bluuebunny/crossref_metadata_2025 huggingface dataset:
The MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity
Authors: CALLAGHAN JAMES P. , MARTIN PARTHENA M. , MASS MARC J.
Published: February 2006

ABSTRACT: Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen‐activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth‐associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug‐induced damage to the CNS, we used the dopaminergic neurotoxicant, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme‐linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10‐fold increase in GFAP mRNA by 1 day and a 4‐fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho‐tyr 204 and phospho‐thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady‐state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Immunoblots of these samples showed a number of phosphotyrosine‐labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho‐MAP kinase was elevated over 1.5‐fold, 3–6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury‐induced glial activation.
GABAergic Mechanisms That Shape the Temporal Response to Odors in Moth Olfactory Projection Neuronsa
Authors: CHRISTENSEN THOMAS A. , WALDROP BRIAN R. , HILDEBRAND John G.
Published: February 2006

ABSTRACT: Mitral/tufted cells in the olfactory bulb and projection neurons (PNs) in the insect antennal lobe are involved in complex synaptic interactions with inhibitory interneurons to help shape their odor‐evoked responses. In the moth Manduca sexta, both γ‐aminobutyric acid (GABA) and the GABAA receptor agonist muscimol hyperpolarize and lower input resistance in many PNs, often blocking ongoing spike traffic. The GABA response mimics a short‐latency, chloride‐mediated inhibitory postsynaptic potential (IPSP) evoked in PNs by electrical or odor stimulation of afferent inputs, and the classical GABAA receptor antagonist bicuculline methiodide (BMI) quickly and reversibly blocks this IPSP. Focal injection of BMI (100 μM) immediately preceding a GABA pulse blocks the hyperpolarization evoked by GABA, but a similar injection of BMI preceding an acetylcholine (ACh) pulse fails to block the depolarization evoked by ACh. Moreover, the temporal pattern of odor‐evoked activity in moth PNs is also strongly and reversibly altered by BMI. Importantly, the temporal pattern of the response depends on the temporal characteristics of the stimulus: continuous stimulation evokes more complex, rhythmic responses, whereas a pulsatile stimulus can be copied with a discrete burst of spikes for each pulse. Collectively our results indicate that PNs in the moth antennal lobe possess GABA receptors that share certain characteristics in common with vertebrate GABAA receptors. These receptors are largely responsible for helping PNs integrate information about both the molecular features and the timing of olfactory input to the brain.
Differential Effects of Psychological and Immunological Challenge on the Hypothalamo‐Pituitary‐Adrenal Axis Function in Adjuvant‐induced Arthritisa
Authors: HARBUZ M. S. , WINDLE R. J. , JESSOP D. S. , RENSHAW D. , INGRAM C. D. , LIGHTMAN S. L.
Published: February 2006

ABSTRACT: Inability to mount a suitable glucocorticoid response to a stressor can be life‐threatening. Rats with hind‐paw inflammation, associated with the development of adjuvant‐induced arthritis (AA), are unable to mount a hypothalamo‐pituitary‐adrenal (HPA) axis response to acute stress. In the present study we have compared the effects of acute psychological stress (noise) and acute immunological challenge (lipopolysaccharide [LPS] injection), on the activation of the HPA axis in rats with the chronic inflammatory stress of AA. We conclude that the increase in HPA axis activity in AA is principally due to an increase in corticosterone pulse frequency and not to any alteration in pulse magnitude. The lack of response to acute stress can be accounted for by the increase in pulse frequency and the associated refractory period following each pulse, producing dramatic but specific changes in basal HPA function. These changes may account for the loss of responsiveness to acute stress, but not to acute immunological challenge, because the HPA axis is able to respond to LPS in male rats with AA. However, there appears to be an impaired adrenal responsiveness in female rats with AA that is not inherent, but occurs as a consequence of the development of inflammation.
Sex Hormones and Glucocorticoids: Interactions with the Immune System
Authors: DA SILVA J. A. P.
Published: February 2006

Gender and sex hormones exert powerful effects in the susceptibility and progression of numerous human and experimental autoimmune diseases. This has been attributed to direct immunological effects of sex hormones that impact a clear gender dimorphism on the immune system. Globally, estrogens depress T cell‐dependent immune function and diseases, but enhance antibody production and aggravate B cell‐dependent diseases. Androgens suppress both T‐cell and B‐cell immune responses and virtually always result in the suppression of disease expression. Defects in the hypothalamic‐pituitary‐adrenal (HPA) axis have been proposed to play an important role in the pathogenesis of autoimmune diseases. Glucocorticoid response to stress, including immune challenge, is strongly inhibited by androgens and enhanced by estrogens. Complex three‐way interactions between these systems appear to be involved in gender dimorphism of the immune system. This paper reviews the mechanisms involved in interactions between sex steroids and the HPA axis, addresses the possibility of similar interactions on immunocompetent cells, and explores an integrated perspective of the impact of these interplays on the immune system.
Prolactin and Neuroimmunomodulation: In Vitro and in Vivo Observations
Authors: CHIKANZA I. C.
Published: February 2006

