Restless legs syndrome in Wilson's disease: frequency, characteristics, and mimics.

OBJECTIVE: To determine characteristics, clinical significance, frequency, and mimics of restless legs syndrome (RLS) in a cohort of Wilson's disease (WD, n = 42/f = 18), compared to healthy, matched controls. MATERIALS AND METHODS: Structured clinical interviews (patients and caregiving family members), repeated neurological examinations (afternoon and presleep), comprehensive laboratory tests, WD-, RLS-, and sleep-specific rating scales, and video-polysomnography. RESULTS: Thirteen patients with WD (13/42 = 31.0%) clearly fulfilled the five diagnostic criteria of RLS; in eight patients (19.1%), the burden of RLS was clinically significant. The RLS was of moderate severity, equally distributed among sexes, manifested mainly in the evening and before falling asleep, and had developed mostly after clinical manifestation of WD (time elapsed 10.2 ± 14.5 years), still at a young mean age (27.5 ± 11.5 years). The known RLS-associated features were absent (normal iron and kidney parameters) or rare (positive family history, polyneuropathy). Compared to WD patients without RLS, patients with RLS were significantly elder and had suffered longer from WD. WD-specific RLS mimics as well as RLS confounding motor comorbidities (dystonia, tremor, chorea) were frequent and a diagnostic challenge; in difficult cases, the differentiation was reached by clinical observation of the motor behavior in the evening or at nighttime. CONCLUSION: RLS was frequent in this cohort of WD and might be causally related to WD. RLS should be included in the diagnostic work-up of WD. In complex motor disorders, differential diagnosis of RLS might require evening/nighttime examination and video-polysomnography. In WD patients with a clinically significant RLS, treatment with dopaminergic substances may be considered.

Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.

Interleukin-10 inhibits polymethylmethacrylate particle induced interleukin-6 and tumor necrosis factor-alpha release by human monocyte/macrophages in vitro.

Periprosthetic membranes commonly observed at sites of total joint implant loosening exhibit abundant macrophages and particulate debris. Macrophages phagocytose orthopedic debris and release the pro-inflammatory mediators interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2. Populations of activated lymphocytes are often seen in periprosthetic membranes. These lymphocytes may modulate the monocyte/macrophage response to particulate debris and influence aseptic loosening. In addition, other immunologic agents, such as interleukin-10, are present in tissues harvested from the bone-implant interface of failed total joint arthroplasties. The present study examined the effects of interleukin-10 on polymethylmethacrylate (PMMA) particle challenged human monocyte/macrophages in vitro. Human monocyte/macrophages isolated from buffy coats of five healthy individuals were exposed to 1-10 microm PMMA particles. Interleukin-10 was added to the monocyte/macrophages with and without the addition of PMMA particles. Interleukin-10-induced alterations in monocyte/macrophage metabolism were determined measuring interleukin-6 and tumor necrosis factor-alpha release by the cells following exposure to PMMA particles. Exposure of the monocyte/macrophages to PMMA particles resulted in a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha at 48 h. Interleukin-10 reduced the levels of interleukin-6 and tumor necrosis factor-alpha release by macrophages in response to PMMA particles in a dose-dependent manner. At 48 h, particle-induced interleukin-6 release was inhibited by 60 and 90% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. At 48 h, particle-induced tumor necrosis factor-alpha release was inhibited by 58 and 88% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. Interleukin-10 challenge alone did not significantly alter basal interleukin-6 or tumor necrosis factor-alpha release relative to control cultures. The data presented in this study demonstrate that the anti-inflammatory cytokine, interleukin-10, inhibits monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.

In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery.

Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.

Induction of interleukin-6 release in human osteoblast-like cells exposed to titanium particles in vitro.

