Hepatoprotective effects of S-adenosylmethionine and silybin on canine hepatocytes in vitro.

Inflammation and oxidative stress are associated with liver injury and development of liver disease. The transcription factors nuclear factor-kappa beta (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) play critical roles in modulating liver injury and damage. Activation of NF-κB induces production of pro-inflammatory molecules including prostaglandin E2 (PGE2 ), interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1). Nrf2 regulates genes controlling antioxidants. Our laboratory previously showed that hepatocytes, the primary functional cell type comprising liver tissue, respond to the cytokine interleukin-1 beta (IL-1ß) by increased production of PGE2 , IL-8 and MCP-1. This increase is associated with nuclear translocation of NF-κB. In this study, we evaluated whether primary canine hepatocytes pre-treated with the combination of S-adenosylmethionine (SAMe; 30 and 2000 ng/ml) and silybin (SB; 298 ng/ml), agents with known anti-inflammatory and antioxidant properties, could attenuate IL-1ß-induced inflammation and oxidative stress. The SAMe and SB combination reduced cytokine-induced PGE2 , IL-8 and MCP-1 production while also inhibiting NF-κB nuclear translocation. These changes were accompanied by increased antioxidant enzyme-reduced glutathione (GSH) comparable to control levels. The study shows for the first time that the SAMe and SB combination inhibits both inflammation and oxidative stress through two separate signalling pathways.

Silybin inhibits interleukin-1ß-induced production of pro-inflammatory mediators in canine hepatocyte cultures.

Hepatocytes are highly susceptible to cytokine stimulation and are fundamental to liver function. We established primary canine hepatocyte cultures to study effects of anti-inflammatory agents with hepatoprotective properties. Hepatocyte cultures were incubated with control media alone, silybin (SB), or the more bioavailable silybin-phosphatidylcholine complex (SPC), followed by activation with interleukin-1 beta (IL-1ß; 10 ng/mL). Inflammatory response was measured by prostaglandin E2 (PGE(2) ), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) production and also nuclear factor-kappa B (NF-κB) translocation. Hepatocyte cultures continued production of the phenotypic marker albumin for more than 7 days in culture. IL-1ß exposure increased PGE(2) , IL-8, and MCP-1 production, which was paralleled by NF-κB translocation from the cytoplasm to the nucleus. Pretreatment with SB and SPC significantly inhibited IL-1ß-induced production of pro-inflammatory markers and attenuated NF-κB nuclear translocation. We demonstrate for the first time that primary canine hepatocyte cultures can be maintained in culture without phenotypic loss. The observation that hepatocyte cultures respond to pro-inflammatory IL-1ß activation indicates hepatocytes as primary cellular targets of extrinsic IL-1ß. The ability of SB and SPC to inhibit hepatocyte culture activation by IL-1ß reinforces the notion of their hepatoprotective effects. Our primary canine hepatocyte culture model facilitates identification of hepatoprotective agents and their mechanism of action.

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate.

OBJECTIVE: To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS: Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS: Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION: The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

Avocado soybean unsaponifiables (ASU) suppress TNF-alpha, IL-1beta, COX-2, iNOS gene expression, and prostaglandin E2 and nitric oxide production in articular chondrocytes and monocyte/macrophages.

