WO2006039819A1 - Marqueurs de la lesion pulmonaire - Google Patents

Marqueurs de la lesion pulmonaire Download PDF

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Publication number
WO2006039819A1
WO2006039819A1 PCT/CA2005/001603 CA2005001603W WO2006039819A1 WO 2006039819 A1 WO2006039819 A1 WO 2006039819A1 CA 2005001603 W CA2005001603 W CA 2005001603W WO 2006039819 A1 WO2006039819 A1 WO 2006039819A1
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WIPO (PCT)
Prior art keywords
lung injury
markers
sample
subject
polynucleotides
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PCT/CA2005/001603
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English (en)
Inventor
Arthur Slutsky
Gregory Downey
Yumiko Imai
Stefan Uhlig
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St. Michael's Hospital
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Publication of WO2006039819A1 publication Critical patent/WO2006039819A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/125—Adult respiratory distress syndrome

Definitions

  • the invention relates to novel markers of lung injury, methods for assessing the status of lung tissue using the markers, and methods for the diagnosis and therapy of lung injuries.
  • ARDS The Acute Respiratory Distress Syndrome
  • ARDS is the most severe form of acute lung injury, affects approximately 1,000,000 people worldwide/year, and has a mortality rate of at least 30% (1).
  • Predisposing factors for ARDS are diverse and include sepsis, pneumonia, aspiration, trauma, and SARS .
  • Mechanical ventilation is life-saving for ARDS patients, but injury due to mechanical ventilation, so-called ventilator-induced lung injury (VILI), can also worsen pre-existing lung injury, and may even contribute to the development of multiple organ failure, possibly through the release of pro-inflammatory mediators from the lung (2,3).
  • VILI ventilator-induced lung injury
  • ARDS lungs are heterogeneously damaged, mechanical ventilation with normal or even low tidal volumes can lead to VILI secondary to alveolar overdistension and/or cycles of recruitment/derecruitment.
  • Overdistension develops because the inspired air preferably distributes to the areas with a higher compliance, that is in the non-atelectatic regions.
  • Recruitment/derecruitment denotes the situation whereby alveolar units open during inspiration and collapse again during expiration (8). This circle of repeated opening and collapse results in high shear stress (9) which can further injure the lungs.
  • Applicants have developed a method for identifying markers associated with lung injury. Using the method they analyzed injured lung tissue from a rodent model of acute lung injury, and identified novel markers for lung injury, in particular markers associated with overdistension and/or derecruitment.
  • the invention relates to a method of characterizing a sample of lung tissue by detecting or quantitating in the sample one or more polynucleotides extracted from the sample that are characteristic of a lung injury the method comprising assaying for differential expression of polynucleotides in the sample. Differential expression of the polynucleotides can be determined by microarray, hybridization or by amplification of the extracted polynucleotides.
  • the invention also relates to a method of characterizing a sample of lung tissue by detecting or quantitating in the sample one or more polypeptides extracted from the sample that are characteristic of a lung injury the method comprising assaying for differential expression of polypeptides in the sample. Differential expression of polypeptides can be assayed by mass spectroscopy of polypeptides extracted from the sample.
  • the invention relates to novel markers for lung injury, and in particular markers associated with overdistension and/or derecruitment, and compositions comprising same.
  • the invention also relates to methods for assessing the status of a lung tissue, and methods for the diagnosis and therapy of a lung injury.
  • Markers associated with lung injury identified in accordance with a method of the invention have application in the determination of the status of lung injury, and in particular in the detection of lung injury.
  • the markers can be used for diagnosis, monitoring (i.e. monitoring progression or therapeutic treatment), prognosis, treatment, or classification of a lung injury, in particular ARDS or VILI, or as markers before or after therapy (e.g. mechanical ventilation).
  • markers identified in accordance with a method of the invention including but not limited to native-sequence polypeptides, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of the markers, including modified forms of the polypeptides and derivatives are referred to herein as "Lung Injury Marker(s)" or "LI Markers”.
  • Lung Injury Markers Polynucleotides encoding Lung Injury Markers are referred to herein as "Lung Injury
  • Polynucleotide Marker(s) "polynucleotides encoding Lung Injury Marker(s)” or "LI
  • the LI Markers and LI Polynucleotides are sometimes collectively referred to herein as "marker(s)”. Table 1 sets out particular markers of the invention.
  • lung tissue can be assessed or characterized, for example by detecting the presence in the sample of (a) a LI Marker or fragment thereof; (b) a metabolite which is produced directly or indirectly by a LI Marker;
  • a transcribed polynucleotide or fragment thereof having at least a portion with which a LI Polynucleotide is substantially identical; and/or (c) a transcribed polynucleotide or fragment thereof, wherein the polynucleotide hybridizes with a LI Polynucleotide.
