Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines

Methotrexate (MTX) is one of the most widely prescribed drugs in the treatment of rheumatoid arthritis (RA). The mechanism by which MTX exerts its anti-rheumatic effect has not yet been defined. The aim of the present study was to investigate the effect of MTX treatment (7.5-15 mg/week) on synovial tissue in RA. For this purpose, synovial biopsies were taken from 11 RA patients before and 16 weeks after initiation of MTX therapy. Immunohistochemistry was performed using monoclonal antibodies (MAb) specific for CD3, CD4, CD8, CD22, CD25, CD38, CD68, MAb67, Ki67, interferon gamma (IFN-gamma), interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor alpha (TNF-alpha), E-selectin, ICAM-1 and VCAM-1. All parameters for disease activity improved during the period of treatment. Immunohistochemical analysis revealed a statistically significant decrease in scores for CD3, CD8, CD38, CD68, Ki67, IL-1beta, TNF-alpha and the adhesion molecules E-selectin and VCAM-1. The observed decrease in synovial scores for inflammatory cells, monokines and adhesion molecules suggests that the anti-inflammatory effect of MTX is, in part, dependent on a reduction in monokine-inducible vascular adhesion molecules and subsequent reduction of cell traffic into joints.     

Modulators of prostaglandin E2 synthesis in human amnion

OBJECTIVE: The purpose of these studies was to determine the effects of the essential fatty acid, linoleic acid, and the commonly used non-steroidal anti-inflammatory agents, aspirin and acetaminophen, on the rate of prostaglandin (PG) biosynthesis by human amnion cells. METHODS: Amnion cells were isolated from term, normal pregnancies and grown to confluence. Cells were incubated with control or medium containing 100 mumol/L linoleic acid. Cells were also incubated with control medium or medium containing 10 or 100 micrograms/mL aspirin or acetaminophen. RESULTS: Following an initial delay, amnion cells exposed to linoleic acid exhibited a significant increase in PGE synthesis. Both aspirin and acetaminophen in clinically relevant concentrations had a significant inhibitory effect on amnion cell PGE synthesis. CONCLUSIONS: Linoleic acid has a stimulatory effect and aspirin and acetaminophen have an inhibitory effect on PGE synthesis in human amnion cells in culture. We speculate that dietary habits, supplement ingestion, and over-the-counter drug use may affect amnion cell PG production. In view of the potential importance of intrauterine PG production in normal and abnormal labor, further study in this area is indicated.     

Effect of cytokines on anticryptococcal activity of human microglial cells

Since blood is a biologic product, it is unlikely that the risk for transfusion-transmitted infection will ever be reduced to zero. The approach to emerging infections associated with transfusion of blood and blood products includes assessing the transmissibility of the agent by this route; developing effective prevention strategies, including screening tests and donor deferral policies; improving viral and bacterial inactivation procedures; and surveillance for known, as well as emerging and poorly characterized, transfusion-transmitted agents. Vigilance is needed to help ensure proper balance between safety and the availability of blood. Finally, vigilance needs to extend to the developing world, where the basic elements to reduce transfusion-transmitted infections and systems of disease surveillance are often not available.     

ATP-mediated cytotoxicity in microglial cells

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.     

Widespread expression of MRP8 and MRP14 in human cerebral malaria by microglial cells

