Showing papers in "Inflammation Research in 1998"

Anti-inflammatory drugs and their mechanism of action

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs) produce their therapeutic activities through inhibition of cyclooxygenase (COX), the enzyme that makes prostaglandins (PGs). They share, to a greater or lesser degree, the same side effects, including gastric and renal toxicity. Recent research has shown that there are at least two COX isoenzymes. COX-1 is constitutive and makes PGs that protect the stomach and kidney from damage. COX-2 is induced by inflammatory stimuli, such as cytokines, and produces PGs that contribute to the pain and swelling of inflammation. Thus, selective COX-2 inhibitors should be anti-inflammatory without side effects on the kidney and stomach. Of course, selective COX-2 inhibitors may have other side effects and perhaps other therapeutic potential. For instance, COX-2 (and not COX-1) is thought to be involved in ovulation and in labor. In addition, the well-known protective action of aspirin on colon cancer may be through an action on COX-2, which is expressed in this disease. Moreover, NSAIDs delay the progress of Alzheimer's disease. Thus, selective COX-2 inhibitors may demonstrate new important therapeutic benefits as anticancer agents, as well as in preventing premature labor and perhaps even retarding the progression of Alzheimer's disease.

Antioxidant and anti-inflammatory properties of C-phycocyanin from blue-green algae

Abstract: Objective: Phycocyanin is a pigment found in blue-green algae which contains open chain tetrapyrroles with possible scavenging properties. We have studied its antioxidant properties.¶Materials and methods: Phycocyanin was evaluated as a putative antioxidant in vitro by using: a) luminol-enhanced chemiluminescence (LCL) generated by three different radical species (O−2, OH•, RO•) and by zymosan activated human polymorphonuclear leukocytes (PMNLs), b) deoxyribose assay and c) inhibition of liver microsomal lipid peroxidation induced by Fe+2-ascorbic acid. The antioxidant activity was also assayed in vivo in glucose oxidase (GO)-induced inflammation in mouse paw.¶Results: The results indicated that phycocyanin is able to scavenge OH• (IC50 = 0.91 mg/mL) and RO• (IC50 = 76 μg/mL) radicals, with activity equivalent to 0.125 mg/mL of dimethyl sulphoxide (DMSO) and 0.038 μg/mL of trolox, specific scavengers of those radicals respectively. In the deoxyribose assay the second-order rate constant was 3.56 × 1011 M−1 S−1, similar to that obtained for some non-steroidal anti-inflammatory drugs. Phycocyanin also inhibits liver microsomal lipid peroxidation (IC50 = 12 mg/mL), the CL response of PMNLs (p < 0.05) as well as the edema index in GO-induced inflammation in mouse paw (p < 0.05).¶Conclusions: To our knowledge this is the first report of the antioxidant and anti-inflammatory properties of c-phycocyanin.

Cyclooxygenase-2 as a therapeutic target.

Abstract: Cyclooxygenase (COX)-2 is the predominant COX isoform present at sites of inflammation, and produces prostaglandins (PG) that cause swelling and pain. However, in situations where the release of protective PGs by COX-1 has been lost, the induction of COX-2 may compensate and reduce inflammatory responses. This is particularly likely in large blood vessels, where, under physiological conditions, the release of prostacyclin by COX-1, present in the endothelium, is an important component of cardiovascular homeostasis. We, and others, have recently shown that COX-2 can be induced by proinflammatory cytokines in human blood vessels, and also in human airway cells. Moreover, recent data from our group have suggested that in these structures, COX-2 is anti-inflammatory at the level of cellular proliferation, adhesion receptor expression, and cytokine release.

The inflammatory basis of trauma/shock-associated multiple organ failure

Abstract: Multiple alterations in inflammatory and immunologic function have been demonstrated in clinical and experimental situations after trauma and hemorrhage, in particular the activation of various humoral (e.g. complement, coagulation) and cellular systems (neutrophils, endothelial cells, macrophages). As a consequence of this activation process there is synthesis, expression and release of numerous mediators (toxic oxygen species, proteolytic enzymes, adherence molecules, cytokines), which may produce a generalized inflammation and tissue damage in the body. Mediators are responsible for ongoing interactions of different cell types and for amplification effects through their networks and feedback cycles, finally leading to a sustained inflammation and multiple organ damage in the body. In the setting of trauma/shock, many activators including bacterial as well as non-bacterial factors may be present that will induce local and systemic inflammatory responses. Although the potential role of bacteria/endotoxin translocation and its clinical relevance remains controversial, many lines of evidence support the concept that the gut may be the reservoir for systemic sepsis and subsequent MOF in a number of pathophysiologic states.

Increased release of ATP from endothelial cells during acute inflammation.