  • Markers or LI Polynucleotides, lung injury, in particular markers associated with overdistension, derecruitment, and/or VILI in a patient comprising: (a). obtaining a sample from a subject;
  • a method for detecting LI Markers or LI Polynucleotides, lung injury, in particular markers associated with overdistension, derecruitment, and/or VILI in a patient comprising:
  • detect includes assaying, imaging or otherwise establishing the presence or absence of the target markers or polynucleotides encoding the markers, subunits thereof, or combinations of reagent bound targets, and the like, or assaying for, imaging, ascertaining, establishing, or otherwise determining one or more factual characteristics of a lung injury or similar conditions.
  • the term encompasses diagnostic, prognostic, and monitoring applications for the LI Markers and LI Polynucleotides.
  • the invention also provides a method of assessing whether a patient is afflicted with or has a pre-disposition for lung injury, in particular ARDS or VILI, the method comprising comparing:
  • lower levels of LI Markers or LI Polynucleotides in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with lung injury.
  • a method for screening a subject for lung injury comprising (a) obtaining a biological sample from a subject; (b) detecting the amount of LI Markers or LI Polynucleotides associated with the injury in said sample; and (c) comparing said amount of LI Markers or LI Polynucleotides detected to a predetermined standard, where detection of a level of LI Markers or LI Polynucleotides that differs significantly from the standard indicates lung injury.
  • a significant difference between the levels of LI Marker or LI Polynucleotides levels in a patient and the normal levels is an indication that the patient is afflicted with or has a predisposition to lung injury.
  • the amount of LI Marker(s) or LI Polynucleotide(s) detected is greater than that of a standard and is indicative of lung injury.
  • the amount of LI Marker(s) or LI Polynucleotide(s) detected is lower than that of a standard and is indicative of lung injury.
  • the invention provides a method for determining acute respiratory distress syndrome (ARDS) development potential in a patient at risk for the development of ARDS comprising the steps of determining the concentration of one or more markers in
  • Table 1 in a sample (e.g. serum, plasma, aspirate or lavage from the lung) from the patient, comparing the concentration of the markers to a cut-off concentration and determining
  • ARDS development potential from the comparison, wherein concentrations of markers above the cut-off concentration are predictive of (e.g., correlate with) ARDS development in the patient.
  • the invention provides a method for determining or monitoring ventilator-induced lung injury (VILI) in a patient comprising the steps of determining the concentration of hemoglobin beta chain or beta-globin, glutathione S-transferase pi- 1 , and/or ferritin light chain 1 in a sample from the patient and comparing the concentration of the markers to a cut-off concentration and determining or monitoring VILI from the comparison, wherein concentrations of markers above the cut-off concentration are predictive of (e.g., correlate with) VILI in the patient.
  • VILI ventilator-induced lung injury
  • the methods are non-invasive (e.g., without lung biopsy) for detecting lung injury, which in turn allows for diagnosis of a variety of conditions or diseases associated with such lung injury.
  • the invention provides a method for monitoring the progression of lung injury in a patient the method comprising:
  • step (a) detecting LI Markers or LI Polynucleotides in a sample from the patient at a first time point; (b) repeating step (a) at a subsequent point in time; and
  • the invention also provides a method for assessing the potential efficacy of a test agent for preventing, inhibiting, or reducing lung injury, and a method of selecting an agent for inhibiting lung injury.
  • the invention also contemplates a method of assessing the potential of a test compound to contribute to a lung injury comprising: (a) maintaining separate aliquots of injured lung cells in the presence and absence of the test compound; and
  • the invention further relates to a method of assessing the efficacy of a therapy for preventing, inhibiting, or reducing lung injury in a patient.
  • a method of the invention comprises comparing: (a) levels of LI Markers or LI Polynucleotides in a sample from the patient obtained from the patient prior to providing at least a portion of a therapy to the patient; and (b) levels of LI Markers or LI Polynucleotides in a second sample obtained from the patient following therapy.
  • a significant difference between the levels of LI Markers or LI Polynucleotides in the second sample relative to the first sample is an indication that the therapy is efficacious for inhibiting lung injury.
  • the method is used to assess the efficacy of a therapy for inhibiting lung injury, where lower levels of LI Markers or LI polynucleotides relative to the first sample, is an indication that the therapy is efficacious for inhibiting the disease.
  • the "therapy” may be any therapy for treating lung injury, in particular, including but not limited to therapeutics, immunotherapy, surgery, and procedures and interventions such as mechanical ventilation.