OBJECTIVE: To evaluate the effects of avocado soybean unsaponifiables (ASU) on proinflammatory mediators in chondrocytes and monocyte/macrophage-like cells. DESIGN: To determine the dose response of ASU, chondrocytes (5 x 10(5) cells/well) were incubated at 5% CO(2), 37 degrees C for 72 h with (1) control media alone or (2) ASU at concentrations of 0.3, 0.9, 2.7, 8.3, and 25 microg/ml. Cells were activated with 20 ng/ml lipopolysaccharide (LPS) for 24 h and cell supernatants were analyzed for prostaglandin E(2) (PGE(2)) and nitrite content. Chondrocytes and THP-1 monocyte/macrophages (5 x 10(5) cells/well) were incubated at 5% CO(2), 37 degrees C for 72 h with (1) control media alone or (2) ASU (25 mug/ml). One set of cells was activated for 1 h with LPS (20 ng/ml) for both reverse-transcriptase PCR and real-time PCR analysis of tumor necrosis factor-alpha (TNF-alpha), interleukin-1-beta (IL-1beta), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression. One set of cells was activated for 24 h to analyze secreted PGE(2) and nitrite levels in the cellular supernatant. RESULTS: ASU reduced TNF-alpha, IL-1beta, COX-2, and iNOS expression in LPS-activated chondrocytes to levels similar to nonactivated control levels. The suppression of COX-2 and iNOS expression was paralleled by a significant reduction in PGE(2) and nitrite, respectively, in the cellular supernatant. ASU also reduced TNF-alpha and IL-1beta expression in LPS-activated monocyte/macrophage-like cells. CONCLUSION: The present study demonstrates that the anti-inflammatory activity of ASU is not restricted to chondrocytes, but also affects monocyte/macrophage-like cells that serve as a prototype for macrophages in the synovial membrane. These observations provide a scientific rationale for the pain-reducing and anti-inflammatory effects of ASU observed in osteoarthritis patients.

Extraction and characterization of metallic wear debris from total joint arthroplasty.

Wear debris generated from total joint arthroplasty may elicit a granulomatous and inflammatory response and has also been implicated in the development of osteolysis. Technical difficulty in retrieval and isolation of wear material from tissues has hindered the study of their physicochemical properties. The purpose of this study was to retrieve and analyze metallic wear debris from periprosthetic tissue obtained during revision arthroplasty. Tissue from six osteoarthritic patients was obtained during revision arthroplasty. The tissue was minced and then heated in a sodium dodecyl sulfate solution. Undigested tissue was incubated sequentially with papain and pepsin solutions. Metallic wear debris retrieved from the digestion procedure was analyzed by scanning electron microscopy. Wear fragments were seen as irregularly shaped flakes, splinters and polyhedral structures ranging from 1 to 100 microns in size. These structures appeared to be free from non-metallic surface-adherent material. Energy dispersion spectroscopy verified the presence of cobalt, chrome and molybdenum which comprised the implant alloy. Fatigue lines were observed on the surface suggesting brittle wear. Our technique for isolating metallic fragments facilitates the retrieval and preparation of wear debris for analysis of physicochemical properties and how wear debris interacts with cellular elements in surrounding tissue.

Identification of caveolae and detection of caveolin in normal human osteoblasts.

Caveolae, specialised regions of the cell membrane which have been detected in a wide range of mammalian cells, have not been described in bone cells. They are plasmalemmal invaginations, 50 to 100 nm in size, characterised by the presence of the structural protein, caveolin, which exists as three subtypes. Caveolin-1 and caveolin-2 are expressed in a wide range of cell types whereas caveolin-3 is thought to be a muscle-specific subtype. There is little information on the precise function of caveolae, but it has been proposed that they play an important role in signal transduction. As the principal bone-producing cell, the osteoblast has been widely studied in an effort to understand the signalling pathways by which it responds to extracellular stimuli. Our aim in this study was to identify caveolae and their structural protein caveolin in normal human osteoblasts, and to determine which subtypes of caveolin were present. Confocal microscopy showed staining which was associated with the plasma membrane. Transmission electron microscopy revealed the presence of membrane invaginations of 50 to 100 nm, consistent with the appearance of caveolae. Finally, we isolated protein from these osteoblasts, and performed Western blotting using anti-caveolin primary antibodies. This revealed the presence of caveolin-1 and -2, while caveolin-3 was absent. The identification of these structures and their associated protein may provide a significant contribution to our further understanding of signal transduction pathways in osteoblasts.

Beta-1 integrin expression by human nasal chondrocytes in microcarrier spinner culture.

