Histamine stimulates the proliferation of human articular chondrocytes in vitro and is expressed by chondrocytes in osteoarthritic cartilage.

OBJECTIVES: To determine the effects of histamine on the proliferative rate of human articular chondrocytes (HAC) in vitro, and to demonstrate whether HAC in osteoarthritic (OA) cartilage express histamine and histidine decarboxylase (HDC). METHODS: HAC in vitro were incubated with and without histamine in 96 well culture plates and the extent of cell proliferation was determined using the naphthol blue-black method. Histamine effects were analysed with the histamine H(1) and H(2) receptor antagonists, mepyramine and ranitidine, respectively. Rabbit polyclonal antibodies and alkaline phosphatase conjugated secondary antibodies were used, and histamine and HDC were demonstrated by immunohistochemistry in OA cartilage tissues. RESULTS: Histamine stimulated the proliferation of HAC in culture. This stimulation was blocked by the addition of mepyramine, but not ranitidine, suggesting that the effect is mediated through H(1) histamine receptors. The addition of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase (the enzyme responsible for histamine production), reduced the rate of proliferation of HAC. Both histamine and histidine decarboxylase were demonstrated in chondrocytes of OA cartilage by immunohistochemistry. CONCLUSION: Changes induced by histamine in the proliferative rate of HAC may contribute to the formation of chondrocyte clusters associated with OA cartilage; an observation supported by the demonstration of histamine and HDC expression by chondrocytes of OA cartilage in situ.

Clinical, biochemical, and immunohistochemical features of necrobiotic xanthogranulomatosis.

AIMS: To describe the clinical features of two patients with paraproteinaemia and necrobiotic xanthogranulomatosis together with detailed immunohistochemistry of the lesions in one. METHODS: The clinical history and results of biochemical investigations of the patients were retrieved from the files. Immunohistochemistry was used to investigate the expression of macrophage and mast cell markers, amyloid A and P, S-100 protein, and apolipoprotein AI and B in xanthogranulomatous skin lesions from patient 2. In addition, protein A-sepharose chromatography was used to separate serum from patient 2 and apolipoprotein B and the IgG paraprotein were measured in the fractions eluted. RESULTS: Monocytes/macrophages comprised the major cellular component of the lesion, and unusually for xanthomata, areas of collagen necrosis were also seen. Activated mast cells were present at the margins of macrophage clusters and adjacent to areas of collagen necrosis. Serum paraprotein was bound to low density lipoproteins as judged by protein A-sepharose chromatography, and was also located within macrophagic foam cells of the lesion on immunohistochemistry. CONCLUSIONS: These observations demonstrate many features similar to atherosclerosis including collagen necrosis and mast cell activation.

Histamine stimulates matrix metalloproteinase-3 and -13 production by human articular chondrocytes in vitro.

OBJECTIVES: To determine the effects of histamine on matrix metalloproteinase (MMP) production by human articular chondrocytes (HAC) in vitro. METHODS: Conditioned culture medium from HAC cultures incubated with and without 20 microM histamine was assayed by enzymne linked immunosorbent assay (ELISA) for MMP-1, MMP-8, MMP-13 (collagenases 1, 2, and 3, respectively) and MMP-3 (stromelysin). Monolayer cultures of HAC were also immunostained for MMP-13 and MMP-3. RESULTS: The HAC cultures showed a significant increase in MMP-13 and MMP-3 production (2.2- and 1.9-fold, respectively) after treatment with 20 microM histamine for 24 hours, but MMP-1 and MMP-8 were unaffected. All cultures showed MMP-13 and MMP-3 detectable by immunolocalisation. MMP-3 was the more prominent enzyme as shown by both ELISA and immunolocalisation techniques. CONCLUSIONS: Histamine exposure increased both MMP-13 and MMP-3 production by HAC in vitro, thereby suggesting a pathophysiological role in the chondrocytic phenotype associated with degenerative changes in osteoarthritis.

Electrochemical monitoring of cell behaviour in vitro: a new technology.