Orthopaedic wear debris induces release of bone-resorbing factors from macrophages and fibroblasts. However, the extent to which elemental metallic particles induce bone cells to express factors contributing to implant loosening remains unclear. This study showed that exposure of MG-63 osteoblast-like cells to titanium particles at a concentration of 0.30% v/v resulted in a 15-fold increase in IL-6 release into the culture medium after 24 hours, when compared with cells without particles. Northern blots revealed that exposure of MG-63 cells to titanium particles at a concentration of 0.30% v/v for 24 hours increased IL-6 mRNA signal levels by 9.6-fold, when compared with control cultures. Pretreatment of MG-63 cells with cytochalasin B prevented the particle-induced increase of IL-6 expression but did not alter the basal level of IL-6 release from cells cultured in the absence of particles. The protein kinase C inhibitor, H7, and the serine/threonine kinase inhibitor, genistein, abolished the particle-induced increase in IL-6 release at a concentration of 100 microM for each compound. In contrast, an inhibitor of protein kinase A, HA1004, had no effect on the particle-induced increase in IL-6 release. The transcription factors, nuclear factor IL-6 and nuclear factor kappa B, translocated into the nucleus within 1 hour of particle exposure. This study showed that osteoblast-like cells respond to titanium particles through increased expression of the proinflammatory cytokine, IL-6, in a process requiring phagocytosis and intracellular signaling pathways. These results suggest that osteoblasts play a direct role in implant loosening because of localized release of soluble mediators such as interleukin-6.

G-protein activity requirement for polymethylmethacrylate and titanium particle-induced fibroblast interleukin-6 and monocyte chemoattractant protein-1 release in vitro.

Periprosthetic granulomatous membranes consisting of fibroblasts, macrophages, lymphocytes, foreign body giant cells, and abundant particulate debris occur at sites of implant loosening. Previous studies demonstrate that fibroblasts respond to particulate debris through the release of interleukin-6 (IL-6), prostaglandin E(2), and matrix metalloproteinases in vitro. C-C chemokines are observed in granulomatous tissue surrounding loosened prosthetic implants and are released by macrophages and fibroblasts in response to particle challenge in vitro. This study tested the hypothesis that G protein activity is required for fibroblast activation by titanium and polymethylmethacrylate (PMMA) particles, and that inhibition of G protein activity would alter IL-6 and and monocyte chemoattractant protein-1 (MCP-1) release from activated fibroblasts. The specific inhibitor of G protein activity, pertussis toxin, was added to the fibroblasts to examine the effects of G protein activity with respect to the production of IL-6 and MCP-1 by orthopedic biomaterial-challenged fibroblasts in vitro. Interleukin-1beta (IL-1beta), a proven activator of MCP-1 and interleukin-6, was used as a positive control. Exposure of fibroblasts to titanium and polymethylmethacrylate (PMMA) particles resulted in a dose-dependent release of MCP-1 and IL-6. Challenge with PMMA particles at doses of 0.150%, 0.300%, and 0.600% vol/vol increased the release of interleukin-6 by 7-, 19-, and 22-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone at 24 h. Challenge with PMMA particles at doses of 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of MCP-1-6 by 2.5-, 3.6-, 4. 3-, and 4.5-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone. Challenge with titanium particles at concentrations of 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of interleukin-6 by 2.6-, 6.4-, 9.6-, and 10. 0-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone at 24 h. Challenge with titanium particles at concentrations of 0.038%, 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of MCP-1 by 2.9-, 3.1-, 5.8-, 5.4-, and 5. 8-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone. Pretreatment of fibroblasts with pertussis toxin inhibited the release of interleukin-6 and MCP-1 from PMMA and titanium particle challenged fibroblasts in a dose-dependent manner. PMMA particle induced fibroblast IL-6 release was inhibited by 23.6% and 35.3% with 20- and 200-ng/mL doses of pertussis toxin, respectively. Titanium particle induced fibroblast IL-6 release was inhibited by 48.2% and 56.3% with 20- and 200-ng/mL doses of pertussis toxin, respectively. PMMA particle-induced fibroblast MCP-1 release was inhibited by 36.0%, 50.4%, and 60.1% with 2-, 20- and 200-ng/mL doses of pertussis toxin, respectively. Titanium particle-induced fibroblast MCP-1 release was inhibited by 15.5%, 53.2%, and 64.6% with 2-, 20-, and 200-ng/mL doses of pertussis toxin, respectively. This study suggests that fibroblasts localized in periprosthetic membranes are a source of macrophage chemoattractant factors and proinflammatory mediators that may influence granuloma formation and lead to periprosthetic bone resorption. Furthermore, this study shows that G proteins are involved in particle-induced fibroblast activation, as evidenced by decrease levels of particle induced IL-6 and MCP-1 release following pertussis toxin treatment. (c) 2000 John Wiley & Sons, Inc.

