoblasts

Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions. Methods: MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or trypan blue exclusion. Expressions of osteoblast-specific genes were quantified with Northern blot hybridization, and the secretions of osteoblast-specific proteins and cytokines were analyzed by enzyme-linked immunosorbent assays. Results: MG-63 osteoblasts phagocytosed particles and became saturated after twenty-four hours. A maximum of forty to sixty particles per cell were phagocytosed. Each type of particle significantly suppressed procollagen a1[I] gene expression (p < 0.05), whereas other osteoblast-specific genes (osteonectin, osteocalcin, and alkaline phosphatase) did not show significant changes. Particle-stimulated osteoblasts released interleukin-6 (p < 0.05) and a smaller amount of transforming growth factor-b1. Particles reduced cell proliferation in a dose-dependent manner without affecting cell viability (p < 0.05). Exogenous tumor necrosis factor-a also enhanced the release of interleukin-6 (p < 0.01) and transforming growth factor-b1 (p < 0.05), whereas the secretion of transforming growth factor-b1 was increased by insulin-like growth factor-I and prostaglandin E2 as well. Insulin-like growth factor-I and transforming growth factor-b1 significantly increased procollagen a1[I] gene expression in osteoblasts (p < 0.05), while tumor necrosis factor-a and prostaglandin E2 significantly suppressed procollagen a1[I] gene expression (p < 0.01). In contrast, neither exogenous nor endogenous interleukin-6 had any effect on other cytokine secretion, on proliferation, or on procollagen a1[I] gene expression. The transcription inhibitor actinomycin D reduced both procollagen a1[I] transcription and interleukin-6 production. Inhibitors of protein synthesis (cyclohexamide) and intracellular protein transport (brefeldin A and monensin) blocked the release of interleukin-6, but none of these compounds influenced the suppressive effect of titanium on procollagen a1[I] gene expression. Conclusions: MG-63 osteoblasts phagocytose particulate wear debris, and this process induces interleukin-6 production and suppresses type-I collagen synthesis. Osteoblast-derived interleukin-6 may induce osteoclast differentiation and/or activation, but the resorbed bone cannot be replaced by new bone because of diminished osteoblast function (reduced type-I collagen synthesis). Exogenous cytokines (tumor necrosis factor-a and interleukin-1b), growth factors (insulin-like growth factor-I and transforming growth factor-b1), and prostaglandin E2 can modify particulate-induced alterations of osteoblast functions. Clinical Relevance: Altered osteoblast functions probably contribute to the progression of periprosthetic osteolysis. Suppressed osteoblast functions, however, could be compensated for by certain growth factors, such as insulin-like growth factor-I or transforming growth factor-b1. These growth factors, if delivered locally, may have therapeutic potential to prevent or reverse periprosthetic osteolysis.

Antigen-Specific B Cells Are Required as APCs and Autoantibody-Producing Cells for Induction of Severe Autoimmune Arthritis

B cells play an important role in rheumatoid arthritis, but whether they are required as autoantibody-producing cells as well as APCs has not been determined. We assessed B cell autoantibody and APC functions in a murine model of autoimmune arthritis, proteoglycan (PG)-induced arthritis, using both B cell-deficient mice and Ig-deficient mice (mIgM) mice that express an H chain transgene encoding for membrane-bound, but not secreted, IgM. The IgH transgene, when paired with endogenous λ L chain, recognizes the hapten 4-hydroxy-3-nitro-phenyl acetyl and is expressed on 1–4% of B cells. B cell-deficient and mIgM mice do not develop arthritis after immunization with PG. In adoptive transfer of PG-induced arthritis into SCID mice, T cells from mIgM mice immunized with PG were unable to transfer disease even when B cells from PG-immunized wild-type mice were provided, suggesting that the T cells were not adequately primed and that Ag-specific B cells may be required. In fact, when PG was directly targeted to the B cell Ig receptor through a conjugate of 4-hydroxy-3-nitrophenyl acetyl-PG, T cells in mIgM mice were activated and competent to transfer arthritis. Such T cells caused mild arthritis in the absence of autoantibody, demonstrating a direct pathogenic role for T cells activated by Ag-specific B cells. Transfer of arthritic serum alone induced only mild and transient arthritis. However, both autoreactive T cells and autoantibody are required to cause severe arthritis, indicating that both B cell-mediated effector pathways contribute synergistically to autoimmune disease.

The Basic Science of Periprosthetic Osteolysis

Despite improvements in the techniques, materials, and fixation of total joint replacements, wear and its sequelae continue to be the main factors limiting the longevity and clinical success of arthroplasty. Since Charnley first recognized aseptic loosening in the early 1960s, a tremendous amount of information has been gained on the basic science of osteolysis. Tissue explant, animal, and cell culture studies have allowed development of an appreciation of the complexity of cellular interactions and chemical mediators involved in these processes. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular interactions and effects on bone primarily include PGE2, TNF-alpha, IL-1, and IL-6. Recent and ongoing work in the field of signaling pathways will continue to advance our understanding of the mechanisms of periprosthetic bone loss. Although initial animal studies are promising for the development of possible pharmacologic agents for the treatment and prevention of osteolysis, well controlled human trials are required.

Suppression of Osteoblast Function by Titanium Particles

In order to understand the effect of particulate debris on osteoblast function, we studied the effect of different particles, including titanium and polystyrene, on bone collagen mRNA (messenger RNA) with the use of Northern blot hybridization analysis, and we studied the effect of the particles on the biosynthesis of bone collagen with analysis of 3H-proline incorporation and with the Western blot technique. The steady-state levels of mRNA for procollagens &agr;1(I) and &agr;1(III) were markedly suppressed in human MG-63 osteoblast-like cells exposed to phagocytosable titanium particles that were smaller than three micrometers. Both titanium and polystyrene particles smaller than three micrometers suppressed the expression of the gene that codes for collagen, and the suppression of the expression of the gene was related to the size but not to the composition of the particles. The biosynthesis of both type-I and type-III collagen also was decreased in cells that had been treated with titanium particles. Neither the viability nor the proliferation of cells was affected by particulate debris. These data indicate that phagocytosable titanium particles can significantly suppress the expression of the gene that codes for collagen in osteoblast-like cells (p < 0.05).