Abstract: Prolactin (PL) is a mammotropic neuropeptide produced by the pituitary and extrapituitary cells existing as several isoforms and belongs to the growth and lactogenic hormone family, which includes growth hormone and placental lactogens. The secretion of pituitary PL is under hypothalamic control. The cytokines IL‐1, IL‐2, and IL‐6 also stimulate production, while IFN‐γ and endothelin‐3 are inhibitory. PL exerts its effects via PL receptors (PLr) which exist as three isoforms. PL regulates reproduction, osmoregulation, and behavior and has potent immunomodulatory effects. PL is structurally related to members of the cytokine/hematopoietic family such as erythropoietin, GM‐CSF, growth hormone, and IL‐2 to IL‐7. The PLr is a member of the cytokine/hematopoietic receptor family. Interaction of PL with PLr activates the Jak kinases which phosphorylate latent STAT proteins resulting in the activation of transcription. PL counteracts the effects of corticosteroids by enhancing Th1 cellular responses.

Perturbations of PL physiology have significant immunologic effects. Hypoprolactinemia impairs immune function while hyperprolactinemia enhances active systemic lupus erythematosus, Reiter's disease, juvenile and adult rheumatoid arthritis (RA), autoimmune thyroiditis, multiple sclerosis, and cardiac allograft rejection. PL gene polymorphisms have now been shown to be linked to RA. Thus, manipulation of PL may have significant clinical utility. Further study of the relationship of the PL/PLr complex, other hormones, and the immune system will provide further insights into the potential therapeutic utility of this complex in immune diseases.
Endocrine End‐Points in Rheumatoid Arthritisa
Authors: CASTAGNETTA LUIGI , CUTOLO MAURIZIO , GRANATA ORAZIA M. , DI FALCO MARCO , BELLAVIA VINCENZO , CARRUBA GIUSEPPE
Published: February 2006

ABSTRACT: Our previous studies are reviewed and at the same time preliminary experimental observation to the topic of endocrine end‐points in autoimmune disease is introduced. To this end, we have used rheumatoid arthritis (RA), including synovial fluids and primary cultures of synovial macrophages, as a model system in order to investigate (a) expression and subcellular localization of high‐affinity sites of steroid binding in immune effector cells; (b) steroid metabolic profiles in both male and female RA patients, as compared to healthy subjects; and (c) activities of key steroid enzymes that govern intratissue accumulation of sex hormones.

In RA tissues and cells, the concurrent evidence for (1) androgen and/or estrogen receptors, (2) high concentrations of biologically active steroids, (3) key enzymes of steroid metabolism, and (4) significant changes of estrogen to androgen ratio, all strongly suggests that individual immune cells, including synovial macrophages, may behave as steroid‐sensitive cells, namely, they may represent a target for sex steroids, supporting the hypothesis of a potential endocrine regulation of the immune response also in RA disease. In this respect, definition of several endocrine end‐points may have important implications for the treatment of rheumatic disease and other immunological disorders.
Possible Pathogenetic Relevance of Interleukin‐1β in “Destructive” Organ‐specific Autoimmune Disease (Hashimoto's Thyroiditis)
Authors: PAOLIERI F. , SALMASO C. , BATTIFORA M. , MONTAGNA P. , PESCE G. , BAGNASCO M. , RICHIUSA P. , GALLUZZO A. , GIORDANO C.
Published: February 2006

ABSTRACT: Thyroid follicular cells (TFC) abundantly express a variety of immunologically relevant surface molecules in Hashimoto's thyroiditis (HT), for example, MHC antigens and adhesion molecules such as ICAM‐1. Cytokines produced by infiltrating type 1 helper and cytotoxic T cells are importantly involved in de novo expression or up‐regulation of such molecules. We recently demonstrated that TFC from HT patients almost invariably bear on their surface two additive functional molecules: Fas/Apo1/CD95, an important participant in apoptosis, and B7.1, a member of a family of “co‐stimulatory” molecules that are crucial for efficient antigen presentation. To date, 12 out of 14 surgical HT thyroid specimens that we studied by immunohistochemistry showed B7.1‐positive TFC, and all showed Fas‐positive TFC, different from Graves' disease (GD) or nonautoimmune specimens. We have investigated the role of a number of cytokines (IL‐1β, TNF‐α, IL‐4, IL‐6, IL‐10, IL‐12, TGF‐β1, IFN‐γ) in regulating B7.1 and Fas expression. The experiments were performed by immunofluorescence flow cytometry on TFC purified from nontoxic goiter specimens which were Fas‐ and B7.1‐negative at baseline, and one B7.1/Fas‐positive HT specimen. IFN‐γ (500 U/mL) and TNF‐α (200 ng/mL) were unable to induce de novo expression of B7.1 or Fas on cultured TFC. At higher doses (2000 U/mL and 800 ng/mL, respectively), they were unable to induce B7.1, but potentiated the spontaneous expression of Fas. Type 2 cytokines (IL‐4 and IL‐10) were unable to induce Fas or B7.1 on TFC at all, or to down‐regulate Fas or B7.1 when expressed. On the other hand, IL‐1β was the only cytokine able to induce Fas expression on Fas‐negative TFC at doses ranging from 100 to 1000 pg/mL. Moreover, at a dose of 400 pg/mL, it was also able to induce B7.1. We demonstrated by immunohistochemistry that IL‐1β is abundantly present on HT thyroids, including follicular structures. It is conceivable that IFN‐γ, or other cytokines secreted by infiltrating T‐lymphocytes, are able to promote IL‐1β secretion by TFC. In conclusion, a crucial role of IL‐1β in “destructive” organ‐specific autoimmunity may be suggested both for the perpetuation of the autoimmune reaction (induction of efficient autoantigen presentation by TFC, via co‐stimulatory molecules) and in induction of tissue damage via “suicide” Fas/FasL‐mediated TFC interaction.
Chronic Administration of UK‐114, a Multifunctional Emerging Protein, Modulates the Th1/Th2 Cytokine Pattern and Experimental Autoimmune Diseases
Authors: PANERAI ALBERTO E. , SACERDOTE PAOLA , BIANCHI MAURO , NICOLETTI FERDINANDO , MANFREDI BARBARA , GASPANI LEDA , BARTORELLI ALBERTO , CECILIANI FABRIZIO , RONCHI SEVERINO
Published: February 2006