  • a method of the invention can be used to evaluate a patient before, during, and after therapy.
  • Certain methods of the invention employ one or more polynucleotides capable of hybridizing to one or more LI Polynucleotides.
  • methods for monitoring a lung injury comprising detecting LI Polynucleotide markers associated with lung injury.
  • the present invention relates to a method for diagnosing and monitoring a lung injury in a sample from a subject comprising isolating polynucleotides, preferably mRNA, from the sample; and detecting LI Polynucleotides in the sample.
  • the presence of different levels of LI Polynucleotides in the sample compared to a Standard or control may be indicative of lung injury, stage of lung injury, and/or a positive prognosis.
  • LI Polynucleotide positive tissues are a negative diagnostic indicator. Positive tissue can be indicative of lung injury, advanced lung injury, or a poor prognosis.
  • LI Polynucleotide negative tissues are a negative diagnostic indicator. Negative tissues can be indicative of lung injury, advanced lung injury, or poor prognosis .
  • the invention provides methods for determining the presence or absence of a lung injury in a subject comprising detecting in the sample levels of polynucleotides that hybridize to one or more LI Polynucleotides, comparing the levels with a predetermined standard or cut-off value, and therefrom determining the presence or absence of lung injury in the subject.
  • the invention provides methods for determining the presence or absence of lung injury in a subject comprising (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to one or more LI Polynucleotides; and (b) detecting in the sample a level of polynucleotides that hybridize to the LI Polynucleotides relative to a predetermined cut-off value, and therefrom determining the presence or absence of lung injury in the subject.
  • the amount of polynucleotides that are mRNA are detected via polymerase chain reaction using, for example, oligonucleotide primers that hybridize to one or more LI Polynucleotides, or complements of such polynucleotides.
  • the amount of mRNA is detected using a hybridization technique, employing oligonucleotide probes that hybridize to one or more LI Polynucleotides, or complements thereof.
  • the method may be carried out by combining isolated mRNA with reagents to convert to cDNA according to standard methods; treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a container along with an appropriate mixture of nucleic acid primers; reacting the contents of the container to produce amplification products; and analyzing the amplification products to detect the presence of one or more LI Polynucleotides in the sample.
  • amplification reaction reagents such as cDNA PCR reaction reagents
  • the analyzing step may be accomplished using Northern Blot analysis to detect the presence of Ll Polynucleotides.
  • the analysis step may be further accomplished by quantitatively detecting the presence of LI Polynucleotides in the amplification product, and comparing the quantity of marker detected against a panel of expected values for the known presence or absence of the markers in normal tissue derived using similar primers.
  • the invention provides a method wherein mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more LI Polynucleotides to produce amplification products; (d) analyzing the amplification products to detect an amount of mRNA encoding the LI Markers; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal tissue derived using similar nucleic acid primers.
  • Certain methods of the invention employ binding agents (e.g. antibodies) that specifically recognize LI Markers.
  • the invention provides methods for determining the presence or absence of lung injury, in a patient, comprising the steps of (a) contacting a biological sample obtained from a patient with one or more binding agent that specifically binds to one or more LI Markers associated with lung injury; and (b) detecting in the sample an amount of marker that binds to the binding agent, relative to a predetermined standard or cut-off value, and therefrom determining the presence or absence of lung injury in the patient.
  • the invention in another embodiment, relates to a method for diagnosing and monitoring lung injury in a subject by quantitating one or more LI Markers associated with lung injury in a biological sample from the subject comprising (a) reacting the biological sample with one or more binding agent specific for the LI Markers (e.g. an antibody) that are directly or indirectly labelled with a detectable substance; and (b) detecting the detectable substance.
  • one or more binding agent specific for the LI Markers e.g. an antibody
  • the invention provides a method for using an antibody to detect expression of one or more LI Marker in a sample, the method comprising: (a) combining antibodies specific for one or more LI Marker with a sample under conditions which allow the formation of antibody:marker complexes; and (b) detecting complex formation, wherein complex formation indicates expression of the marker in the sample. Expression may be compared with standards and is diagnostic of a lung injury.
  • Embodiments of the methods of the invention involve (a) reacting a biological sample from a subject with antibodies specific for one or more LI Markers which are directly or indirectly labelled with an enzyme; (b) adding a substrate for the enzyme wherein the substrate is selected so that the substrate, or a reaction product of the enzyme and substrate forms fluorescent complexes; (c) quantitating one or more LI Markers in the sample by measuring fluorescence of the fluorescent complexes; and (d) comparing the quantitated levels to levels obtained for other samples from the subject patient, or control subjects.