Abstract: OBJECTIVE AND DESIGN: The effects of lipopolysaccharide (LPS), a potent inflammatory mediator, on the shear stress stimulated release of adenosine triphosphate (ATP) were investigated on endothelial cells from human umbilical vein in primary culture. METHODS: Human umbilical vein endothelial cells (HUVEC) in primary cultures were subjected to shear stress using a cone and plate apparatus. ATP released by the cells was measured by luminometry, using a luciferin-luciferase assay. RESULTS: Under conditions of shear stress alone (25dyn/cm2), ATP accumulates into the culture medium and reaches a maximum after 3 to 5 min of stimulation (121.7+/-13.2 pmol/ml). The shear stress-stimulated release of ATP was significantly increased after a 4 h pre-incubation of endothelial cells with 50 microg/ml (314.4+/-26.7 pmol/ml) and 10microg/ml lipopolysaccharide (207.7+/-22.2 pmol/ml). Dexamethasone, an anti-inflammatory glucocorticoid, inhibited the effects of lipopolysaccharide. CONCLUSIONS: These results show that non-damaged endothelial cells release ATP under experimental inflammatory conditions and support an early role of extracellular ATP in the inflammatory process.

Estrogen-mediated immunosuppression in autoimmune diseases.

Abstract: Gender affects the susceptibility to many autoimmune diseases. Women have an increased risk of developing diseases such as rheumatoid arthritis and multiple sclerosis compared with men. The female preponderance is believed to depend in part on the influence of sex hormones on the immune system. The mechanism of estrogen-induced immune suppression both in human autoimmune diseases and their experimental animal model counterparts is discussed. In addition, the mechanisms of estrogen and anti-estrogens are discussed in relation to their possible use as future therapeutic anti-inflammatory agents.

Further studies on anti-inflammatory activity of phycocyanin in some animal models of inflammation

Abstract: Objective: To examine the effects of C-phyco- cyanin, a pigment found in blue-green algae which acts as an antioxidant in vitro and in vivo, in different animal models of inflammation. Material: Male Sprague Dawley rats and OF1 mice were used. Treatments: Oedema was induced by: a) AA (0.5 mg/ear) or TPA (4 mg/ear) in the mouse ear b) carrageenan injection (0.1 mL of 1% suspension) in the rat paw (6 adrenalectomy) and c) cotton pellet implantation in the rat axilla. Phyco- cyanin (50-300 mg/kg, p.o.) or indomethacin (1 mg/ear or 3-10 mg/kg, p.o.) as control were tested in the four animal models. Methods: Measurement of the increase in the weight (mg) of 6 mm ear punch biopsies from treated ears were made in comparison to control ears, together with myeloperoxidase (MPO) activity as an index of neutrophil infiltration. The increase in the paw thickness (mm) was measured with a dial caliper. Cotton pellet was implanted and seven days afterwards the granuloma was removed and the dry weight was determined. Acute toxicity was studied in mice and rats. Statistics were performed using one-way analysis of variance with the Duncan Multirange test. Results: Phycocyanin reduced significantly (p < 0.05) and in a dose-dependent manner ear oedema induced by AA and TPA in mice as well as carrageenan-induced rat paw oedema (both in intact and adrenalectomized animals). In the TPA test, phycocyanin also reduced MPO content. Phycocyanin also exerted an inhibitory effect in the cotton pellet granuloma test. In the acute toxicity test in rats and mice, even at the highest dose tested (3 000 mg/kg, p.o.), no toxicity was found. Conclusions: Phycocyanin shows anti-inflammatory activity in four experimental models of inflammation. Its anti- oxidative and oxygen free radical scavenging properties may contribute, at least in part, to its anti-inflammatory activity.

Macrophage NRAMP1 and its role in resistance to microbial infections.

Abstract: The identification and characterization of genetic factors influencing natural susceptibility to infectious diseases in humans and in model organisms, such as the laboratory mouse, can provide new insight into the basic mechanisms of host defense against infections. In the mouse, resistance or susceptibility to infection with intracellular pathogens such as Salmonella, Mycobacterium and Leishmnania is controlled by the Natural resistance associated macrophage protein (Nramp1) gene on chromosome 1, which influences the rate of intracellular replication of these parasites in macrophages. Nramp1 codes for an integral membrane protein, which is expressed exclusively in macrophage/monocytes and polymorphonuclear leukocytes. The protein is localized to the endosomal/lysosomal compartment of the macrophage and is rapidly recruited to the membrane of the particle-containing phagosome upon phagocytosis. Nramp defines a novel family of functionally related membrane proteins including Nramp2, which was recently shown to be the major transferrin-independent uptake system of the intestine in mammals. This observation supports the hypothesis that the phagocyte-specific Nramp1 protein may regulate the intraphagosomal replication of antigenically unrelated bacteria by controlling divalent cation concentrations at that site. Recent genetic studies have found that allelic variants at the human NRAMP1 locus are associated with susceptibility to leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis) and possibly with the onset of rheumatoid arthritis.

IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis

Abstract: In the context of joint biology, insulin-like growth factor-1 (IGF-1) is the most likely candidate to affect the anabolism of cartilage matrix molecules. Mechanisms for controlling the effects of IGF-1 include alterations in the level of this growth factor, its receptor and/or the IGF-1 affinity or availability to its receptor. Disturbance of any one of the above elements may induce a disregulation of the mechanisms involved in the local control of joint tissue integrity. This review focuses on recent studies of the IGF system, and the potential relevance of these results to in vivo effects in osteoarthritic (OA) tissues. It has been shown that, although the IGF-1's expression and synthesis are increased in OA cartilage, chondrocytes are hyporesponsive to IGF-1 stimulation. This phenomenon appears to be related, at least in part, to an increased level of IGF-binding proteins (IGFBP). The IGFBP have a high affinity for IGF-1, and appear to be important biomodulators for IGF action. Though to date seven IGFBP have been cloned and sequenced, disregulation in IGFBP-3 and -4 appears instrumental to arthritic disorders. Proteolytic activity directed against IGFBP has been found in both cartilage and bone; this activity appears to belong to serine- and/or metallo-proteases families. It has been suggested that a thickening of the subchondral bone participates in OA pathophysiology, and that IGF-1 production by bone and/or subchondral bone cells may contribute to these changes. An abnormal regulation of subchondral bone formation via an increase in the local activation of IGF-1 in bone cells, possibly via abnormal IGFBP synthesis due to aberrant PA/plasmin regulation of the IGF-I/IGFBP system, is believed to be a plausible hypothesis.

Antinociceptive effect of meloxicam, in neurogenic and inflammatory nociceptive models in mice

Abstract: Objective: The antinociceptive effect of the new cyclooxygenase (COX)-2 inhibitor, meloxicam, given intraperitoneally (i.p.), was assessed in different models of chemical and thermal nociception in mice.¶Material and Methods: The analgesic effect was analysed using acetic acid-induced abdominal constriction (AA), formalin and capsaicin-induced licking, and hot-plate tests.¶Results: The treatment of animals with meloxicam or diclofenac (2.8–94.3 μmol/kg, i.p. 30 min prior) caused graded and significant inhibition of AA, with mean ID50 values of 7.4 and 38.0 μmol/kg, respectively. At the ID50 level, meloxicam was about 5-fold more potent than diclofenac. In the formalin test, meloxicam or diclofenac (0.8–94.3 μmol/kg, i.p. 30 min prior) also caused significant inhibition of both the early (neurogenic pain) and the late (inflammatory pain) phases of formalin-induced licking. The calculated mean ID50 values for the early phase were: 7.1 and > 94.3 μmol/kg, while for the late phase they were 2.8 and 34.5 μmol/kg, respectively, for meloxicam and diclofenac. Meloxicam also caused significant inhibition of formalin-induced oedema ( p < 0.05). Meloxicam and diclofenac (0.8–314.4 μmol/kg, i.p. 30 min prior) produced significant and dose-related inhibition of neurogenic nociception caused by topical injection of capsaicin, with mean ID50 values of 4.0 and 47.4 μmol/kg, respectively, but were ineffective in the hot-plate model of nociception.¶Conclusions: The present study shows that meloxicam dose-dependently exhibited systemic antinociceptive action when assessed against neurogenic and inflammatory pain caused by acetic acid, formalin and capsaicin models. In contrast, when assessed in the hot-plate test, meloxicam had no significant effect. Thus, meloxicam and other COX-2 inhibitors might be useful for therapeutic intervention in the management of neurogenic and inflammatory pain.

Involvement and dual effects of nitric oxide in septic shock.

Abstract: Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.

Molecular mechanism of vitamin D receptor action.

Abstract: The vitamin D system is unique in that distinct calcium homeostatic functions and cell growth regulatory activities are mediated through a single ligand, calcitriol, acting through a specific receptor exhibiting ubiquitous tissue expression, the vitamin D receptor (VDR). The VDR is a member of a superfamily of nuclear steroid hormone receptors which regulate gene transcription by interacting with response elements in gene promoters. Structure-function analysis of the VDR protein has defined distinct domains involved in DNA binding, ligand binding, receptor dimerisation and gene transactivation, including a C-terminal activation function domain (AF-2) that is important for cofactor interaction. A model for regulation of gene transcription by the VDR is evolving and proposes VDR interaction with various components of the basal transcriptional machinery, including newly defined coactivators and corepressors, which may act to regulate gene transcription by altering histone acetylation and chromatin structure. This review describes the vitamin D endocrine system and the role of the VDR in regulating this system, including the molecular basis for the diverse actions of synthetic calcitriol analogues in the treatment of autoimmune disease and cancer.

Expression of cyclooxygenase isoforms in the rat spinal cord and their regulation during adjuvant-induced arthritis

Abstract: Objective and Design: Spinal regulation of cyclooxygenase (COX) isoforms was investigated in the animal model of peripheral inflammation induced by injection of complete Freund's-type adjuvant (CFA) in the rat hindpaw.¶Subjects and Treatment: Peripheral inflammation was induced by intraplantar injection of CFA in one hind footpad of male Sprague Dawley rats (n = 3 per time point).¶Methods: Spinal cord was removed after different times (3 h to 22 d). mRNA and protein were isolated and analyzed by comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.¶Results: Under the acute inflammatory stimulus 6 h after CFA application, RT-PCR revealed a twofold increase in COX-2 mRNA that reached baseline again at day 3. This transient increase occurred in the lumbar spinal cord, but changes in COX-2 mRNA expression were also registered in RNA preparations from cervical sections, spinal COX-2 induction thus not being a spatially confirmed phenomenon. Western blot analysis of spinal membrane preparations reflected the transient COX-2 mRNA induction at protein levels. During the chronic phase of arthritis at day 22, COX-2 levels were again raised significantly (1.6 fold) over baseline. Spinal levels of COX-1 were not altered at any time point of the peripheral inflammation.¶Conclusion: These data imply a regulatory role for COX-2 but not COX-1 in the spinal modulation under acute and chronic peripheral inflammation.