ABSTRACT: UK‐114 is a 14‐kDa ubiquitous protein recently sequenced by several groups throughout the world. Its activity ranges from being a tumor antigen, a protein synthesis inhibitor or a specific μ‐calpain activator. UK‐114 shows structural homologies also with proteins of the MHC‐1 binding proteins, and heat shock proteins (HSPs). We investigated the possible effects of UK‐114 on T helper cells cytokine profile and the development and progression of experimental autoimmune diseases. Homogeneous recombinant UK‐114 was used in all experiments. Treatment of Balb/c male mice for two weeks resulted in the increase of IL‐4, and the decrease of TNF‐α, IFN‐γ, and IL‐2 release from stimulated splenocytes, suggesting that UK‐114 modulates the Th1/Th2 cytokine profile toward Th2. Similar to that observed with HSP60/65, a single pretreatment of Lewis rats with UK‐114 significantly blunted the development of adjuvant‐induced arthritis, whereas chronic treatment of 4‐week‐old female NOD mice dose dependently inhibited the development of diabetes.
In Vitro Superfusion Method to Study Nerve‐Immune Cell Interactions in Human Synovial Membrane in Long‐standing Rheumatoid Arthritis or Osteoarthritisa
Authors: MILLER L. E. , WESSINGHAGE D. , MÜLLER‐LADNER U. , SCHÖLMERICH J. , FALK W. , KERNER T. , STRAUB R. H.
Published: February 2006

ABSTRACT: Reports on patients with hemiparalysis indicate the importance of the nervous system for the pathophysiology of rheumatoid arthritis (RA) or osteoarthritis (OA). Norepinephrine (NE) and opioids seem to be more anti‐inflammatory neurotransmitters whereas substance P is proinflammatory. The study aimed to investigate the direct noradrenergic nerve‐immune cell interaction in human synovial membrane. We used a recently developed superfusion technique with electrical stimulation of synovial membrane to elicit local NE from synovial membrane slices. The readout parameter of synovial immune cells was interleukin‐6 (IL‐6). IL‐6 was spontaneously secreted from RA and OA synovial membranes. Electrical field stimulation intensively reduced IL‐6 secretion. In patients with OA or RA, this electrically induced reduction of IL‐6 secretion was not significantly changed by α‐ or β‐adrenergic antagonists. The study demonstrates that local endogenous NE seem to play a minor role, which may be due to a depletion of NE or loss of noradrenergic fibers during chronic RA and OA.
Disease Activity Related Catecholamine Response of Lymphocytes from Patients with Rheumatoid Arthritis
Authors: WAHLE M. , KRAUSE A. , ULRICHS T. , JONAS D. , von WICHERT P. , BURMESTER G. R. , BAERWALD C. G. O.
Published: February 2006

ABSTRACT: In patients with chronic rheumatic diseases, a decreased density of β2‐adrenergic receptors (β2R) on peripheral blood mononuclear cells (PBMC) could be demonstrated negatively correlating with various disease activity parameters. The aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 6 healthy blood donors (HD) were investigated. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with phytohemagglutinin (PHA) and monoclonal anti‐CD3‐antibodies (OKT3), respectively. The results revealed that lymphocytes of patients with RA showed a significantly reduced influence of catecholamines on PBMC function. In RA patients with high disease activity only, a shift to α1‐adrenergic‐mediated catecholamine effects upon PBMC reactivity could be observed. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that is mediated via α‐ and β‐adrenergic receptors due to disease activity. In this respect the altered catecholamine response of PBMC from patients with RA may contribute to the pathogenic process of RA.
Hormonal Pertubations in Fibromyalgia Syndrome
Authors: NEECK GUNTHER , RIEDEL WALTER
Published: February 2006