This article describes a novel electrochemical technique for the real-time monitoring of changes in the behaviour of adherent human cells in vitro: i.e., a biosensor that combines a biological recognition mechanism with a physical transduction technique, described collectively as Oncoprobe. Confluent viable cells adherent to gold electrodes (sensors) modify the extracellular microenvironment at the cell:sensor interface to produce a change in the electrochemical potential compared to that measured in the absence of cells. The potential was measured as an open circuit potential (OCP) with respect to a saturated calomel reference in the bulk culture medium. Typical OCP values for confluent cultures of human breast carcinoma cells, 8701-BC, approximated -100 mV compared with cell-free values of approximately -15 mV. The OCP for 8701-BC cells was modified in response to temperature changes over the range 32 to 40 degrees C and also to treatments with phytohemagglutinin (PHA, 25 microg/mL), cycloheximide (30 microM) and interleukin-1 beta (IL-1, 0.5 ng/mL) over 24 h. Cultures of synovial fibroblasts also responded to the same treatments with similar responses, producing negative shifts in the OCP signal with PHA and IL-I, but a positive shift in OCP signal with cycloheximide, all relative to the untreated control cultures. From experimental data and theoretical considerations it is proposed that the cell-derived signals are mixed electrode potentials reflecting a "conditioned," more reducing environment at the cell:sensor interface. Only viable cells caused a negative shift in the OCP signal, this being lost when cells were rendered nonviable by formalin exposure. This technology appears unique in its ability to passively "listen in" on cell surface activities, suggesting numerous applications in the fields of drug discovery, chemotherapy, and cell behaviour.

Electrochemical monitoring of anticancer compounds on the human ovarian carcinoma cell line A2780 and its adriamycin- and Cisplatin-resistant variants.

A novel electrochemical technique which detects and monitors real-time changes in cell behavior in vitro has been used to examine the effects of recognized anticancer drugs on the human ovarian carcinoma cell line A2780 and its adriamycin (A2780adr)- and cisplatin (A2780cispt)-resistant variants. These cells, adherent to gold electrodes or sensors, modify the extracellular microenvironment at the cell:sensor interface, producing an electrochemical potential that is different from that of the bulk culture medium. Confluent, adherent A2780 cells produced an electrochemical signal, measured as an open circuit potential (OCP), of approximately -100 mV compared to a cell-free value of approximately -15 mV. Exposure of A2780 cells to cisplatin (range 10(-4) to 10(-6) M), adriamycin (range 10(-5) to 10(-7) M), and vinblastine (10(-6) M) all produced positive shifts in the OCP signal relative to untreated control cells during 24 h of culture, but Taxotere (range 10(-5) to 10(-7) M) had no effect. These positive shifts in OCP signal were evident well before observations of reduced cellular adhesion and viability after 24 h, as judged in parallel cultures with a plastic substratum and by scanning electron microscopy. By contrast, the same treatments applied to the A2780adr and A2780cispt variants showed that each demonstrated different sensitivities to the same drugs applied to the parental A2780 cells. The effects of the same four anticancer drugs on ovarian carcinoma (A2780) and breast carcinoma (8701-BC) cell lines showed that the former was far more responsive to adriamycin and cisplatin. Such differences in drug sensitivities between the two cell lines were subsequently confirmed using the conventional MTT assay over 5 days. Although this electrochemical technology readily detects changes in cell adhesion and viability, the modified OCP signals recorded within a few hours of anticancer drug treatments are evident well before microscopic morphological changes become apparent. It is proposed that these early changes in OCP signals, relative to control untreated cells, reflect modifications of physiological/behavioral processes manifested at the cell surface.

Distribution and activation of eosinophils in inflammatory bowel disease using an improved immunohistochemical technique.

