Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-18
2002-04-02
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06365352
ABSTRACT:
TECHNICAL FIELD
This invention relates to compositions and methods useful to identify agents that modulate the response of granulocytes to inflammatory and infectious conditions.
BACKGROUND OF THE INVENTION
Granulocytes (i.e., neutrophils, eosinophils and basophils) are involved in the immune response elicited by inflammation and infection.
Inflammation
Inflammation is a localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute or wall off both the injurious agent and the injured tissue. It is characterized by fenestration of the microvasculature, leakages of the elements of blood into the interstitial spaces, and migration of leukocytes into the inflamed tissue. On a macroscopic level, this is usually accompanied by the familiar clinical signs of erythema, edema, tenderness (hyperalgesia), and pain. During this complex response, chemical mediators such as histamine, 5-hydroxytryptamine, various chemotactic factors, bradykinin, leukotrienes, and prostaglandins are released locally. Phagocytic cells migrate into the area, and cellular lysosomal membranes may be ruptured, releasing lytic enzymes. All of these events may contribute to the inflammatory response.
Inflammation is initiated by, among other things, trauma, tissue necrosis, infection or immune reactions. The immediate response is temporary vasoconstriction. Vasoconstriction is followed within seconds by the acute vascular response resulting in increased blood flow (hyperemia) and edema. The acute phase is also characterized by the margination of polymorphonuclear white blood cells (neutrophils) next to endothelial cells, followed by emigration of neutrophils into the adjacent tissue. Margination is recognized by the lining up of neutrophils along the endothelium of vessels. Emigration occurs by passage of the inflammatory cells between endothelial cells.
Neutrophils
Neutrophils are the first wave of cellular attack on invading organisms and are the characteristic cells of acute inflammation. The appearance of neutrophils in areas of inflammation may be caused by chemicals released from bacteria, factors produced nonspecifically from necrotic tissue or antibody reacting with antigen. Neutrophils use an actin-rich cytoskeleton to move in a directed manner along a chemotactic gradient from the bloodstream to an inflammatory site where they ingest particles (e.g,. bacteria) and immune complexes bearing IgG (via FcR) and/or breakdown products of the complement component C3.
Neutrophils belong to a category of white blood cells known as polymorphonuclear white blood cells. The blood cells with single nuclei (mononuclear cells) form the white blood cell population that includes macrophages, T and B cells. White blood cells that contain segmented nuclei are broadly classified as polymorphonuclear. Polymorphonuclear white blood cells (or “granulocytes”) are further subdivided into three major populations on the basis of the staining properties of their cytoplasmic granules in standard hematologic smears or tissue preparations: neutrophils staining pink, eosinophils staining red and basophils staining blue.
Neutrophils (also referred to as polymorphonuclear neutrophils-PMNs) make up 50% to 70% of the white blood cells (WBCs) of the peripheral blood and may be found scattered diffusely in many tissues, although they are most frequently found in areas of acute inflammation or acute necrosis. Like other WBCs, neutrophils are produced from precursor cells in the bone marrow and released into the blood when mature. After entering the circulation, neutrophils are thought to last only 1 or 2 days.
Neutrophils are characterized by numerous cytoplasmic granules that contain highly destructive enzymes that must be kept isolated from the cytoplasm. These granules contain a number of oxygen-independent enzymes as well as oxygen-dependent mechanisms of killing. Upon attraction to sites of inflammation, neutrophils attempt to engulf and digest bacteria coated with antibody and complement. Phagocytosis by neutrophils is also usually accompanied by release of the lysosomal enzymes into the tissue spaces, particularly if the organism is difficult for the neutrophil to digest
At least three cytoplasmic granules are identifiable in neutrophils: specific granules containing lactoferrin, B cytochrome, the complement receptor CR3 and &mgr;
2
-integrin; azurophilic granules containing acid hydrolases and other enzymes; and a third granule containing gelatinase.
In addition to the role neutrophils and other granulocytic cells play in immune response to pathogens, including bacterial infection, neutrophils and other granulocytic cells play an unwanted role in many chronic inflammatory diseases. There are many disease states in which excessive or unregulated granulocytic cell infiltration and activation are implicated in exacerbating and/or causing the disease. For instance, many inflammatory diseases are characterized by massive neutrophil infiltration, such as psoriasis, inflammatory bowel disease, Crohn's disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, rheumatoid arthritis, thrombosis and glomerulonephritis. All of these diseases are associated with increased IL-8 production which may be responsible for the chemotaxis of neutrophils into the inflammatory site.
While the role of neutrophil infiltration and activation in inflammation is well known, the biosynthetic responses of neutrophils to pathogens, chemotactic agents, proinflammatory molecules, etc. are not as well understood. Neutrophils were once thought to be in a state of terminal differentiation, thereby lacking biosynthetic ability. This view is consistent with the relative scarcity in mature circulating neutrophils of ribosomes and endoplasmic reticulum and with the ability of neutrophils to ingest particles when RNA and/or protein synthesis has been inhibited. More recently it has been demonstrated that neutrophils perform more active roles in their response to environmental stimuli.
It has thus recently been established that neutrophils synthesize de novo important macromolecules including, but not limited to interleukin (IL) 1, I1-6, I1-8, tumor necrosis factor (TNF&agr;), granulocyte and macrophage colony-stimulating factors, interferon &agr; (IFN&agr;), intercellular adhesion molecule (ICAM-1) and membrane and cystoskeletal molecules, such as major histocompatibility class I antigens and actin (Beaulieu et al (1992)
J. Biolog. Chem
. 267(1):426-432; Arnold et al. (1993)
Infect. Immun
. 61(6):2545-2552; and Elsner et al. (1995)
Immunobiol
193:456-464). No study, however, has taken a systematic approach to assess the transcriptional response during neutrophil activation via contact with a pathogen or from neutrophils isolated from a subject with a sterile inflammatory disease.
Eosinophils and Basophils
Eosinophils are another granulocytic or polymorphonuclear white blood cell that are involved in the inflammatory response. Eosinophils are found predominately in two types of inflammation: allergy and parasite infections.
The role of eosinophils in the host response to parasites is thought to be mediated through the components of the eosinophilic granules. Eosinophils are cytotoxic to schistosome larvae through an antibody-dependent cell-mediated mechanism. Eosinophil cationic proteins are highly toxic for schistosomes and may be responsible for binding of eosinophils to parasitic worms as well as fragmentation of the parasite.
The role of eosinophils in acute inflammation is not fully understood. On one hand, there is evidence that enzymes in eosinophils may serve to limit the extent of inflammation by neutralizing mediators of anaphylaxis, such as LTC4, histamine and platelet-activating factor. On the other hand, there is increasing evidence that cationic proteins in eosinophilic granules are mediators of acute inflammation. Eosinophil activation is associated with acute tissue injury and cause an intense vasoconstriction in lung microvasculature, followed by increased pulmonary vascular
Goguen Jon
Newburger Peter
Prashar Yatindra
Weissman Sherman M.
Yerramilli Subrahmanyam V.
Loeb Bronwen M.
Morgan & Lewis & Bockius, LLP
Schwartzman Robert A.
Yale University
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