Recently, laser treatment of the prostate has been added to the urologist's armamentarium for the treatment of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). Until now, limited data on long-term outcome are available notwithstanding the fact that such information is crucial in determining the ultimate role of laser prostatectomy in the treatment of BPH. We now have 3-year data of a comparative study using the Urolase and Ultraline fiber in Nd:YAG sidefiring laser prostatectomy. The study was performed to compare laser prostatectomy using a pure coagulation (Urolase fiber) and a combination of a coagulation and vaporization (Ultraline fiber). In a period of 15 months, 93 men were randomized for laser treatment with the Ultraline fiber (N = 44) or the Urolase fiber (N = 49). Symptom scores, maximal uroflow, postvoiding residual volume, and sexual history were noted over a 3-year period. Adverse events and retreatments were also recorded. The mean postoperative catheterization time was 18 days, without significant difference between the two groups. After 3 years, we demonstrated a durable improvement in maximal flow rate, from 7.8 to 13.9 mL/sec in the Urolase group and from 7.9 to 13.6 mL/sec in the Ultraline group. In both groups, however, a considerable decrease in the maximal flow rate was noted after 3 years compared with 3 months after treatment, from 18.7 to 13.9 mL/sec in the Urolase group and from 20.0 to 13.6 mL/sec in the Ultraline group. The symptom scores showed marked and lasting improvement. The postvoiding residual urine volume became very low in the early postoperative period but did significantly increase after 3 years; nevertheless, it was still only 50% of the preoperative value. Although after 3 years, the maximal uroflow rate was still significantly improved compared with baseline, a considerable decrease was noted when compared with the early postoperative value. The same considerable and lasting improvement in subjective outcome (symptom scores) was seen in both groups. Although the Ultraline fiber also causes vaporization of prostatic tissue, no differences could be noted in the clinical outcome obtained with the two fibers.     

Cloning and characterization of a novel promiscuous human beta-chemokine receptor D6

Members of the chemokine family of chemotactic peptides interact with their target cells through heptahelical cell surface receptors. An understanding of the biochemistry and expression patterns of these receptors is therefore central to our overall understanding of the roles played by chemokines in both physiological and pathological processes. To date, eight receptors for the beta-chemokine subfamily have been described. We have recently cloned a novel murine beta-chemokine receptor and report here the identification and characterization of a highly homologous human gene termed human D6 (hD6). This is a promiscuous beta-chemokine receptor and appears to be able to bind the majority of members of the beta-chemokine family. It is, however, specific for this family and shows no detectable affinity for members of the alpha-chemokine or the C or CXXXC chemokines. Unlike the majority of other chemokine receptors, human D6 does not appear to be able to flux calcium following ligand binding, thus it is currently not clear if this novel receptor is indeed a signaling receptor. Human D6 is expressed in a range of tissues including hemopoietic cells although it appears not to be ubiquitously expressed in hemopoietic cells. Human D6, unlike some other beta-chemokine receptors, appears not to be able to function as an entry co-factor for human immunodeficiency virus type 1 (HIV-)1 on CD4-expressing cells.     

The "interleukin 1 receptor antagonist" is a partial agonist of prostaglandin synthesis by human decidual cells

In many systems the interleukin-1 receptor antagonist opposes the effects of interleukin-1 beta. We considered that it might block interleukin-1 beta-stimulated prostaglandin production from human decidual cells. Very high levels of interleukin-1 receptor antagonist (> 1000 pg/ml) had limited inhibitory effects on IL-1 beta-stimulated PGE2 synthesis, and lower levels of antagonist (< 1000 pg/ml) increased the effects of IL-1 beta. Low concentrations of the antagonist alone (1-100 pg/ml) increased basal PGE2 production, whereas higher levels (10-100 ng/ml) had less effect. It seems, therefore, that in human decidua the "antagonist" is more accurately described as a partial agonist. It has been suggested that the IL-1 receptor antagonist could be used to inhibit decidual prostaglandin synthesis and thereby prevent preterm labor, but this report shows that caution should be exercised before using the receptor antagonist.     

Expression of prostaglandin receptors EP4 and FP in human lens epithelial cells

Physicians have at their disposal a great number of established diagnostic tests, and new ones continue to be developed that are potentially helpful in diagnosing and establishing the prognosis of disease. Many of these tests were either inadequately evaluated or were found, on more careful scrutiny, to be less helpful than first believed. To ensure optimal patient care as well as appropriate use of health care resources, practitioners must be adept in understanding the true efficacy of diagnostic tests that they ask patients to undergo. They must be able to understand the basic language applied in evaluating tests and be able to determine if and when tests are applicable. (J Am Assoc Gynecol Laparosc 6(1):105-112, 1999)     

Negative feedback control of the spermatogenic progression by testicular oestrogen synthesis: insights from the shark testis model