Eosinophils are a recognized feature of inflammatory bowel disease (IBD), but their tissue distribution and functional importance in Crohn's disease (CD) and ulcerative colitis (UC) remain obscure. This study describes an improved immunohistochemical protocol to identify eosinophils in full thickness bowel wall specimens of IBD (n=40) and their in situ relationships with the chemoattractants eotaxin and RANTES. Eosinophils were identified using immunohistochemistry with a combination of monoclonal antibodies (EG1+EG2+MBP), an ultrasensitive technique superior to other methodologies, and their tissue distributions were related to those for eotaxin, RANTES, mast cells and neutrophils. Increased numbers of eosinophils (up to 400 cells/mm(2)) were observed in active, fulminant inflammation in both CD and UC, this being related to the severity of inflammation and not the diagnosis of the two disorders. The chemoattractants eotaxin (CCL11) and RANTES (CCL5) were upregulated in IBD tissues showing eosinophilia. Neutrophils and mast cells were commonly associated with eosinophil accumulations. Eosinophil numbers and their in situ activation are increased in active rather than chronic IBD. The observations strongly suggest a pivotal role for the eosinophil and its potent mediators in many pathophysiological symptoms of CD and UC, where it represents the major proportion of all granulocytic cells in active inflammatory bowel disease.

Expression of vitamin D receptors and matrix metalloproteinases in osteoarthritic cartilage and human articular chondrocytes in vitro.

OBJECTIVES: To examine the in situ distributions of vitamin D receptors (VDR) and matrix metalloproteinases (MMPs) in osteoarthritic cartilage for comparison with non-arthritic, normal cartilage; and to assess the in vitro effects of 1alpha,25 dihydroxyvitaminD(3)(1alpha,25(OH)(2)D(3)) on MMPs-1, -3 and -9 and prostaglandin E(2)(PGE(2)) production by cultures of human articular chondrocytes (HAC) shown to be VDR-positive. METHODS: Using immunohistochemistry VDR expression in different specimens of osteoarthritic cartilage (N=11) was compared to that in normal cartilage (N=6), along with the immunodetection of MMPs-1, -3 and -9. The effects of 1alpha25(OH)(2)D(3)on MMP and PGE(2)production by HAC in vitro, with and without stimulation by TNFalpha or phorbol myristate acetate (PMA), was evaluated using ELISA methodology. RESULTS: VDR was demonstrated in HAC of all specimens of osteoarthritic cartilage, especially the superficial zone, whereas only two of five normal cartilage specimens were VDR(+)for a minor proportion of HAC. Immunolocalization of MMPs-1, -3 and -9 was often seen in areas where chondrocytes were VDR(+), and dual immunolocalization has demonstrated individual chondrocytes positive for both VDR and MMP-3 in situ. In vitro, 1alpha25(OH)(2)D(3)alone had no effect on MMP-1, -9 and PGE(2)production by HAC, but MMP-3 production was up-regulated by 1alpha25(OH)(2)D(3)either with or without stimulation with TNFalpha or PMA. By contrast the increased production of MMP-9 and PGE(2)induced by PMA was significantly suppressed by concomitant treatment with 1alpha25(OH)(2)D(3). CONCLUSIONS: The demonstration of VDR expression by HAC in osteoarthritic cartilage was often associated with sites where MMP expression was prevalent, observations in contrast to their virtual absence in normal age-matched cartilage. Together with HAC in vitro studies, the data suggests that 1alpha25(OH)(2)D(3)contributes to the regulation of MMP and PGE(2)production by HAC in osteoarthritic cartilage.

Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes.

OBJECTIVE: To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in osteoarthritic (OA) cartilage compared with normal, age-matched articular cartilage. METHODS: Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL-1beta, TNFalpha, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography. RESULTS: Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP-3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL-1beta- and TNFalpha-positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, full-thickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL-1beta, TNFalpha, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL-1 with MMP-8 by individual chondrocytes in situ. CONCLUSION: These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL-1beta, TNFalpha, and 6 different MMPs. These observations support the concept that cytokine-MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.

Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion.

INTRODUCTION: Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident. Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis o RA, especially its ability to regulate interleukin (IL)-1beta expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been assigned numerous biological effects and has been implicated as an important factor in TNF-alpha expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-alpha in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint. AIMS: To examine the in situ relationships of TNF-alpha, IL-1beta and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue. MATERIALS AND METHODS: Samples of rheumatoid synovial tissue and cartilage-pannus junction were obtained from patients (n=15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-alpha and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1beta, TNF-alpha and IL-15 using enzyme-linked immunosorbent assay methodology. RESULTS: Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-alpha, IL-1alpha and IL-1beta production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-alpha and IL-1beta were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens. MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-alpha and IL-1beta released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect of the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-alpha and IL-1beta. Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-alpha and IL-1beta, but not of IL-15. Four preparations of primary rheumatoid synovial cell cultures produced more IL-1beta than TNF-alpha, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ. DISCUSSION: The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-alpha and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.