ABSTRACT: The symptomatology characterizing fibromyalgia (FM) comprises three systems: the musculoskeletal system with widespread muscular pain, neuroendocrine disorders, and psychological distress including depression. Though the most prominent symptom of FM is pain in defined points of the musculoskeletal system, the numerous other somatoform and psychological disorders suppose a common primary disturbance which we consider to originate within higher levels of the central nervous system. Recent studies of the entire endocrine profile of FM patients following a simultaneous challenge of the hypophysis with corticotropin‐releasing hormone (CRH), thyrotropin‐releasing hormone, growth hormone‐releasing hormone, and luteinizing hormone‐releasing hormone support the hypothesis that an elevated activity of CRH neurons determines not only many symptoms of FM but may also cause the deviations observed in the other hormonal axes. Hypothalamic CRH neurons thus may play a key role not only in “resetting” the various endocrine loops but possibly also nociceptive and psychological mechanisms as well.
Can We Use Steroid Hormones to Immunomodulate Rheumatic Diseases? Rheumatoid Arthritis as an Example
Authors: BIJLSMA J.W. J.
Published: February 2006

ABSTRACT: This review discusses the available clinical trials in which steroids have been used in the (adjuvant) treatment of rheumatoid arthritis. Glucocorticosteroids have a positive effect on symptoms and signs of inflammation, but probably not on structural damage. Therefore glucocorticosteroids should be used as part of a long‐term treatment strategy, including disease modifying drugs. Preventive measures regarding osteoporosis and peptic ulcer disease are now possible, and an active screening for potential adverse effects is advisable. The benefit of adjuvant treatment with sex hormones is limited. Estrogens have a slight positive effect in postmenopausal women on disease activity and bone mass. Androgens have a slight positive effect in men and postmenopausal women, especially on general well‐being and bone mass.
Field Research on the Relationship between Stress and Disease Activity in Rheumatoid Arthritisa
Authors: ZAUTRA ALEX J. , HAMILTON NANCY A. , POTTER PHILLIP , SMITH BRUCE
Published: February 2006

ABSTRACT: We review empirical evidence from two field studies for the role of stressful life events in disease flare‐ups among patients with rheumatoid arthritis (RA). RA patients were expected to be more vulnerable psychologically, and physiologically, to stressful events in their everyday lives than other arthritis patients without an autoimmune disease. Findings from two studies are reviewed both for their substantive contribution, but also to provide guidance on measurement issues in future field research of this kind. One study included 41 patients with RA, who were interviewed weekly and called to a clinic for blood work and joint examinations when their levels of interpersonal stress increased significantly. A second study used a similar design but included comparison samples of patients with osteoarthritis (OA) and healthy controls of similar age and the same gender as the RA sample. The findings provided support for the proposition that interpersonal stressors are predictive of increases in disease activity. Not all RA patients, however, showed these relationships, and there was evidence that some participants with OA who were depressed also showed higher disease activity following interpersonal stressors. Significant individual differences in the stress‐disease relationship were uncovered that deserve greater attention in future studies. Important improvements in the assessment of stressful events and refinements in panel study design are also presented as guides to research on the role of stress in disease processes.
Stress and Immune Dysfunction in Gulf War Veteransa
Authors: EVERSON MICHAEL P. , KOTLER SUSAN , BLACKBURN WARREN D.
Published: February 2006

ABSTRACT: The role of stress and immunological abnormalities, as well as the neuroendocrine regulation of these two variables, in illnesses in Gulf War veterans (GWVs) is unknown. Many GWV patients complain of skin and joint problems, that is, disorders that may have a common immunological basis. Post‐traumatic stress disorder (PTSD), an anxiety disorder associated with chronic stress, is diagnosed in approximately 10% of the Alabama GWVs. Chronic stress can lead to a reduced capacity to resist disease. Recent work suggests that a dysregulated balance of cytokines produced by T helper cells of the immune system can play a significant role in stress‐related illnesses. Indeed, a balanced immune response (cell‐mediated and humoral immunity) is an important defense mechanism, and cytokines can regulate this balance. It is therefore plausible that stress‐induced changes in hormones (such as cortisol and catecholamines) and cytokines, both of which have been implicated in altering levels of cellular or humoral immunity, may play a role in GWV illnesses.
Engineering of Selective TIMPs
Authors: NAGASE HIDEAKI , MENG QI , MALINOVSKII VLADIMIR , HUANG WEN , CHUNG LINDA , BODE WOLFRAM , MASKOS KLAUS , BREW KEITH
Published: February 2006

ABSTRACT: Differences in proteinase susceptibility between free TIMP‐1 and the TIMP‐1‐MMP‐3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP‐1 may interact with MMPs. The crystal structure of the complex between TIMP‐1 and the catalytic domain of MMP‐3 has revealed that the α‐amino group of Cys1 bidentately chelates the catalytic zinc of MMP‐3 and the Thr2 side chain occupies the S1′ pocket. Generation of the N‐terminal domain of TIMP‐1 (N‐TIMP‐1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N‐TIMP‐1 for three different MMPs (MMPs‐1, ‐2 and ‐3). The results also demonstrate that the mode of binding of N‐TIMP‐1 residue 2 differs from the binding of the P1′ residue of a peptide substrate.
Role of Matrix Metalloproteinases and Their Inhibition in Cutaneous Wound Healing and Allergic Contact Hypersensitivity
Authors: PILCHER BRIAN K. , WANG MIN , QIN XUE‐JIN , PARKS WILLIAM C. , SENIOR ROBERT M. , WELGUS HOWARD G.
Published: February 2006