  • the quantitated levels are compared to levels quantitated for control subjects (e.g. normal) without a lung injury wherein an increase in LI Marker levels compared with the control subjects is indicative of lung injury.
  • quantitated levels are compared to levels quantitated for control subjects (e.g. normal) without a lung injury wherein a decrease in LI Marker levels compared with the control subjects is indicative of lung injury.
  • a particular embodiment of the invention comprises the following steps
  • LI Markers which are directly or indirectly labeled with a detectable substance, and second antibodies specific for one or more LI Markers which are immobilized; (b) detecting the detectable substance thereby quantitating LI Markers in the biological sample; and (c) comparing the quantitated LI Markers with levels for a predetermined standard.
  • the standard may correspond to levels quantitated for samples from control subjects without lung injury (normal), with a different stage of lung injury, or from other samples of the subject.
  • increased levels of LI Markers as compared to the standard may be indicative of lung injury.
  • lower levels of LI Markers as compared to the standard may be indicative of lung injury.
  • the invention also contemplates a method comprising administering to cells or tissues imaging agents that carry labels for imaging and bind to LI Markers and optionally other markers of lung injury, and then imaging the cells or tissues.
  • the invention provides an in vivo method comprising administering to a subject an agent that has been constructed to target one or more LI Markers.
  • the invention contemplates an in vivo method comprising administering to a mammal one or more agent that carries a label for imaging and binds to one or more LI Marker, and then imaging the mammal.
  • an in vivo method for imaging a lung injury comprising:
  • the agent is an antibody which recognizes a LI Marker. In another embodiment of the invention the agent is a chemical entity which recognizes a LI Marker.
  • An agent carries a label to image an LI Marker and optionally other markers.
  • labels useful for imaging are radiolabels, fluorescent labels (e.g. fluorescein and rhodamine), nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase.
  • fluorescent labels e.g. fluorescein and rhodamine
  • nuclear magnetic resonance active labels e.g. fluorescein and rhodamine
  • PET positron emission tomography
  • chemiluminescers such as luciferin
  • enzymatic markers such as peroxidase or phosphatase.
  • Short-range radiation emitters such as isotopes detectable by short-range detector probes can also be employed.
  • the invention also contemplates the localization or imaging methods described herein using multiple markers for lung injury.
  • kits for carrying out the methods of the invention are for carrying out the methods of the invention.
  • the kit is for assessing whether a patient is afflicted with a lung injury and it comprises reagents for assessing one or more LI Markers or LI Polynucleotides.
  • the invention also provides a diagnostic composition comprising an LI Marker or a
  • a composition is also provided comprising a probe that specifically hybridizes to LI Polynucleotides, or a fragment thereof, or an antibody specific for LI Markers or a fragment thereof.
  • a composition is provided comprising one or more LI Polynucleotide specific primer pairs capable of amplifying the polynucleotides using polymerase chain reaction methodologies.
  • the probes, primers or antibodies can be labeled with a detectable substance.
  • the invention relates to therapeutic applications for lung injury employing LI Markers and LI polynucleotides, and/or binding agents for the markers.
  • the invention relates to compositions comprising LI Markers or parts thereof associated with a lung injury, or binding agents (e.g antibodies) specific for LI Markers associated with a lung injury, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the invention provides a method of treating or preventing lung injury in a subject afflicted with or at risk of developing a lung injury comprising administering to the subject an effective amount of an antagonist of a LI Marker or LI Polynucleotide, in particular an antagonist of NPY or IP-IO.
  • an antagonist or antagonizing is used in its broadest sense.
  • Antagonism can include any mechanism or treatment that results in inhibition, inactivation, blocking or reduction or alteration of the presence of a LI Marker or LI Polynucleotide.
  • Examples of antagonists are antibodies specific for LI Markers, binding agents for LI Markers, and inhibitors of LI Polynucleotides (e.g. antisense).
  • a method for treating or preventing a lung injury in a subject comprising administering to a subject in need thereof, LI Markers or parts thereof, antibodies specific for LI Markers, or a composition of the invention.
  • the invention provides a method of treating a subject afflicted with or at risk of developing a lung injury comprising inhibiting expression of one or more LI Marker or LI Polynucleotide.
  • the invention provides antibodies specific for LI Markers associated with a lung injury that can be used to inhibit LI Marker or LI Polynucleotide expression.
  • the invention contemplates a method of using antagonists of LI Markers or LI Polynucleotides or parts thereof in the preparation or manufacture of a medicament for the prevention or treatment of a lung injury.