Monocyte migration inhibitory factor synthesis and gene expression in particle-activated macrophages.

This study analysed MIF mRNA and protein expression in human macrophages exposed in vitro to polymethylmethacryate and titanium alloy particles. MIF levels released from macrophages without exposure to particles were in the range of 2-4 ng/ml. Exposure of macrophages to particles as demonstrated increased MIF release at 0. 075%-0.225% v/v particle concentration, which was maximal at 12-24 h. MIF mRNA signal levels in cells with and without particles at a concentration of 0.075% showed no significant differences in a time course experiment. The profile of MIF release in response to increasing particle concentration coincided with increased release of lactate dehydrogenase. The viability of the cells was unchanged by the addition of particles as determined by 3H-thymidine uptake. These data suggest that MIF expression may represent an independent macrophage response to locally high particle concentrations.

Effects of shear stress on articular chondrocyte metabolism.

The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.

Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression.

The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.

Interferon-gamma exacerbates polymethylmethacrylate particle-induced interleukin-6 release by human monocyte/macrophages in vitro.

Periprosthetic membranes commonly observed at sites of total joint implant loosening exhibit abundant macrophages and particulate debris. Macrophages phagocytose orthopedic debris and release the pro-inflammatory mediators interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2. In addition, other immunologic agents, such as interferon-gamma, are present in tissues harvested from the bone-implant interface of failed orthopedic implants. The present study examined the effects of interferon-gamma on polymethylmethacrylate (PMMA) particle-challenged monocyte/macrophages in vitro. The effects of interferon-gamma were determined by measuring interleukin-6 and tumor necrosis factor-alpha release by primary human monocyte/macrophages following exposure to PMMA particles. Exposure of the monocyte/macrophages to PMMA particles resulted in a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha at 48 h. The interleukin-6 release in response to PMMA particle challenge was stimulated by 76% and 127% in the presence of 1.0 and 10.0 ng/mL of interferon-gamma, respectively. Interferon-gamma challenge alone did not alter interleukin-6 release relative to controls. In contrast to interleukin-6, interferon-gamma challenge stimulated tumor necrosis factor-alpha release in a dose-dependent manner. In the presence of particles, addition of 1.0 and 10.0 ng/mL of interferon-gamma resulted in 17% and 171% increases in the levels of tumor necrosis factor-alpha release, respectively, relative to cultures challenged solely with particles. Blocking antibody to IFN-gamma inhibited the effect of IFN-gamma on particle-induced interleukin-6 and tumor necrosis factor-alpha release. The data presented in this study demonstrate that the immunologic modulator interferon-gamma exacerbates monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.

Chemotaxis and activation of particle-challenged human monocytes in response to monocyte migration inhibitory factor and C-C chemokines.

Cytokines that regulate monocyte migration were found in membrane tissue surrounding loosened prosthetic implants. Monocyte migration inhibition factor (MIF) is able to inhibit macrophage migration. Monocyte chemoattractant protein (MCP) and macrophage inflammatory protein (MIP) are potent macrophage chemoattractants. These cytokines may be expressed as part of the foreign body response to prosthetic particulate debris. Chemotaxis analysis and macrophage activation experiments were performed to determine the effects of MIF, MCP-1, and MIP-1alpha on macrophage migration and activation in vitro. We demonstrated that MIF had its maximal migration inhibitory effect for unchallenged and particle challenged macrophages at 1 ng/mL. MCP-1 and MIP-1alpha stimulated macrophage chemotaxis maximally at 1 to 10 ng/mL. Dose-response studies with MIF, MCP-1, and MIP-1alpha demonstrated that these cytokines did not modulate activation of unchallenged or particle challenged macrophages as evaluated by IL-6 and TNF-alpha release. However, these cytokines do not appear to affect macrophage release of proinflammatory mediators in vitro.

Proinflammatory mediator release in response to particle challenge: studies using the bone harvest chamber.