Beta-1 integrin plays a major role in cell attachment and is believed to be involved in mediating the interactions of chondrocytes with their environment. We previously reported that articular chondrocytes propagated in microcarrier spinner culture proliferated and reexpressed their chondrocytic protein. The goal of the present study was to investigate the expression of beta-1 integrin by chondrocytes growing on the surface of microcarriers. Nasal chondrocytes (4 x 10(3)/cm(2)) were seeded on microcarriers and incubated at 37 degrees C, 5% CO(2), 60 rpm. Expression of chondrocyte markers and beta-1 integrin was determined using reverse transcriptase-polymerase chain reaction and immunocytochemical analyses. De novo synthesis of sulfate-containing proteoglycans was studied using 35SO(4) incorporation techniques. Like articular chondrocytes propagated in microcarrier spinner culture, nasal chondrocytes expressed high levels of collagen type II mRNA, whereas collagen type I mRNA levels were low. Aggrecan mRNA was detectable and levels of de novo 35SO(4) incorporation were high. Chondrocytes immunostained intensely for collagen type II and keratan sulfate but did not stain for collagen type I. beta-1 integrin mRNA levels were high, and the protein was immunolocalized to regions of cell-to-cell or cell-to-microcarrier contact. The fact the chondrocytes expressed high levels of beta-1 integrin raises the possibility that this integrin molecule has a role in the maintenance of the chondrocytic phenotype.

Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes.

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

Proinflammatory cytokine expression of IL-1beta and TNF-alpha by human osteoblast-like MG-63 cells upon exposure to silicon nitride in vitro.

This study was designed to determine the effect of Si(3)N(4) disks and particulates on human osteoblast-like MG-63 cells in vitro. The MG-63 (10(5)/mL) cells were plated onto 24-well polystyrene plates fitted with either sintered reaction-bonded (SRBSN) or reaction-bonded (RBSN) 15-mm disks. Controls consisted of wells without Si(3)N(4) disks. Cells propagated at 37 degrees C, 5% CO(2) for 48 h on Si(3)N(4) disks and control polystyrene surfaces exhibited similar proliferative capacities (7000 and 4000 cpm/10(5) cells, respectively, p > 0.05). Cells incubated with 1, 10, or 100 microgram/ml of Si(3)N(4) particles (<1.00 to 5.00 micrometer) for 24 h did not exhibit a decrease in DNA synthetic activity: 12 +/- 1.3 x 10(4), 10.5 +/- 1.5 x 10(4), and 11.0 +/- 1.7 x 10(4) cpm, respectively, compared to 11.6 +/- 2.6 x 10(4) cpm/10(5) for the control cells, as indicated by (3)H-thymidine uptake. Cells propagated on RBSN displayed increased expression of cytokines IL-1beta and TNF-alpha compared to the control cells, as shown by reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, cells propagated on SRBSN surfaces expressed the same level of IL-1beta and TNF-alpha as that of control cells. Incubation of MG-63 cells with 1-10 microgram/mL of particles did not increase IL-1beta expression. However, at 100 microgram/mL, TNF-alpha expression was greater than that of the control cells. Silicon nitride, evaluated here as disks or as particulates (1-10 microgram/mL), is biocompatible and does not hinder the proliferation or induce proinflammatory cytokine expression of human osteoblast-like MG-63 cells in vitro.

Effect of prosthetic titanium wear debris on mitogen-induced monocyte and lymphoid activation.