ABSTRACT: Normal wounds can heal by secondary intention (epidermal migration to cover a denuded surface) or by approximation of the wound edges (e.g., suturing). In healing by secondary intention, epidermis‐derived MMPs are important. Keratinocyte migration begins within 3‐6 hr post injury, as basal cells detach from underlying basal lamina and encounter a dermal substratum rich in type I collagen. Cell contact with type I collagen in vitro stimulates collagenase‐1 expression, which is mediated by the α2β1 integrin, the major keratinocyte collagen‐binding receptor. Collagenase‐1 activity alone is necessary and sufficient for keratinocyte migration over a collagen subsurface. Stromelysins‐1 and ‐2 are also found in the epidermis of normal acute wounds. Stromelysin‐2 co‐localizes with collagenase‐1 and may facilitate cell migration over non‐collagenous matrices of the dermis. In contrast, stromelysin‐1 is expressed by keratinocytes behind the migrating front and which remain on basal lamina, i.e., the proliferative cell population. Studies with stromelysin‐1‐deficient mice that suggest this MMP plays a role in keratinocyte detachment from underlying basement membrane to initiate cell migration. In chronic ulcers, MMP levels are markedly elevated, in contrast to their precise temporal and spatial expression in acute wounds. Both collagenase‐1 and stromelysin‐1 are found in fibroblasts underlying the nonhealing epithelium, and stromelysin‐1 expression is especially prominent. Two key questions underlie the use of MMP inhibitors and wound healing: (1) will these agents impair normal re‐epithelialization in wounds that heal by secondary intention; and (2) can MMP inhibitors be effective therapy for chronic ulcers? The answer to neither is known. Batimastat and marimastat appear not to interfere with normal wound healing, but only in sutured surgical wounds, a situation in which MMP expression has practically no role. We also show the first example of an in vivo immune response, contact hypersensitivity, which is dependent upon MMP activity. Using gene‐deficient mice, we demonstrate that stromylysin‐1 (MMP‐3) is required for sensitization, whereas gelatinase B (MMP‐9) is required for timely resolution of the reaction to antigenic challenge.
Design and Synthetic Considerations of Matrix Metalloproteinase Inhibitors
Authors: SKOTNICKI JERAULD S. , ZASK ARIE , NELSON FRANCES C. , ALBRIGHT J. DONALD , LEVIN JEREMY I.
Published: February 2006

ABSTRACT: Experimental evidence confirms that the matrix metalloproteinases (MMPs) play a fundamental role in a wide variety of pathologic conditions that involve connective tissue destruction including osteoarthritis and rheumatoid arthritis, tumor metastasis and angiogenesis, corneal ulceration, multiple sclerosis, periodontal disease, and atherosclerosis. Modulation of MMP regulation is possible at several biochemical sites, but direct inhibition of enzyme action provides a particularly attractive target for therapeutic intervention. Hypotheses concerning inhibition of specific MMP(s) with respect to disease target and/or side‐effect profile have emerged. Examples are presented of recent advances in medicinal chemistry approaches to the design of matrix metalloproteinase inhibitors (MMPIs), approaches that address structural requirements and that influence potency, selectivity, and bioavailability. Two important approaches to the design, synthesis, and biological evaluation of MMPIs are highlighted: (1) the invention of alternatives to hydroxamic acid zinc chelators and (2) the construction of nonpeptide scaffolds. One current example in each of these two approaches from our own work is described.
Aggrecanase: A Target for the Design of Inhibitors of Cartilage Degradation
Authors: ARNER ELIZABETH C. , PRATTA MICHAEL A. , DECICCO CARL P. , XUE CHU‐BIAO , NEWTON ROBERT C. , TRZASKOS JAMES M. , MAGOLDA RONALD L. , TORTERELLA MICKY D.
Published: February 2006

ABSTRACT: In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341‐Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373‐Ala374, which is attributed to a novel protease, “aggrecanase.” We have generated aggrecanase in conditioned media from IL‐1‐stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL‐1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.
INHIBITION OF MATRIX METALLOPROTEINASES THERAPEUTIC APPLICATIONS
Authors: BLAVIER LAURENCE , HENRIET PATRICK , IMREN SUZAN , DECLERCK YVES A.
Published: February 2006

ABSTRACT: Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in the homeostasis of the extracellular matrix (ECM) by controlling the activity of matrix metalloproteinases (MMPs). Some TIMPs have a second function as well, unrelated to their antiMMP activity, which affects cell proliferation and survival. The role of these inhibitors in cancer has been the subject of extensive investigations that have examined their biological activity in tumor growth, invasion, metastasis and angiogenesis, as well as their potential use in the diagnosis and treatment of human cancer.
The Regulation of MMPs and TIMPs in Cartilage Turnover
Authors: CAWSTON TIM , BILLINGTON CARON , CLEAVER CATHERINE , ELLIOTT SARAH , HUI WANG , KOSHY PAUL , SHINGLETON BILL , ROWAN ANDREW
Published: February 2006