This study reports on the effects of phagocytosable particles on proinflammatory mediator release in an animal model. Bone harvest chambers (BHCs) were implanted bilaterally into mature rabbits; phagocytosable ultrahigh molecular weight polyethylene (UHMWPE) and polystyrene (PS) particles, and the carrier sodium hyaluronate (HE) were tested for their ability to stimulate proinflammatory mediator release. Tissues were harvested after 3, 4, or 6 weeks. Retrieved tissues were placed into culture medium. The release of the proinflammatory mediators interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) into the culture medium was assessed using bioassays. DNA content and dry weights were also measured. The maximal biological response to the PE particles with respect to TNF-alpha and IL-1beta was observed at three weeks with 11- and fivefold stimulations over controls, respectively. The maximal response to PE particles with respect to IL-6 was observed at 4 weeks with a twofold stimulation over controls. Similar patterns were seen with PS particles; however, PE particles stimulated higher cytokine release. PE particles stimulated the expression of IL-1beta, IL-6, and TNF-alpha in the BHC model. Cell culture and human retrieval studies also implicate these proinflammatory mediators in loosening and osteolysis of total joint replacements. Thus, the BHC is a useful in vivo model to document the effects of particles on the evolution of biological responses to particulate debris.

Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro.

BACKGROUND: Loosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. METHODS: Macrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-kappaB) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. RESULTS: Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-alpha and a sevenfold increase in the release of IL-6 (p<0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha and IL-6. Antibodies to CD11b/ CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p<0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-kappaB and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-kappaB and NF-IL-6. CONCLUSIONS: These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of

Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles.

Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.

Interleukin-4 inhibits granulocyte-macrophage colony-stimulating factor, interleukin-6, and tumor necrosis factor-alpha expression by human monocytes in response to polymethylmethacrylate particle challenge in vitro.

The outcome of total joint arthroplasty is determined by biological events at the bone-implant interface. Macrophages phagocytose implant or wear debris at the interface and release proinflammatory mediators such as interleukins 1 and 6, tumor necrosis factor-alpha, and prostaglandin E2. These mediators are thought to contribute to the resorption of periprosthetic bone. Previous studies of tissues harvested from the bone-implant interface of failed orthopaedic implants demonstrated a possible role for two other cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-4. The present study examined the effects of in vitro challenge with polymethylmethacrylate particles on the expression of granulocyte-macrophage colony-stimulating factor by primary human monocytes/macrophages and the role of interleukin-4 in regulating this expression. The polymethylmethacrylate particles caused a dose-dependent release of granulocyte-macrophage colony-stimulating factor at 48 hours. This release was accompanied by increased expression of interleukins 6 and 1beta and tumor necrosis factor-alpha. Release of the lysosomal enzyme hexosaminidase also increased in response to the particles. Interleukin-4 inhibited the expression of granulocyte-macrophage colony-stimulating factor, interleukin-6, and tumor necrosis factor-alpha at 48 hours in a dose-dependent manner. The data presented in this study confirm the hypothesis that interleukin-4 downregulates particle-induced activation of macrophages, as demonstrated by the decreased release of proinflammatory mediators.

Effects of particulate debris on macrophage-dependent fibroblast stimulation in coculture.

The interactions between the different cell types in periprosthetic tissue are still unclear. We used a non-contact coculture model to investigate the effects of polymethylmethacrylate (PMMA) particles and human macrophage-derived soluble mediators on fibroblast activation. Macrophages were either exposed or not exposed to phagocytosable PMMA particles, but fibroblasts were not. Increasing numbers of macrophages were tested in cocultures in which the fibroblast cell number was held constant and cultures of macrophages alone were used for comparison of cytokine release. We used the release of interleukin-1 beta (IL-1beta), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha), lysosomal enzyme and metalloproteinase activity to assess the cultivation of macrophages and fibroblasts. In cocultures, IL-6 release was increased 100-fold for both unchallenged and particle-challenged cultures when compared with macrophage cultures alone. Furthermore, particle-challenged cocultures had threefold higher IL-6 levels than unchallenged cocultures. Release of TNF-alpha was similar in cocultures and in macrophage cultures. IL-1beta release in cocultures was independent of the macrophage-fibroblast ratio. Lysosomal enzyme activity and metalloproteinase activity were increased in cocultures. Our data show that macrophages and fibroblasts in coculture significantly increase the release of IL-6 and to a less degree other inflammatory mediators; particle exposure accentuates this effect. This suggests that macrophage accumulation in fibrous tissue may lead to elevated IL-6 levels that are much higher than those caused by particle activation of macrophages alone. This macrophage-fibroblast interaction represents a novel concept for the initiation and maintenance of the inflammatory process in periprosthetic membranes.