Differential effects of inhibition of isoforms of cyclooxygenase (cox-1, cox-2) in chronic inflammation

Abstract: Objective and Design: The anti-inflammatory effects of therapeutic dosing of drugs with greater selectivity for the inhibition of the constitutive (COX-1) or inducible isoform (COX-2) of cyclooxygenase were assessed in a model of chronic inflammation.¶Methods: The murine chronic granulomatous tissue air pouch model involves the subcutaneous injection of air into the dorsum of mice followed 24 h later by the intrapouch injection of an inflammatory stimulus (0.5 ml of Freund's complete adjuvant containing 0.1% croton oil). Aspirin, more selective in vitro for the inhibition of COX-1 (10,200 mg/kg) and nimesulide, a selective in vitro inhibitor of COX-2 (0.5, 5 mg/kg) were dosed p.o. daily from 3 days after injection of the inflammatory stimulus. Granuloma dry weight, vascularity and COX activity (measured as PGE2) were assessed at various time points throughout the inflammatory lesion to resolution at day 28. A second COX-2 inhibitor, NS 398 (0.1, 1, 10 mg/kg), was dosed p.o. daily from 3 days after the injection of the inflammatory stimulus and its effects on granuloma dry weight, vascularity and COX activity were measured at 7 days.¶Results: Aspirin (200 mg/kg) significantly inhibited levels of PGE2 throughout the time course and at the lower dose (10 mg/kg) from day 14. Nimesulide (5 mg/kg) however, significantly increased levels of PGE2 at days 5 and 21, but at 0.5 mg/kg was without effect. Aspirin (200 mg/kg) significantly reduced granuloma dry weight at day 14 but had no effect on granuloma vascularity at day 7. In contrast, nimesulide (5 mg/kg) significantly increased granuloma vascularity at day 7 and granuloma dry weight at day 14. NS-398 at all doses had no effect on granuloma dry weight, vascularity or COX activity 7 days after the injection of the inflammatory stimulus.¶Conclusion: In this model of chronic inflammation, aspirin, more selective for the inhibition of COX-1 is more effective than the selective COX-2 inhibitors nimesulide and NS-398 at inhibiting granuloma dry weight, vascularity and COX activity.

Novel pathways for glucocorticoid effects on neutrophils in chronic inflammation

Abstract: Neutrophils have been implicated in mediating much of the tissue damage associated with chronic inflammatory diseases such as rheumatoid arthritis, where they are involved in destruction of both cartilage and bone. Glucocorticoids are powerful anti-inflammatory agents, often used in the treatment of this autoimmune disease. They exert significant inhibitory effects on neutrophil activation and functions, such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis. The mechanisms by which glucocorticoids exert these effects on neutrophils are unclear. Evidence from studies of inflammation in human subjects and animal models suggests that annexin-I, an endogenous, glucocorticoid-induced protein also known as lipocortin-1, has a pivotal role in modulating neutrophil activation, transmigratory, and phagocytic functions. Furthermore, we present evidence for altered neutrophil functions in rheumatoid arthritis that correspond to a significantly reduced capacity of these cells to bind annexin-I. A proposed novel pathway for glucocorticoid actions on neutrophils involving annexin-I could explain the development of chronic neutrophil activation in diseases such as rheumatoid arthritis.

Inhibition of neutrophil-mediated acute inflammation injury by an antibody against interleukin-8 (IL-8)

Abstract: Chemokines are a family of cytokines regulating the migration and functions of leukocytes in a cell-type specific manner. A prototype of C-X-C chemokines, interleukin-8 (IL-8), chemoattracts and activates neutrophils in vitro, and IL-8 concentrations in body fluids are markedly increased in several neutrophil-mediated acute inflammation. Moreover, we previously reported that the administration of a neutralizing antibody to IL-8 prevented neutrophil-mediated tissue injury, as well as neutrophil infiltration, in several animal disease models. These observations implicate IL-8 as a major mediator of neutrophil-mediated tissue injury. Furthermore, we recently showed that an anti-IL-8 antibody effectively prevented two models that are very relevant to clinical situations; endotoxemia-induced acute respiratory distress syndrome (ARDS)-like lung injury and cerebral reperfusion injury. These results raise the possibility that IL-8 is a novel target for therapeutic intervention in neutrophil-mediated acute inflammation.