ABSTRACT: The treatment of cartilage with mediators initiates the breakdown of proteoglycan followed by collagen. This is accompanied by the modulation of different proteinases and inhibitors that include members of the MMP family and TIMPs. We have evidence that a chondrocyte membrane‐associated metalloproteinase cleaves aggrecan. This activity is rapidly induced after stimulation with IL‐1 and OSM and is not inhibited by TIMPs‐l and ‐2 but is inhibited by synthetic MMP inhibitors. This same combination of cytokines also upregulates the collagenases with the subsequent release of collagen fragments, and there is a close correlation between the amount of collagen released and collagenase activity produced. Collagen release can be prevented after treatment with specific inhibitors of MAP kinases, inhibitors of MMP transcription, synthetic metalloproteinase inhibitors, TIMPs and treatment of cartilage with agents that upregulate TIMPs. The results from bovine cartilage culture models show that collagen release occurs when TIMP levels are low, collagenases are upregulated and then subsequently activated.
Scientific Basis of a Matrix Metalloproteinase‐8 Specific Chair‐side Test for Monitoring Periodontal and Peri‐implant Health and Disease
Authors: SORSA TIMO , MÄNTYLÄ PÄIVI , RÖNKÄ HANNE , KALLIO PEKKA , KALLIS GUN‐BRITT , LUNDQVIST CHRISTINA , KINANE DENIS F. , SALO TUULA , GOLUB LORNE M. , TERONEN OLLI , TIKANOJA SARI
Published: February 2006

ABSTRACT: Matrix metalloproteinases (MMPs), especially collagenase‐2 (MMP‐8), are key mediators of irreversible tissue destruction associated with periodontitis and peri‐implantitis. MMP‐8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri‐im‐plant sulcular fluid (PISF) from progressing periodontitis and peri‐implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112‐131 [1994]; Teronen et al. J. Dent. Res. 76: 1529‐1537 [1997]). We have developed monoclonal antibodies to MMP‐8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504‐31509 [1997]) that can be used in a chair‐side dipstick test to monitor the course and treatment of periodontitis and peri‐implantitis. Monoclonal and polyclonal antibody tests for MMP‐8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont.Res. 22: 386‐393 [1988]) in periodontal and peri‐implant health and disease. In future a chair‐side functional and/or immunological MMP‐test can be useful to diagnose and monitor periodontal and peri‐implant disease and health.
Fluorogenic MMP Activity Assay for Plasma Including MMPs Complexed to α2‐Macroglobulin
Authors: BEEKMAN B. , DRIJFHOUT J. W. , RONDAY H. K. , TeKOPPELE J. M.
Published: February 2006

ABSTRACT: Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor α2‐macroglobulin (α2M). Reconstituted MMP‐13/α2M complex was unable to degrade collagen (MW 300,000) in contrast to the low‐molecular‐weight fluorogenic substrate (MW < 1500). Limited access of high‐MW substrates to the active site of MMPs captured by α2M presents the most likely explanation. Consistently, the high‐MW inhibitor TIMP (MW ∼ 28,000) was unable to inhibit MMP/α20M enzyme activity, whereas the low‐MW inhibitor BB94 (MW ∼ 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin‐ation, sensitive MMP‐activity assays in plasma were achieved. Spiking of active MMP‐13 and MMP‐13/α2M complex, and inhibitor studies with TIMP‐1 and BB94, indicated that active MMPs are efficiently captured by α2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.
MMP Inhibition in Abdominal Aortic Aneurysms: Rationale for a Prospective Randomized Clinical Trial
Authors: THOMPSON ROBERT W. , BAXTER B. TIMOTHY
Published: February 2006

ABSTRACT:Abdominal aortic aneurysms (AAAs) represent a chronic degenerative condition associated with a life‐threatening risk of rupture. The evolution of AAAs is thought to involve the progressive degradation of aortic wall elastin and collagen, and increased local production of several matrix metalloproteinases (MMPs) has been implicated in this process. We have previously shown that tetracycline derivatives and other MMP inhibitors suppress aneurysm development in experimental animal models of AAA. Doxycycline also reduces the expression of MMP‐2 and MMP‐9 by human vascular wall cell types and by AAA tissue explantsin vitro. To determine whether this strategy might have a role in the clinical management of small AAA, we examined the effect of doxycycline on aortic wall MMP expressionin vivo. Patients were treated with doxycycline (100 mg p.o. bid) for 7 days prior to elective AAA repair, and aneurysm tissues were obtained at the time of surgery (n= 5). Tissues obtained from an equal number of untreated patients with AAA were used for comparison. By reverse transcription‐polymerase chain reaction and Southern blot analysis, MMP‐2 and MMP‐9 were both found to be abundantly expressed in the aneurysm wall. Preoperative treatment with doxycycline was associated with a 3‐fold reduction in aortic wall expression of MMP‐2 and a 4‐fold reduction in MMP‐9 (p< 0.05compared to untreated AAA). These preliminary results suggest that even short‐term treatment with doxycycline can suppress MMP expression within human AAA tissues. Given its pleiotropic effects as an MMP inhibitor, doxycycline may be particularly effective in suppressing aortic wall connective tissue degradation. While it remains to be determined whether MMP inhibition will have a clinically significant impact on aneurysm expansion, it is expected that this question can be resolved by a properly designed prospective randomized clinical trial.
Effect of Matrix Metalloproteinase Inhibition on Progression of Atherosclerosis and Aneurysm in LDL Receptor‐Deficient Mice Overexpressing MMP‐3, MMP‐12, and MMP‐13 and on Restenosis in Rats after Balloon Injury
Authors: PRESCOTT MARGARET FORNEY , SAWYER WILBUR K. , VON LINDEN‐REED JEAN , JEUNE MICHAEL , CHOU MARY , CAPLAN SHARI L. , JENG ARCO Y.
Published: February 2006