Phenotype of mast cells in the brain tumor. Capillary hemangioblastoma.

Mast cells (MC) are heterogenous cell population. In normal human brain they are not numerous. Increases in number of mast cells within CNS occur in certain disease states including neoplasms. In capillary hemangioblastoma several authors reported mast cells as a fourth cell type of the tumor. The aim of the present study was to examine phenotype and distribution of MC in cerebellar capillary hemangioblastoma by means of specific immunological markers. Study was performed on the tumor of ten affected individuals. Tumor specimens of seven cases were fixed in formalin and embedded in paraffin wax. Additional three tumours were fresh-frozen samples. Mast cells were identified with two monoclonal antibodies generated against tryptase and chymase. In all capillary hemangioblastomas mast cells were numerous exclusively in the tumor mass and only occasionally found in adjacent or far from the tumor located areas of the cerebellum. The cells contained tryptase and chymase. At periphery of hemangioblastomas some mast cells underwent degeneration and calcification. Our results confirm previous observations that mast cells are numerous in the capillary hemangioblastoma and show that most of these cells are tryptase/chymase phenotype (MCTC).

Menstruation: induction by matrix metalloproteinases and inflammatory cells.

Menstruation occurs at the end of a normal reproductive cycle in the human female, following the fall in progesterone resulting from the demise of the corpus luteum. Current data support a central role for the matrix metalloproteinases in menstruation but their focal pattern of expression within peri-menstrual and menstrual endometrium suggests local rather than hormonal regulation. This review emphasizes the similarities between menstruation and an inflammatory process and examines the relationship between cells of hemopoietic lineage, particularly mast cells, eosinophils, neutrophils and macrophages, and the local production and activation of matrix metalloproteinases within the endometrium. It proposes a complex of critical regulatory circuits, initially activated by the withdrawal of progesterone, which provide interactions between the migratory cells that produce a myriad of important regulatory molecules and endometrial stromal and epithelial cells which produce both chemokines and matrix metalloproteinases. These mechanisms could account for the focal nature of the tissue degradation at menstruation.

Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development.

Neovascularization is a prominent feature of late-stage atherosclerotic lesions and their complications but is generally regarded as an insignificant, undetectable component of the earliest stages of plaque development, probably because of relatively poor histological techniques. Using an improved vascular staining procedure, we have examined the extent of neovascularization in the earliest plaque lesions. Combined monoclonal antibodies to CD31, CD34, and von Willebrand factor have provided an ultrasensitive technique with which to visualize blood vessels in early atherosclerotic lesions (n = 55) of human carotid arteries obtained through surgical procedures. Capillary-like microvessels were shown in very early atherosclerotic lesions (type II), where they were associated with the distribution of macrophages and a few immature mast cells. Neovascularization was more prominent in type III lesions with vessels of variable size, often providing a focus around which local accumulations of macrophages and apolipoproteins A-I and B were visualized. Thickened type III lesions usually showed an intricate network of microvessels, together with numerous mast cells. These studies have shown neovascularization as a prominent feature of early stages of atherosclerotic plaque development. Whereas distribution of apolipoproteins A-I and B were observed in the very earliest stages of the plaque intima, these lipids, together with macrophages, foam cells, and mast cells, were observed as perivascular accumulations in a proportion of type II and III lesions. Such findings indicate that neovascularization is an important feature of early plaque development and may provide an additional or alternative source of leukocyte and lipid accumulations relative to the arterial lumen.

Local neovascularization and cellular composition within vulnerable regions of atherosclerotic plaques of human carotid arteries.