Differential inhibition of cyclooxygenases-1 and -2 by meloxicam and its 4′-isomer

Abstract: Objective and Design: Two structurally related compounds, meloxicam (Mel) and its structural 4′-isomer (4′-Mel), were compared to examine the role of a slightly different chemical structure on cyclooxygenase (COX) selectivity in in vitro and in vivo experimental models.¶Material or Subjects: In vitro studies were performed using human whole blood obtained from healthy volunteers, in vivo studies were performed in rats.¶Treatment: A concentration-response curve was obtained in the whole blood assay for Mel, 4′-Mel, indomethacin, piroxicam and diclofenac. These were used to calculate the respective IC50 values of either prostaglandin E2 (PGE2) or thromboxane B2 (TxB2). Similarly, a dose-response curve was obtained for Mel, 4′-Mel and piroxicam when measuring in vivo prostaglandin production, anti-inflammatory activity and gastric tolerance to determine the dose resulting in a 50% reduction of the each parameter.¶Methods: COX selectivity was investigated in vitro using a human whole blood assay. PGE2 synthesis in vivo was measured in inflammatory exudate, in the stomach and kidneys of rats. Anti-inflammatory effects were measured in an adjuvant arthritis model and gastric tolerance was tested in an ulcerogenicity model in vivo in rats.¶Results: In the human whole blood assay, the ratio of IC50 values for COX-1 vs. COX-2 inhibition was 13 for Mel and 1.8 for 4′-Mel. In inflammatory exudate in rats, Mel and 4′-Mel inhibited PGE2 synthesis to a similar extent, ID50 values ∼ 0.3 mg/kg. In contrast, Mel was a weaker inhibitor of PG synthesis than 4′-Mel in the rat stomach and in the rat kidney. Paw swelling was reduced by 50% with 0.1 and 0.2 mg/kg for Mel and 4′-Mel, respectively, in the rat adjuvant arthritis model. Gastric tolerance (UD50) was 2.4 mg/kg for Mel and 0.4 mg/kg for 4′-Mel.¶Conclusions: These data demonstrate that the in vitro and in vivo pharmacological profile of meloxicam is structurally dependent and that minor structural changes can lead to significant differences in the selectivity for COX-1 and COX-2 in vitro and to different profiles in vivo suggesting different therapeutic potential.

Experimental models used to investigate the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2 by non-steroidal anti-inflammatory drugs

Abstract: Numerous in vitro assays have been developed for testing and comparing the relative inhibitory activities of non-steroidal anti-inflammatory drugs against cyclooxygenase (COX)-1 and COX-2. Despite variability among these systems, which precludes direct comparison of data, analysis of the ratio of inhibition of COX-1 to COX-2 by non-steroidal anti-inflammatory drugs, suggests inhibitors can be classified based on their COX selectivity. Standard non-steroidal anti-inflammatory drugs can be considered nonselective; compounds such as meloxicam and nimesulide can be classified as COX-2 preferential; and compounds such as SC 58125 and L-754,337 are selective for COX-2. Although in vitro systems are important for characterizing COX-1 and COX-2 inhibitory activity, the clinical relevance of these data should be considered carefully. The level of inhibition of COX-1 and COX-2, in vivo at a given dose in patients, cannot be predicted from in vitro data alone. The pharmacokinetic properties of each compound, including plasma levels, distribution and binding to plasma proteins, have to be taken into account. Human pharmacology studies concentrating on the inhibition of prostanoid synthesis in target tissues are of paramount importance in determining the clinical relevance of COX-2 selectivity.

Analysis of anti-IgE and allergen induced human basophil activation by flow cytometry. Comparison with histamine release

Abstract: Objective and Design: On the basis of flow cytometric methods previously described for the analysis of human basophil activation, we present here a bi-color anti-IgE FITC, anti CD63 PE method and the correlation with histamine release.¶Materials and Subjects: Subjects allergic to grass pollen were selected by their clinical history, skin tests and specific IgE.¶Methods: Basophils gated in the lymphocyte region of the side scatter (SSC)/forward scatter (FSC) pattern were selected by their high IgE epitope density. Percentage of cells expressing CD63 marker, upregulated on activated basophil membrane, was calculated by the cytometer. Histamine released into the supernatants was measured by RIA.¶Results: In these conditions, flow cytometric analysis of blood leukocytes showed that the selected cells had the phenotype CD14−, CD19−, CD45+, IgE++ and CD63− or + which is related to human basophil phenotype, the isotype controls being negative. The use of an anti-CD41 FITC antibody also showed the presence of aggregated platelets on the basophil membrane, CD63 antigen being, however, expressed by basophils themselves and not by platelets. Moreover, no statistical difference was observed between histamine release and flow cytometry after passive sensitization of blood donor leukocytes.¶Conclusion: Flow cytometry, as a popular method often used in the immunology and haematology departments of clinical laboratories may represent a new alternative for allergy diagnosis and basophil pharmacology.