Wear debris generated by joint implant components has been reported to activate inflammatory and immune cells. Particulate debris derived from prosthetic material induces monocytes/macrophages, lymphocytes, synoviocytes, and fibroblasts to secrete cellular products, such as cytokines, which mediate inflammation. It has been speculated that degradation products impair the ability of inflammatory and immune cells to mount a protective response against noxious agents and infectious organisms by interfering with cell activation. Recent in vitro studies suggest that soluble metal ions inhibit T and B cell activation, but it is not known whether insoluble metal particles generated by prosthetic wear in tissue have the same effect. The purpose of the present study was to determine whether titanium wear debris retrieved from periprosthetic tissues surrounding a failed knee prosthesis suppresses activation of human monocytic and lymphoid cells. Peripheral blood monocytes and lymphocytes were incubated with the nonspecific activator pokeweed mitogen (PWM) in the presence or absence of titanium particles. Cell proliferative capacity and production of interleukins IL-1beta and IL-2 were determined as measures of activation. Titanium wear debris induced monocyte secretion of IL-1beta at levels comparable to those induced by PWM alone. In combination with PWM, titanium wear debris stimulated monocytes to secrete higher concentrations of IL-1beta than is stimulated by titanium itself or by PWM alone. Titanium wear debris did not activate lymphocytes, as indicated by marginal changes in DNA synthesis and IL-2 secretion, nor did it suppress the PWM-induced stimulation of DNA synthesis and IL-2 secretion. Our study suggests that nonspecific mitogen activators in spite of exposure to titanium wear debris can stimulate monocytic and lymphoid cells.

Glass peek composite promotes proliferation and osteocalcin production of human osteoblastic cells.

An isoelastic intramedullary implant has been developed using a composite of polyetheretherketone and 10% random, chopped E-glass fibers (GPEEK). The effect of this novel material on human bone cells has not been defined. The objective of this study was to test whether GPEEK supported the proliferation of the human bone cell line MG63, which exhibits osteoblastlike characteristics. Cells (1 x 10(5)/mL) were propagated on GPEEK discs with three different surface roughnesses (3, 6, and 9 microns) and on polystyrene plates, for comparison. The reaction of MG63 osteoblastlike cells to the GPEEK polymer composite was analyzed by determination of cell yield, osteocalcin production, and levels of alkaline phosphatase. The viable cells that were retrieved from the GPEEK discs of all three surface roughness had an approximate sixfold increase in number. Osteoblastic function of the cells, indicated by osteocalcin production, was unimpaired after a 5-day culture on the three surfaces of GPEEK. The highest level of osteocalcin was produced by osteoblastic cells propagated on GPEEK with a 9 microns surface roughness. The levels of alkaline phosphatase of these cells were similarly greater for the different degrees of surface roughness. Overall, this study demonstrates that GPEEK supported proliferation of osteoblastlike cells and provided a favorable environment for the continued production of osteocalcin in vitro.

Silicone gel and octamethylcyclotetrasiloxane (D4) enhances antibody production to bovine serum albumin in mice.

The purpose of the present study was to determine whether components of silicone breast implants [silicon (polydimethylsiloxane) oil, silicone gel, and octamethylcyclotetrasiloxane (D4)] potentiate the antibody response to bovine serum albumin (BSA) in mice. Seventy A/J mice were divided into seven groups which received the following: group I--phosphate buffer solution (PBS); group II--20 centistoke (cs) silicone oil; group III--50% silicone gel homogenized in silicone oil; group IV--incomplete Freunds' adjuvant (IFA); group V--IFA mixed with an equal volume of silicone oil; group VI--D4; and group VII--IFA mixed with an equal volume of DA. Each 0.1 mL of treatment material(s) was mixed or emulsified with an equal 0.1-mL volume of 250 micrograms/mL BSA in PBS solution. Antibodies to BSA were measured using an enzyme-linked immunosorbent assay. Our study demonstrates for the first time that both D4 and the silicone gel potentiate antibody production to BSA in mice. Histopathologic evaluation of the injection sites reveals granulomas for mice injected with IFA and D4 preparations. Whether D4 or silicone gel acts as an adjuvant against self-antigens has yet to be determined.

The effects of ciprofloxacin on human chondrocytes in cell culture.