ABSTRACT: The broad‐spectrum MMP inhibitor CGS 27023A was tested to determine its potential as a therapy for atherosclerosis, aneurysm, and restenosis. LDL receptor‐deficient (LDLr −/−) mice fed a high‐fat, cholic acid‐enriched diet for 16 weeks developed advanced aortic atherosclerosis with destruction of elastic lamina and ectasia in the media underlying complex plaques. Lesion formation correlated with a 4.6‐ to 21.7‐fold increase in MMP‐3, ‐12, and ‐13 expression. Treatment with CGS 27023A (p.o., b.i.d. at 50 mg/kg) had no effect on the extent of aortic atherosclerosis (36 ± 4% versus 30 ± 2% in controls), but both aortic medial elastin destruction and ectasia grade were significantly reduced (38% and 36%, respectively, p < 0.05). In the rat ballooned‐carotid‐artery model, CGS 27023A (12.5 mg/kg/day via osmotic minipump) reduced smooth muscle cell migration at 4 days by 83% (p < 0.001). Intimal lesions were reduced by 85% at 7 days (p < 0.001), but intimal smooth muscle proliferation was unaffected, and inhibitory efficacy was lost with time. At 12 days, intimal lesion reduction was less potent (52%, p < 0.01). At 3 and 6 weeks, reductions of 11% and 4%, respectively, were not significant. This demonstrates that it is essential to include late time points when the ballooned‐carotid‐artery model is employed to ensure that lesion size does not “catch up” when a compound solely inhibits smooth muscle cell migration. In summary, MMP inhibitor therapy delayed but did not prevent intimal lesions, thereby demonstrating little promise to prevent restenosis. In contrast, MMP inhibitor therapy may prove useful to retard progression of aneurysm.
Treatment of Osteoporosis with MMP Inhibitors
Authors: WILLIAMS S. , BARNES J. , WAKISAKA A. , OGASA H. , LIANG C. T.
Published: February 2006

ABSTRACT: In the current study, we examined the effects of minocycline on the osteopenia of ovariectomized (OVX) aged rats using the marrow ablation model. This injury induces rapid bone formation followed by bone resorption in the marrow cavity. Old female rats were randomly divided into five groups: sham, OVX, OVX+minocycline (5‐15 mg/day, orally), OVX+17β‐estradiol (25 μg/day, subcutaneously), and OVX+both agents. Rats were OVX, treated with minocycline and/or estrogen, followed by marrow ablation. Bone samples were collected 16 days post‐marrow ablation. X‐ray radiography of bones operated on showed that treatment of OVX old rats with minocycline increased bone mass in diaphyseal region. Diaphyseal bone mineral density (BMD) was measured by DEXA scan. Diaphyseal BMD of OVX rats was increased 17‐25% by treatment with 5‐15 mg of minocycline or 17β‐estradiol. The effects of minocycline and estrogen treatments on the expression of osteoblast and osteoclast markers were also examined. Northern and dot blot analysis of RNA samples showed that treatment of OVX aged rats with minocycline increased the expression of type I collagen (COL I) (49%) and decreased that of interleukin‐6 (IL‐6) (31%). In contrast, estrogen treatment decreased the expression of interleukin‐6 (IL‐6) (39%), carbonic anhydrase II (CA II) (36%), and osteopontin (OP) (37%). Neither minocycline nor 17β‐estradiol had an effect on the expression of osteocalcin (OC) and alkaline phosphatase (AP). To elucidate the mechanism by which minocycline prevented the loss of bone in OVX aged rats, we examined the colony‐formation potential of bone marrow stromal cells in ex vivo cultures. Minocycline stimulated the colony‐forming efficiency of marrow stromal cells derived from old animals. We have therefore concluded that the modest increase in BMD noted in OVX aged rats, in response to minocycline treatment, may be due to a change in bone remodeling that favors bone formation; and the anabolic effect of minocycline is likely due to its effect on the expression of COL I and/or the metabolism of osteoprogenitor cells.
Broad Antitumor and Antiangiogenic Activities of AG3340, a Potent and Selective MMP Inhibitor Undergoing Advanced Oncology Clinical Trials
Authors: SHALINSKY D. R. , BREKKEN J. , ZOU H. , MCDERMOTT C. D. , FORSYTH P. , EDWARDS D. , MARGOSIAK S. , BENDER S. , TRUITT G. , WOOD A. , VARKI N. M. , APPELT K.
Published: February 2006

ABSTRACT: We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP‐2 and ‐9), MT‐MMP‐1 (MMP‐14), and collagenase‐3 (MMP‐13) in many tumor models. AG3340 produced dose‐dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose‐response relationship was explored in three models: COLO‐320DM colon, MV522 lung, and MDA‐MB‐435 breast. Dose‐dependent inhibitions of tumor growth (over 12.5‐200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen‐independent PC‐3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD‐31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16‐F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front‐line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.
MMP Inhibition in Prostate Cancer
Authors: LOKESHWAR BAL L.
Published: February 2006