Modulation of peripheral inflammation by locally administered hemorphin-7

Abstract: Objectives: Neurogenic inflammation is mediated via sensory peptides released from the peripheral terminals of sensory nerves and can be modulated by locally released opioid peptides at the site of injury. Endomorphins are recently discovered endogenous opioid peptides with high selectivity and affinity for the μ-opioid receptor. The aim of this study was to examine the ability of endomorphin-1 (EM-1) to modulate the inflammatory response under different injury conditions. ¶Methods: A vacuum-induced blister model in anaesthetised rats (nembutal 60 mg/kg i.p.) was used to examine the effect of EM-1 on the acute inflammatory response induced by; (1) electrical stimulation (ES) of the distal portion of the exposed/cut sciatic nerve at 20 V, 5 Hz, 2 ms for 1 min or; (2) superfusion of substance P (SP) over the blister base. In addition, the effect of EM-1 on the inflammatory response to SP was examined under acute, recurrent (repeated blister induction) and chronic (chronic sciatic nerve lesion) injury conditions. ¶Results: Prior and concomitant perfusion of EM-1 (100 μM) significantly inhibited the vascular response to ES by 58% compared to controls. EM-1 also inhibited the inflammatory response to SP (both vasodilatation and plasma extravasation) in a dose-dependent manner. Significant inhibition was achieved at 100 μM and 1 mM concentrations of EM-1. Naloxone (1 mg/kg i.v.) reversed the inhibitory effect of EM-1 on the inflammatory response to SP. EM-1 (100 μM) was equally potent in inhibiting the inflammatory response to SP under acute (34% inhibition) recurrent (39%) and chronic (42%) injury conditions. ¶Conclusions: The current results demonstrate a greater inhibitory effect of EM-1 on the inflammatory response to electrical nerve stimulation (58% inhibition) compared to SP (34% inhibition) suggesting the involvement of both pre- and post-terminal mechanisms in the inhibitory modulatory actions of EM-1. Evidence is provided for the involvement of opioid receptors in this inhibitory effect. The results also suggest that EM-1 is equipotent in inhibiting the inflammatory response under different injury conditions.

Activation of nuclear factor kappa b in crohn's disease

Abstract: Objectives and Design: The location and degree of activation of nuclear factor kappa (NFκB), a primary transcription factor that plays a regulating role in immune and inflammatory responses, was determined in Crohn's disease using full thickness specimens of bowel collected at surgery.¶Materials and Methods: Resected specimens of inflamed and non-inflamed bowel were collected from thirteen patients with Crohn's disease and non-inflamed bowel from eleven control subjects. Prepared frozen sections were immunostained using a monoclonal antibody to the activated form of the p65 subunit of NFκB and the number of positive staining cells counted using a Lennox graticule.¶Results: The number of cells positive for activated NFκB was significantly increased (p = 0.001) in all layers of inflamed Crohn's disease bowel, compared to non-inflamed bowel from controls. There was also a significant increase (p = 0.009) in the number of positive cells, when compared to non-inflamed bowel from control subjects, in the submucosa of non-inflamed areas of Crohn's disease bowel. Cells positive for activated NFκB were provisionally identified by morphological criteria as mostly macrophages with some lymphocytes. There was no activation in endothelia.¶Conclusion: NFκB is activated within large mononuclear cells in all layers of inflamed areas of the bowel in Crohn's disease and may represent key events in the inflammatory process. Increased activation in the submucosa of non-inflamed Crohn's disease bowel provides further evidence of early immunological activation in macroscopically and microscopically uninvolved areas and an underlying abnormal immune system in Crohn's disease.

Antagonism of the IL-6 cytokine subfamily - a potential strategy for more effective therapy in rheumatoid arthritis

Abstract: Pro-inflammatory cytokines, some of which have the capacity to modulate cartilage and bone metabolism, are important mediators of the frequently sustained and destructive inflammation that characterises rheumatoid arthritis (RA). Tumour necrosis factor alpha (TNFα) and interleukin-1 (IL-1) have been studied extensively in this regard. That these proteins are important is no longer in doubt following the demonstration that the IL-1 receptor antagonist and neutralising antibodies directed against TNFα are clinically effective. Recent studies suggest that interleukin-6 (IL-6) and other members of the IL-6 cytokine subfamily are also potentially important cytokines in the pathogenesis of RA. The recognition of shared molecular subunits in the receptors for these cytokines raises the possibility that components of these receptors or their derivatives, either alone or in combination, may be useful for antagonising members of the IL-6 cytokine subfamily. Effective antagonism could be therapeutically beneficial in respect to attenuating inflammation and protecting critically important chondral and skeletal tissue. In this review the rationale and possible strategies for such antagonism are discussed.

Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells

Abstract: Objective and Design: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide homologous with vasoactive intestinal polypeptide (VIP) which is known to induce histamine release in human skin mast cells. PACAP has not been detected in human skin. The purposes of the study were to investigate the occurrence of PACAP in human skin and to evaluate the histamine releasing activity of the two common pro-PACAP products, PACAP-27 and PACAP-38.¶Material: Fourteen human surgical skin samples were obtained. PACAP and VIP were visualized by immunohistochemistry. A microdialysis technique was used to measure histamine release in intact skin samples following intradermal injections of the peptides.¶Results: PACAP and VIP were localized in dermal nerves in connection with sweat glands. Intradermal injection of 3 or 10 μm PACAP significantly released histamine. Kinetics of histamine release showed peak release 2–4 min after skin challenge. Ten μm of PACAP-27, VIP and somatostatin caused histamine release with similar efficacy, whereas PACAP-38 was less effective. Substance P was twice as efficient as PACAP-27, whereas calcitonin gene-related peptide did not release histamine.¶Conclusions: PACAP is found in human skin and is capable of releasing histamine from skin mast cells.