Ciprofloxacin is a highly potent antibacterial agent that is used extensively in bone and joint infections. Because of reports of potential chondro-toxicity in animals, the effects of this drug on cells derived from human cartilage were tested in liquid micromass and agarose gel cultures. An inhibition of cell proliferation as indicated by a decrease in [3H]-thymidine uptake and bromodeoxyuridine labeling at ciprofloxacin concentrations of 0.5 and 50 mg/l was found which corresponded to the therapeutic and toxic serum levels. There was no effect on proteoglycan synthesis as indicated by 35SO4 incorporation. Immunocytochemistry showed no changes in morphology or staining patterns for type-I procollagen, type-II collagen, keratan sulfate and unsulfated chondroitin. Because the amount of inhibition of DNA synthesis varied with different ciprofloxacin concentrations, this data suggests that this agent has a differential effect on newly differentiating cells and might be the basis for contraindication in pediatric patients.

Silicone gel and octamethylcyclotetrasiloxane potentiate antibody production to bovine serum albumin in mice.

Earlier studies, in the rat, have shown that silicone gel acts as a potent adjuvant for antibody production against bovine serum albumin (BSA). The purpose of the present study is to determine whether components of the silicone breast implant (silicone oil, silicone gel, and octamethylcyclotetrasiloxane [D4]) potentiate the antibody response to BSA in mice. Groups of ten A/J mice received a single subcutaneous injection of 0.1 ml of BSA emulsified with the following: a) silicone preparations, b) incomplete Freund's adjuvant, or c) saline alone. Antibodies to BSA were measured using an enzyme-linked immunosorbant assay. The results indicate that both D4 and the silicone gel potentiate antibody production to BSA in mice. Known adjuvants have been shown to induce autoimmune syndromes in animal models. Whether silicones can act in a similar mechanism is still unclear.

Isolation and characterization of metallic wear debris from a dynamic intervertebral disc prosthesis.

A dynamic intervertebral disc prosthesis (DIDP) has been developed. It consists of a CoCrMo body and uses Ti6Al4V springs to replicate the mechanical function of the lumbar joint. Wear studies have been performed previously on the DIDP using two specialized simulators to test the wear properties of the moving parts of the disc prosthesis. A pin-in-slot simulator generates wear that would occur in the hinge-pin assembly of the prosthesis. A spring-in-pocket simulator approximates the conditions under which the springs would wear against the body of the prosthesis. The spring-pocket interface is responsible for the production of approximately 90% of the total wear occurring in the prosthesis, and is therefore the main focus of this study. Bovine serum with a preservative has been used as a lubricant in both simulators. The spring-in-pocket simulator compares the effects of two different manufacturing techniques of CoCrMo (HIPing and forging) on their wear characteristics against Ti6Al4V springs. Debris from the spring-in-pocket simulator has been isolated from the serum lubricant and characterized using scanning electron microscopy techniques. The morphology of the Ti6Al4V fragments is rough and irregularly shaped. The size of these fragments ranges from < 1 microns to > 30 microns. The forged CoCrMo alloy debris has an irregular polyhedral shape, with sizes in the same range as the spring fragments. The morphology of the HIPed CoCrMo debris is spherical with a size range < 5 microns to > 30 microns. Length and width measurements of micron-size particles were made with the particle measurements feature of the scanning electron microscope. Micron-size particles were found in all stations. This article provides a unique way to isolate and analyze debris from serum lubricants used in simulators.

Polymethylmethacrylate particles enhance DNA and protein synthesis of human fibroblasts in vitro.