An improved immunohistochemical method has been used to assess neovascularization within the vulnerable 'shoulder' regions of atherosclerotic plaques from carotid arteries. A combination of monoclonal antibodies (CD31, CD34, +/- von Willebrand factor) was shown to be far more effective than conventional techniques in demonstrating extensive vascularizations within the 'shoulder' and cap regions of late-stage plaques. Such sites were shown to be microfocal, often appearing as a plexus of both large and small vessels which occupied a significant proportion of the 'shoulder' area. These regions of marked neovascularization were commonly associated with accumulations of macrophages, mast cells, and T-cells, indicative of local inflammatory reactions. The matrix components elastin and collagen type VI showed variable distributions which suggested extensive tissue remodelling, whereas collagen type IV was recognized as a basement membrane protein of most blood vessels, as well as being associated with 'stellate' smooth muscle cells. Evidence of local microvascular damage within the shoulder regions of some specimens was demonstrated by extravascular red blood cells, macrophages containing haemosiderin, and perivascular fibrin deposition. These local haemorrhages derived from microvessels beneath the lining of the arterial lumen are a further indication of how the microfocal vascularization of the plaque 'shoulder' might contribute to further complications of inflammation and plaque destabilization in late-stage disease.

Vitamin D receptors in the rheumatoid lesion: expression by chondrocytes, macrophages, and synoviocytes.

OBJECTIVES: The active form of vitamin D3, 1 alpha,25 dihydroxyvitamin D3 (1,25D3), through its interaction with vitamin D receptors (VDR), is reported to effect a variety of anabolic and catabolic events, especially in bone and cartilage tissues. As cartilage degradation and tissue remodelling are characteristic features of the rheumatoid lesion, the distribution and expression of VDR at sites of cartilage erosion was examined. METHODS: Immunolocalisation techniques using a rat monoclonal antibody to VDR and an alkaline phosphatase conjugated avidin/biotin detection system were used to examine VDR in 18 specimens of cartilage-pannus junction, 10 specimens of rheumatoid synovium or cartilage tissue, and four primary cultures of adherent rheumatoid synovial cells (RSC). For comparison, VDR expression was examined in 10 specimens of normal, healthy age matched articular cartilage. RESULTS: VDR was demonstrated in 15 of 18 cartilage-pannus junctions either at the interface (8 of 18), within the pannus tissue (12 of 18), and by chondrocytes often close to the erosive lesion (10 of 18). All the rheumatoid synovial tissue and 5 of 10 cartilage specimens showed cells with positive staining, but the extent of this was variable. Negligible VDR staining was observed for normal cartilage. Primary cultures of RSC also showed variability in both the numbers and proportions of macrophages or synovial fibroblasts stained for VDR (range 10-50%), this being more common in cultures with a high proportion of macrophages. CONCLUSIONS: VDR expression has been demonstrated by most specimens of cartilage-pannus junction; was associated with various cell types, including chondrocytes, but not exclusively with CD68+ macrophages. The focal nature of VDR expression within the rheumatoid lesion suggests a contributory role for 1 alpha,25D3 in the pathophysiological processes of rheumatoid arthritis.

The short-acting anesthetic propofol produces biphasic effects-depression and withdrawal rebound overshoot-on some (but not all) limbic evoked potentials in the behaving rat.