ABSTRACT: Matrix metalloproteinases (MMPs) play a significant role during the development and metastasis of prostate cancer (CaP). CaP cells secrete high levels of MMPs and low levels of endogenous MMP inhibitors (TIMPs), thus creating an excess balance of MMPs. Established CaP cell lines that express high levels of MMPs frequently metastasize to the bone and the lungs. Drugs such as Taxol and alendronate that reduce cell motility and calcium metabolism reduce bony metastasis of xenografted CaP tumors. We tested several synthetic, nontoxic inhibitors of MMPs that can be administered orally, including doxycycline (DC) and chemically modified tetracyclines (CMTs) on CaP cells in vitro and on a rat CaP model in vivo. Among several anti‐MMP agents tested, CMT‐3 (6‐deoxy, 6‐demethyl,4‐de‐dimethylamino tetracycline) showed highest activity against CaP cell invasion and cell proliferation. Micromolar concentration of CMT‐3 and DC inhibited both the secretion and activity of MMPs by CaP cells. When tested for in vivo efficacy in the Dunning rat CaP model by daily oral gavage, CMT‐3 and DC both reduced the lung metastases (> 50%). CMT‐3, but not DC, inhibited tumor incidence (55 ± 9%) and also reduced the tumor growth rate (27 ± 9.3%). More significantly, the drugs showed minimum systemic toxicity. Ongoing studies indicate that CMT‐3 may inhibit the skeletal metastases of CaP cells and delay the onset of paraplegia due to lumbar metastases. These preclinical studies provide the basis for clinical trials of CMT‐3 for the treatment of metastatic disease.
A Chemically Modified Nonantimicrobial Tetracycline (CMT‐8) Inhibits Gingival Matrix Metalloproteinases, Periodontal Breakdown, and Extra‐Oral Bone Loss in Ovariectomized Rats
Authors: GOLUB LORNE M. , RAMAMURTHY NUNGAVARUM S. , LLAVANERAS ANALEYDA , RYAN MARIA E. , LEE HSI MING , LIU Y. , BAIN STEPHEN , SORSA TIMO
Published: February 2006

ABSTRACT:Estrogen deficiency in the postmenopausal (PM) female is the major cause of osteoporosis and may contribute to increased periodontal disease, including alveolar bone loss, seen in these women. In the current study, an animal model of PM osteoporosis, the OVX adult female rat, was studied to determine: (i) the relationship between periodontal breakdown and skeletal bone loss, and (ii) the effect of CMT‐8 on gingival collagenase and bone loss. OVX rats were daily gavaged with CMT‐8 (1, 2, or 5 mg/rat) for 28 or 90 days; non‐OVX rats and those gavaged with vehicle alone served as controls. Elevated collagenase activity, assessed using [3H‐methyl] collagen as substrate in the presence or absence of APMA, was seen in the gingiva of the OVX rats, and CMT‐8 therapy suppressed this effect. Western blot revealed a similar pattern for MMP‐8 and MMP‐13 concentrations. The changes in the gingival collagenase activity paralleled changes in periodontal bone loss, which, in turn, reflected trabecular bone density changes. Preliminary studies on PM humans administered sub‐antimicrobial tetracycline as a matrix metalloproteinase inhibitor are under way.
Clinical Trials of a Matrix Metalloproteinase Inhibitor in Human Periodontal Disease
Authors: ASHLEY ROBERT A. ,   THE SDD CLINICAL RESEARCH TEAM
Published: February 2006

ABSTRACT: After demonstration by Golub et al. of the ability of the tetracyclines to inhibit elevated collagenolytic activity in animal models of periodontal diseases, a clinical development program was initiated to demonstrate the potential of a subantimicrobial dose of doxycycline (SDD) to augment and maintain the improvements in clinical parameters of adult periodontitis (AP) afforded by conventional nonsurgical periodontal therapy. Clinical trials were carried out in which a number of different SDD dosing regimens and placebo were compared in patients administered a variety of adjunctive nonsurgical therapies. Measured parameters included levels of collagenase activity in gingival crevicular fluid (GCF) and gingival specimens, clinical attachment levels (cALv), probing pocket depths (PD), bleeding on probing (BOP), and subtraction radiographic measurements of alveolar bone height. When used as an adjunct to either scaling and root planing or supragingival scaling and dental prophylaxis, SDD was shown to reduce collagenase levels in both GCF and gingival biopsies, to augment and maintain cALv gains and PD reductions, to reduce BOP, and to prevent loss of alveolar bone height. These clinical responses arose in the absence of any significant effects on the subgingival microflora and without evidence of an increase in the incidence or severity of adverse reactions relative to the control groups. It is proposed that one of the mechanisms of action of SDD is as an inhibitor of pathologically elevated MMPs, including neutrophil and bone cell collagenases (MMP‐8 and MMP‐13), which are associated with the host response in chronic AP, and that SDD provides a novel systemic approach to the management of AP.