Aseptic loosening of polymethylmethacrylate (PMMA) fixed prosthesis is characterized by formation of a radiolucent fibrous membrane, accumulation of inflammatory cells and osteolysis. Since this membrane is produced by fibroblasts, it is likely that these connective tissue cells play a critical role in the loosening process. Whether fibroblasts form the radiolucent membrane in response to stimulation by PMMA has not yet been established, nor is it known whether fibroblasts play a role in attracting inflammatory cells to the bone-cement interface by secreting chemical mediators. To address this question, we analyzed the in vitro response of normal human fibroblasts to PMMA. Cells were plated with 10(5)/mL Dulbecco minimal essential medium and were incubated 4 h later with PMMA particles, polystyrene (PS) particles, or medium alone. Proliferative capacity monitored by incorporation of 3H-thymidine was significantly increased following a 48 h exposure to PMMA. Protein synthesis determined by incorporation of 14C-leucine and 14C-proline was also increased. In contrast, levels of secreted prostaglandin (PG) E2 assayed immunoenzymatically was not altered by PMMA. Fibroblasts exposed to control PS did not change their proliferative or protein synthetic activity. Fibroblasts internalized PMMA and PS particles without detectable ultrastructural damage. PMMA enhancement of fibroblast proliferative capacity and protein synthetic ability observed in our in vitro assay system suggests similar effects on fibroblasts in vivo.

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry.

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage exposure to polymethyl methacrylate leads to mediator release and injury.

To understand further the role of macrophages in the loosening of cemented arthroplasty, several in vitro effects of polymethyl methacrylate (PMMA) particle exposure in these cells were studied. The kinetics of arachidonic acid and derived inflammatory mediator release was characterized following macrophage exposure to either PMMA or control polystyrene particles. Temporal release of radiolabeled products by [14C]arachidonate-labeled cells was determined by sequential scintillation counting. Significant dose-dependent release of arachidonic acid mediators by macrophages was observed within half an hour of exposure to either PMMA or styrene particles. Unexposed control cells incubated in media alone did not release detectable amounts of radiolabeled products. The leakage of intracellular lactate dehydrogenase (LDH), a marker of cell injury, was detected spectrophotometrically 4 h following exposure to PMMA but not styrene. PMMA-induced LDH release was dose dependent. In contrast, polystyrene exposure failed to increase LDH release above unexposed control cells. These in vitro studies reveal that macrophages rapidly released arachidonic acid and derived inflammatory mediators in response to both PMMA and styrene particles. However, cells exposed to PMMA are lethally damaged, as reflected by the subsequent leakage of their intracellular LDH. We propose that a similar sequence of events may occur when macrophages encounter PMMA particles at the bone-cement interface. This is characteristic of a foreign body granulomatous response.

Immunocytochemical localization of N-acetyl-aspartate with monoclonal antibodies.

N-Acetyl-aspartate is found in high concentrations in all areas of the brain, but is undetectable in non-neuronal tissue. In order to characterize the cellular localization of N-acetyl-aspartate in brain, highly specific monoclonal antibodies against N-acetyl-aspartate were produced by fusing spleen lymphocytes obtained from mice immunized with N-acetyl-aspartate conjugated to thyroglobulin by carbodiimide with P3/x63-Ag8.653 mouse myeloma cells. Clones were selected which secrete IgG2a(k) antibodies highly specific for conjugated N-acetyl-aspartate. Only 3-6% cross-reactivity with conjugated N-acetyl-aspartate-glutamate was observed at high antibody concentrations, whereas no cross-reactivity (less than 1%) was observed with conjugated N-acetyl-glutamate or aspartate. Preincubation of the antibodies with 0.5 mg/ml conjugated N-acetyl-aspartate blocked immunoreactivity more than 90%, while preincubation with conjugated N-acetyl-aspartate-glutamate and free N-acetyl-aspartate had no effect. Immunocytochemical staining has shown that N-acetyl-aspartate-like immunoreactivity is localized in neurons, which are widely distributed throughout the brain. The immunoreactive neurons exhibited intense staining of the perikarya, proximal dendrites and axons. No consistent pattern of distribution of immunoreactivity was observed with regard to primary neurotransmitter characteristics of stained neurons although neurons with long projections or extensive arbors, such as pyramidal cells in cortex, locus coeruleus, motor neurons and Purkinje cells, stained much more intensively than local circuit neurons.