Propofol, the relatively new, short-acting general anesthetic, markedly enhances the action of GABA at the GABAA receptor. To evaluate its effects on field potentials evoked in the dentate gyrus (DG) during the anesthetic and recovery periods, propofol was administered intraperitoneally to behaving rats bearing stimulating electrodes in the dorsal perforant path (DPP), where medial perforant path fibers predominate, and in the anterior piriform cortex (PC; i.e., olfactory cortex), and recording electrodes in the DG. Input from the PC reaches the DG via the lateral perforant path. Population slow waves (SWs) were evoked by paired-pulse stimulation of the PC at a 32 ms interstimulus interval (ISI) to produce paired-pulse facilitation in the awake animal. We had previously demonstrated that amplitude of SW2 (produced by the second stimulus) was greatly decreased by GABAergic drugs and increased by antiGABAergic convulsant agents. After administration of propofol, mean amplitude of SW2 decreased immediately and remained low for 30-60 min during propofol-induced sleep (as expected), then unexpectedly increased to about 1.5- to 2-fold above pretreatment levels at 2-4 h before gradually returning to pretreatment levels. In addition, the DPP was stimulated to produce either paired-pulse inhibition (20 ms ISI) or facilitation (32 ms ISI) of DG population spikes (PSs) in the awake animal. PS2 was much more inhibited during propofol-induced sleep, than during the pretreatment period, consistent with an expected marked increase in recurrent inhibition. An overshoot in PS2 amplitude was observed only occasionally during recovery, suggesting that withdrawal overshoot in amplitudes is more characteristic of PC-evoked DG SW2 potentials. The overshoot in SW2 amplitude during recovery may have been related to propofol's 'rapid on-rapid off' actions on the GABAA receptor, perhaps resulting in a phenomenon like the 'GABA withdrawal syndrome'. Such an effect, if true, may help explain the rare occurrence of seizures, especially during recovery, associated with its use clinically.

The effects of 1 alpha,25-dihydroxyvitamin D(3) on matrix metalloproteinase and prostaglandin E(2) production by cells of the rheumatoid lesion.

INTRODUCTION: 1alpha,25-dihydroxyvitamin D(3)[1alpha,25(OH)(2)D(3)], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1alpha,25(OH)(2)D(3); and cell cultures have suggested that 1alpha,25(OH)(2)D(3) may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1alpha,25(OH)(2)D(3) within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1alpha,25(OH)(2)D(3) at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1alpha,25(OH)(2)D(3) modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E(2) (PGE(2)) production in two cell types known to express chondrolytic enzymes. AIMS: To investigate VDR expression in rheumatoid tissues and to examine the effects of 1alpha,25-dihydroxyvitamin D(3) on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE(2) production. METHODS: Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chrondrocytes were treated with either 1alpha,25(OH)(2)D(3) or interleukin (IL)-1beta, or both. Conditioned medium was assayed for MMP and PGE(2) by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values. RESULTS: The rheumatoid synovial tissue specimens (n=18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1alpha,25(OH)(2)D(3) alone, but when added simultaneously with IL-1beta the stimulation by IL-1beta was reduced from expected levels by up to 50%. In contrast, 1alpha,25(OH)(2)D(3) had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1beta was not affected by the simultaneous addition of 1alpha,25(OH)(2)D(3) whilst MMP-3 production was enhanced (Table 1). The production of PGE(2) by RSFs was unaffected by 1alpha,25(OH)(2)D(3) addition, but when added concomitantly with IL-1beta the expected IL-1beta-stimulated increase was reduced to almost basal levels. In contrast, IL-1beta stimulation of PGE(2) in HACs was not affected by the simultaneous addition of 1alpha,25(OH)(2)D(3)(Table 2). Pretreatment of RSFs with 1alpha,25(OH)(2)D(3) for 1h made no significant difference to IL-1beta-induced stimulation of PGE(2), but incubated for 16h suppressed the expected increase in PGE(2) to control values. This effect was also noted when 1alpha,25(OH)(2)D(3) was removed after the 16h and the IL-1beta added alone. Thus it appears that 1alpha,25(OH)(2)D(3) does not interfere with the IL-1beta receptor, but reduces the capacity of RSFs to elaborate PGE(2) after IL-1beta induction. Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1apha,25(OH)(2)D(3) in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1alpha,25(OH)(2)D(3) had little effect of basal MMP production by RSFs, although more pronounced differences were noted when IL-1beta-stimulated cells were treated with 1alpha,25(OH)(2)D(3), with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1alpha,25(OH)(2)D(3) to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1alpha,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1alpha,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1beta stimulation of these cells was markedly suppressed by the concomitant addition of 1alpha,25(OH)2D3. As with MMP production, there are disparate effects of 1alpha,25(OH)2D3 on IL-1beta stimulated PGE2 production by the two cell types; 1alpha,25(OH)2D3 added concomitantly with IL-1beta had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1alpha,25(OH)2D3 may have a functional role in the joint disease process. 1 alpha,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.