The role of radiotherapy in soft tissue sarcomas. Retrospective study of 115 cases treated from 1979 to 1988.

In this study was analysed the results of a group of patients with soft tissue sarcomas treated at the IPOFG Center of Lisbon from January 1979 to December 1988. All these cases were referred to the Radiotherapy Department for radiation treatment, which was given in 84% of the patients as a complement to surgery and in 16% was either combined with chemotherapy or given as exclusive therapy. The immediate results showed complete remission in 79% and partial remission in 7% of the cases. Seventeen per cent of the patients developed relapses and 28% distant metastases after a free interval of 27.7 and 19.6 months respectively. The five year survival rates were 73.3% for the patients without recurrences, 55.4% for the patients with relapses and 21.4% for disseminated disease. The long term results varied according to the localization of the tumor, the stage of the disease, the age of the patient, the extension of surgery, the histologic type and the radiation dose. Five year survival rates also varied with the type of treatment (63.8% for surgery followed by radiotherapy with or without chemotherapy and 50.8% for preoperative radiotherapy and surgery with or without adjuvant chemotherapy). Radiotherapy with or without chemotherapy, in inoperable patients, was generally useful because it improved the quality of life, saving about 25% of the inoperable cases. Treatment related complications were minimal.

[The role of radiotherapy in soft tissue sarcomas. Retrospective analysis of 115 cases treated from 1979 to 1988]. / O papel da radioterapia nos sarcomas dos tecidos moles. Análise retrospectiva de 115 casos tratados de 1979 a 1988.

In this study was analysed the results of a group of patients with soft tissue sarcomas treated at the IPOFG Center of Lisbon from January 1979 to December 1988. All these cases were referred to the Radiotherapy Department for radiation treatment which was given in 84% of the patients as a complement to surgery and in 16% was either combined with chemotherapy or given as exclusive therapy. The immediate results showed complete remission in 79% and partial remission in 7% of the cases. Seventeen per cent of the patients developed relapses and 28% distant metastases after a free interval of 27.7 and 19.6 months respectively. The five year actuarial survival rates were 73.3% for the patients without recurrences, 55.4% for the patients with relapses and 21.4% for disseminated disease. The long term results varied according to the localization of the tumor, the stage of the disease, the age of the patient, the extension of surgery, the histologic type and the radiation dose. Five year actuarial survival rates also varied with the type of treatment (63.8% for surgery followed by radiotherapy with or without chemotherapy and 50.8% for preoperative radiotherapy and surgery with or without adjuvant chemotherapy). Radiotherapy with or without chemotherapy, in inoperable patients, was generally useful because it improved the quality of life, saving about 25% of the inoperable cases. Treatment related complications were minimal.

A model clinical program for patients with diabetes and vision impairment.

A program was developed to improve independence, self-esteem, and glycemic control in patients with diabetes and blindness. Twenty-nine individuals with both insulin-dependent and noninsulin-dependent diabetes mellitus entered 12-week programs that included education focusing on diabetes self-management skills for the visually impaired, monitored exercise sessions, and group support. Glycated hemoglobin values fell from 13.0% +/- 0.6% (SEM) to 11.4% +/- 0.5% (P = .001). Exercise tolerance in a 12-minute walk test improved from 0.48 +/- 0.04 to 0.64 +/- 0.05 miles (P = .001). Marked improvements occurred in psychosocial indices, demonstrated through changes in the Rand Mental Health Index from 155 +/- 6 to 174 +/- 5 (P = .0001), the Rosenberg Self-Esteem Scale from 22 +/- 1 to 19 +/- 1 (P = .001), the Zung Depression Scale from 0.50 +/- 0.02 to 0.44 +/- 0.02 (P = .001), and the Diabetes Self-Reliance Test from 60 +/- 3 to 74 +/- 2 (P = .0001).