The organisation of the testis of the dogfish shark is technically advantageous for stage-by-stage analysis of spermatogenesis in vivo and in vitro. Prior studies using this model show that total oestrogen receptors (ER) are concentrated in regions where spermatocysts ("follicle-like" germ cell-Sertoli cell units) are in stem cell and spermatogonial stages: respectively, germinal zone (GZ) and premeiotic (PrM) regions. By contrast, key enzymes regulating oestrogen (E) concentrations (aromatase, 17 alpha-hydroxylase) are maximal in meiotic (M) and postmeiotic (PoM) regions, respectively, which are upstream in the intratesticular vascular pathway. To investigate the hypothesis that E is part of a signalling mechanism between stages of development, studies were undertaken to test direct effects of oestradiol-17 beta (E2) on processes in ER-rich regions. As measured by [3H]thymidine (-Tdr) incorporation. DNA synthesis in GZ and PrM regions was inhibited by E2 (0-1000 nM) in a dose-response fashion. The maximal response (30-40%) was significant, reproducible and observed within 72 hr of treatment. Insulin differentially affected DNA synthesis and the response to E2 in GZ in GZ and PrM regions. As measured by [3H]Tdr release after prelabelling spermatocysts of GZ regions, apoptosis progressively decreased with increasing concentrations of E2. At the maximal dose of E2 used, there was no effect on total protein synthesis or secretion in combined GZ/PrM cysts, indicating that effects on DNA synthesis and cell death were authentically physiological, not pharmacological, and consistent with a state of developmental arrest. These results support the hypothesis that E synthesised within the testis is part of a negative feedback regulatory mechanism whereby more mature stages regulate the developmental advance of less mature stages. A growth control mechanism of this type could explain the strict temporal, spatial and quantitative order of succeeding stages characteristic of normal spermatogenesis in all vertebrates. Further study is required to determine whether E signalling in this model is restricted to Sertoli cells or has a germ cell component.     

Actions of interleukin-4 on prostaglandin biosynthesis by human amnion cells

Acute spinal cord injury (SCI) is associated with a marked propensity to thromboembolism and a variety of coagulation abnormalities. However, data on blood coagulation profiles in patients with uncomplicated long-standing SCI are limited. These data were studied here. Eight men with uncomplicated chronic SCI and nine able-bodied normal men were studied. Plasma activities and/or antigen concentrations of high molecular weight kininogen (HMWK) and of factors XII, XI, IX, VIII, VII, X, V, II and XIII as well as von Willebrand factor (vWF), fibrinogen and fibronectin were measured by appropriate functional and or immunological assays. The SCI group exhibited normal values for factors XII, IX, VIII, vWF, VII, X and V as well as HMWK, vWF and fibronectin concentration. However, they showed slight reductions in plasma factor XI activity, factor XIII antigen concentration and modest increases in fibrinogen and factor II concentrations. No correlation was found between the parameters studied and either the duration or the level of injury. In conclusion, in contrast to acute SCI, the coagulation profile in uncomplicated chronic SCI is noted to be largely normal with only a few minor alterations of questionable clinical significance.     

Neurons induce the activation of microglial cells in vitro

Although microglial cells are well known to become activated in the pathological brain, mechanisms underlying the microglial activation are not fully understood. In the present study, with an aim to elucidate whether neurons are involved in the microglial activation, we compared the morphology and the superoxide anion (O2-)-generating activity of rat microglial cells in pure culture with those of cells cocultured with rat primary cortical neurons. Microglial cells in pure culture in serum-free Eagle's minimum essential medium on poly-L-lysine-coated coverslips displayed ramified morphology and suppressed activity of O2- generation. In contrast, microglial cells in neuron-microglia coculture under the same conditions as those for the pure culture displayed ameboid shape and upregulated activity of O2- generation. Electron microscopic observation revealed that microglial cells in coculture were more abundant in Golgi apparatus and secretory granules than those in pure culture and that some of microglial cells in the vicinity of neurites exhibited membrane specialization reminiscent of a junctional apparatus with high electron density between a microglial soma and a neurite. Microglial cells in coculture tended to tie neurites in bundles by extending processes. Medium conditioned by neurons significantly enhanced O2- generation by microglia, but microglial cells in contact with or in close apposition to cocultured neurons were much more intensely activated than those remote from the neurons. Furthermore, the membrane fraction of cortical neurons activated microglial cells, and this effect was abolished by treating the neuronal membrane with trypsin or neuraminidase. In conclusion, neuronal-microglial contact may be necessary to mediate microglial activation. The present findings suggest that the contact of microglia with damaged neurons in the brain is a plausible cause to activate microglia in the neuropathological processes.     