COX-2 and colon cancer.

Abstract: The role of cyclooxygenase-2 (COX-2) in colorectal tumorigenesis in mice was studied by Oshima et al. to determine the effects of COX-2 gene knockouts and a new COX-2 inhibitor. In the study, heterozygous Apcdelta716 knockout mice, a mouse model of human familial adenomatous polyposis (FAP), were either crossed to COX-2 gene knockout mice, or fed chow containing the COX-2-selective inhibitor. Apcdelta716 litter mates were used as positive controls, which developed 652+/-198 (SD) polyps at 10 weeks. Introduction of a COX-2 gene mutation, or feeding with the COX-2-selective inhibitor to the Apcdelta716 knockout mice, reduced the number and size of intestinal polyps dramatically. The results provide direct genetic evidence that COX-2 plays a key role in tumorigenesis, and indicate that COX-2-selective inhibitors can be a new class of therapeutic agents for colorectal polyposis and cancer.

Cyclooxygenase selectivity and the risk of gastro-intestinal complications of various non-steroidal anti-inflammatory drugs: A clinical consideration

Abstract: Severe gastro-intestinal complications are a major cause of NSAID-induced deaths in cases of rheumatoid arthritis. We measured COX selectivity by using an intact cell assay system, and found that NS-398 is a highly COX-2-selective inhibitor. Meloxicam, etodolac and diclofenac also showed high COX-2 selectivity. Zaltoprofen, loxoprofen-SRS (active metabolite of loxoprofen), 6-MNA (active metabolite of nabumetone) and ibuprofen showed intermediate COX-2 selectivity. The lowest COX-2 selectivities, which means the highest COX-1 selectivities, were observed in indomethacin, aspirin, and oxaprozin. There appears to be a good relationship between our data and some clinical data of severe gastro-intestinal toxicity. The more a given NSAID is selective for COX-2, the safer it is for clinical use. In conclusion, to anticipate the safety of NSAIDs, we find that an intact cell assay system, using human cells for measurement of COX selectivity, may be more useful than using direct enzyme assay systems.

Interleukin-15 and its role in chronic inflammatory diseases.

Abstract: This review focuses on the biological effects of the newly discovered cytokine, interleukin 15 (IL-15), in chronic inflammatory disorders. IL-15 shares biological activities with IL-2, and like IL-2 it is a member of the four-helix bundle cytokine family. IL-15 interacts with a heterotrimeric receptor that consists of the β and γ subunits of the IL-2 receptor (IL-2R) as well as a specific, high-affinity IL-15-binding subunit, IL-15Rα. IL-15 is produced by macrophages and various other cells in response to environmental stimuli and infectious agents, and it is important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monocytes as well as it activates a number of important intracellular signaling molecules, including the Janus kinases and members of the transcription factor family of signal transducers and activators of transcription. These facts suggest that IL-15 may play a pivotal role both in protective immune responses and in the pathogenesis of various chronic immuno-inflammatory disorders. The important new insight into the role of IL-15 in diseases such as rheumatoid arthritis, sarcoidosis, chronic hepatitis C, and ulcerative colitis are reviewed in this paper.

Antiinflammatory potency of dehydrocurdione, a zedoary-derived sesquiterpene.

Abstract: Objective and Design: Dehydrocurdione, a sesquiterpene isolated from zedoary, was tested for in vivo and in vitro antiinflammatory actions.¶Materials: Analgesic effect was tested in ICR mice by the acetic acid-induced writhing method. Antipyretic effect was studied in Sprague-Dawley rats treated with baker's yeast. Antiinflammatory activities were tested in Wistar rats with carrageenan-induced paw edema and adjuvant-induced chronic arthritis. In vitro analyses included the capabilities to inhibit cyclooxygenase activity, and to scavenge free radicals as determined by electron paramagnetic resonance (EPR).¶Results: Oral administration of dehydrocurdione (40 to 200 mg/kg) mitigated the writhing reflex induced by acetic acid and the fever elicited by baker's yeast. A higher dose (200 mg/kg) of dehydrocurdione was required to inhibit the carrageenan-induced paw edema. Oral administration of dehydrocurdione at 120 mg/kg/day for 12 days significantly reduced chronic adjuvant arthritis. Unlike indomethacin (IC50: 0.1 μM), dehydrocurdione showed minimal cyclooxygenase inhibition. However, dehydrocurdione (100 μM to 5 mM) significantly reduced free radical formation from hydrogen peroxide and ferrous iron determined by EPR spectrometry using 5,5′-dimethyl-l-pyrroline-N-oxide as a spin trap agent.¶Conclusion: In addition to the well-known effect of zedoary as a stomachic, dehydrocurdione, the major component of Curcuma zedoaria Roscoe has antiinflammatory potency related to its antioxidant effect.