Formation of glutathione conjugates of prostaglandin A1 in human red blood cells

When prostaglandin A1 was incubated with a Tris/saline suspension of washed human red blood cells, a substantial amount was converted to polar metabolites. These were purified by solvent extraction, XAD-2 column, and cellulose thin layer chromatography and characterized by chromatography, amino acid analysis, and mass spectrometry. The polar metabolites were a mixture of two glutathione conjugates of prostaglandin A1. The first (approximately 40%) was identical with the product of the nonenzymic reaction of glutathione with prostaglandin A1. The second (approximately 60%) was formed from the first by reduction of the 9-keto group of the prostaglandin moiety. The latter compound was also prepared synthetically by treating the glutathione conjugate of prostaglandin A1 with sodium borohydride.     

Endothelial prostaglandin and nitric oxide synthesis in atherogenesis and thrombosis

Human arterial thrombotic disorders are triggered by many agents, with participation of platelets and monocytes, blood coagulation factors and vascular cells. Platelet hyperaggregability appears to be an important risk factor for these disorders. Vascular endothelium possesses several properties to defend against vascular insults and thrombotic atherosclerotic lesions. Two molecules, prostacyclin (PGI2) and nitric oxide (NO), are of particular importance. The rate-limiting step of PGI2 synthesis is cyclooxygenase (COX). Constitutive and upregulated constitutive COX (COX-1) expression and inducible COX (COX-2) expression are important in PGI2 production required for the physiologic and pathologic defense of blood vessels and blood fluidity. NO synthesis is catalyzed by endothelial nitric oxide synthase (eNOS), which can be stimulated by lipid mediators. Virus or non-virus mediated transfer of COX-1 and eNOS are accompanied by augmented PGI2 and NO synthesis, respectively. In animal angioplasty models, it has been shown that transfer of these two genes has a dramatic antithrombotic and anti-intimal hyperplastic effect. Transfers of these two enzymes may have potential therapeutic uses.     

The origin and differentiation of microglial cells during development

Some authors claim that microglia originate from the neuroepithelium, although most now believe that microglial cells are of mesodermal origin, and probably belong to the monocyte/macrophage cell line. These cells must enter the developing central nervous system (CNS) from the blood stream, the ventricular space or the meninges. Afterward microglial cells are distributed more or less homogeneously through the entire nervous parenchyma. Stereotyped patterns of migration have been recognized during development, in which long-distance tangential migration precedes radial migration of individual cells. Microglial cells moving through the nervous parenchyma are ameboid microglia, which apparently differentiate into ramified microglia after reaching their definitive location. This is supported by the presence of cells showing intermediate features between those of ameboid and ramified microglia. The factors that control the invasion of the nervous parenchyma, migration within the developing CNS and differentiation of microglial cells are not well known. These phenomena apparently depend on environmental factors such as soluble or cell-surface bound molecules and components of the extracellular matrix. Microglial cells within the developing CNS are involved in clearing cell debris and withdrawing misdirected or transitory axons, and presumably support cell survival and neurite growth.     

Comparative study of astrocytes in human and rabbit retinae

Immunohistochemical location of Glial Fibrillary Acidic Protein (GFAP) was used to compare the morphology of astrocytes in vascularized and partially vascularized retinae (human and rabbit, respectively). Astrocytes in human and rabbit retinae were found in the same regions as the blood vessels. These cells in partially vascularized retinae differed from those in vascularized retina in several respects. Firstly, there were six morphological types in rabbit retina and only two in human retina. Secondly, in rabbit retinae there were astrocytes only related to blood vessels called "perivascular astrocytes" which were absent in human retinae. Thirdly, the astrocytes were located in the nerve fiber layer and ganglion cell layer in both types of retinae, but in human retinae these cells could also be seen in the internal nuclear layer. These observations demonstrate that there are many differences between astrocytes in human and rabbit retina, suggesting that rabbit retina could be used with caution as an experimental model in comparative studies with human retina.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.