Analysis of tissue and subcellular localization of mammalian diamine oxidase by confocal laser scanning fluorescence microscopy

Abstract: oxidase by confocal laser scanning fluorescence microscopy H. G. Schwelberger, A. Hittmair 2 and S. D. Kohlwein Labor für Theoretische Chirurgie, II. Univ.-Klinik fu ̈r Chirurgie, Universita ̈t Innsbruck, Scho ̈pfstr. 41, A-6020 Innsbruck, Austria, Fax +43 512 507 2871, e-mail: hubert.schwelberger@uibk.ac.at Institut für Pathologische Anatomie, Universita ̈t Innsbruck, Müllerstr. 44, A-6020 Innsbruck, Austria SFB Biomembrane Research Center, Institut fu ̈r Biochemie, Technische Universita ̈t Graz, Petersgasse 12, A-8010 Graz, Austria

Development of pollen-induced allergic rhinitis with early and late phase nasal blockage in guinea pigs.

Abstract: Objective and Design: Development of nasal blockage and sneezing during repeated inhalation challenges with Japanese cedar pollens was evaluated in guinea pigs.¶Subjects: Male Hartley guinea pigs.¶Treatment: Guinea pigs were sensitized by intranasal instillation of cedar pollen extracts + Al(OH)3 2 times a day for 7 days. The animal was then forced to inhale the pollens for challenge, which was restrictively trapped in the upper airways, once a week.¶Methods: Change of specific airway resistance (sRaw), sneezing frequency, and titers of anaphylactic antibodies in the serum were measured after each of the 30 challenges.¶Results: At the first challenge, no obvious increase in sRaw was observed. However, the second and third challenges to the animals caused modest biphasic elevations of sRaw, with peaks at the first and the fourth to sixth hour. At the fourth to tenth challenges, marked elevations of sRaw were observed. However, with repetition of the inhalation challenge, the early and the late responses became almost indistinguishable because of partial overlapping as the responses expanded. All guinea pigs sneezed immediately after each pollen inhalation challenge. Apparent increases of both circulating γ1 and IgE antibodies were seen after the seventh challenge.¶Conclusions: These results indicate that the experimental allergic rhinitis established in the present study can be a valuable model for analyzing the pathogenesis of the disease and developing new therapeutic drugs.

Anti-inflammatory activity of myricetin-3-O-beta-D-glucuronide and related compounds.

Abstract: Objective and Design: The anti-inflammatory effect of myricetinglucuronide (MGL) was investigated and structurally-related compounds were compared to examine the structure/activity-relationship in carrageenan-induced rat paw edema.¶Materials and Subjects: In vitro studies were performed using rat basophilic leukemia (RBL-1) cells, human polymorphonuclear leukocytes (PMNL), COX-1 from ram seminal vesicle, COX-2 from sheep placenta and human venous blood. For the in vivo tests male Wistar rats were used, for the ex vivo test perfused rabbit ears.¶Treatment: 1–300 μg/kg MGL or myricetinmethylglucuronate and 0.1–5 mg/kg other related compounds administered p.o. (carrageenan edema). 5, 50 and 150 μg/kg MGL p.o. for 14 days (Freund's adjuvant arthritis), 5 and 50 μg/kg p.o. for 6 days (ulceration).¶Methods: Anti-inflammatory effects were measured in carrageenan edema and in adjuvant arthritis. Incidence of gastric lesions was tested in an ulcerogenicity model in vivo. Influence on COX was determined in the perfused rabbit ear, in PMNL and in a test assay using COX-1 and COX-2. 5-LOX activity was studied using PMNL and RBL-1. The influence on platelet aggregation was evaluated measuring light transmission.¶Results: MGL exerted a marked and dose-dependent anti-inflammatory effect in acute (carrageenan edema, ED5 15 μg/kg, indomethacin ED50 10 mg/kg) and chronic (adjuvant arthritis, inhibition at 150 μg/kg 18.1% left paw, 20.6% right paw, indomethacin 3 mg/kg 18.0% and 19.4%)) models of inflammation. In the perfused rabbit ear 1 μg MGL inhibited the release of PGI2, PGD2 and PGE2 to the same extent as 1 μg indomethacin. The inhibition of COX-1 in the intact cell system was IC50 = 0.5 μM, that of indomethacin 0.0038 μM. In the isolated enzyme preparations of COX-1 and COX-2 the IC50 was 10 μM and 8 μM, that of indomethacin 9.2 mM and 2.4 μM. In the RBL-1 and PMNL test assay the inhibition of 5-LOX was 0.1 μM and 2.2 μM. An orally administered dose of 50 μg/kg/day induced no gastric ulcers in rats treated for 6 days. The investigations on carrageenan edema showed a close relationship between the structure of MGL and the anti-inflammatory effect.¶Conclusions: MGL is a COX-1, COX-2 and 5-LOX inhibitor. In view of the moderate in vitro activity and the very potent in vivo activity an additive mechanism must be involved. Small changes in the molecular structure lead to the loss or reduction of the anti-inflammatory activity.