  • the invention contemplates a method of using LI Markers or parts thereof, antibodies specific for LI Markers, or inhibitor of LI Polynucleotides (e.g. antisense) in the preparation or manufacture of a medicament for the prevention or treatment of a lung injury.
  • the invention also provides a method for stimulating or enhancing in a subject production of antibodies directed against one or more LI Marker.
  • the method comprises administering to the subject one or more LI Marker, peptides derived therefrom, or chemically produced (synthetic) peptides, or any combination of these molecules of the invention in a dose effective for stimulating or enhancing production of the antibodies.
  • the invention contemplates the methods, compositions, and kits described herein using additional markers associated with lung inj ury .
  • the methods described herein may be modified by including reagents to detect the additional markers, or polynucleotides for the markers.
  • the invention contemplates the methods described herein using multiple markers for lung injury. Therefore, the invention contemplates a method for anaylzing a biological sample for the presence of LI Markers and LI Polynucleotides, and other markers that are specific indicators of lung injury.
  • the methods described herein may be modified by including reagents to detect the additional markers, or nucleic acids for the additional markers.
  • the methods, compositions and kits use one or more of the markers listed in Table 1. In another embodiment, they use a panel of markers selected from the markers listed in Table 1, in particular a panel comprising two or more of the markers in Table 1.
  • the marker is one or more of IP-lO/CXCL-10, galanin, ' Fra-1, NGFI-B, SOCS-3, Robo-1, adrenomedullin, neuropeptide Y, hemoglobin beta chain, beta-globin, glutathione S-transferase-pi-1, ferritin light chain 1 , or oxidative-low density lipoprotein [LDL] , preferably IP- 10 and neuropeptide Y.
  • the LI marker is NPY and/or IP- 10 or related peptides or coding polynucleotides.
  • the invention provides a method for diagnosing lung injury or ARDS (Acute Respiratory Distress Syndrome) by monitoring or measuring for NPY and/or IP-IO levels.
  • ARDS acute Respiratory Distress Syndrome
  • the methods of the invention as described above can be used in such diagnostic methods.
  • said diagnosis is made by comparing NPY and/or IP- 10 levels with known normal levels of said markers or by comparing to a control or baseline levels of said markers, in one embodiment the lung injury is ventilaoty induced lung injury.
  • NPY levels can be used to diagnose or detect injury due to overdistension.
  • IP-10 levels can be used to diagnose or detect injury due to both overdistension and recruitment/de-recruitment.
  • inhibitors of IP-10 or NPY levels can be used in the treatment and/or prevention of lung injury, ventilator induced lung injury and/or ARDS.
  • IP-IO inhibitor such an IP-IO antibody, can be used to treat lung injury, ventilator-induced lung injury, or ARDS.
  • NPY and IP-IO or coding polynucleotides can be used to design or screen for potential inhibitors of NPY and IP-10 and drugs that can be used in the treatment or prevention of lung injury, ventilator- induced lung injury or ARDS.
  • NPY and IP- 10 levels can be assessed directly or indirectly or through monitoriy polynucleotide expression levels (e.g., niRNA levels) or other means known in the art.
  • genes in blue or red circles are (gene symbols in brackets): a, neuropeptide Y (Npy); b, galanin (Gal); c, CD14 (Cdl4); d, MIP-3 ⁇ (Scya20); e, plasminogen-activator inhibitor-1 (Pail); f, urokinase- type plasminogen activator receptor (Plaur); g, adrenomedullin (adm); h, IP-10 (Cxcl 10); i, MlP-l ⁇ (Scya4); j, oxidised low density lipoprotein (Olrl); k, guanylate binding protein 2 (Gbp2); m, tissue plasminogen activator (Plat); n, E-selectin (SeIe); o, aminolevulinate synthase 2 (Alas2); p, c-fos (c-fos); q, immediate early gene transcription
  • FIG. 4 IP-10 antagonism attenuates VILI by overdistension and derecruitment.
  • (a) IP-10 concentration in lung tissue. **, P ⁇ 0.01 (two-sided t-test) vs. [b l].
  • Methods are provided for detecting the presence of a lung injury in a sample, the absence of lung injury, the stage of a lung injury, and other characteristics of a lung injury that are relevant to prevention, diagnosis, characterization, and therapy of a lung injury in a patient. Methods are also provided for assessing the efficacy of one or more test agents for preventing, inhibiting, or reducing a lung injury, assessing the efficacy of a therapy for a lung injury, monitoring the progression of a lung injury, selecting an agent or therapy for a lung injury, treating a patient afflicted with a lung injury, preventing, inhibiting, or reducing a lung injury in a patient, and assessing the potential of a test compound to cause lung injury. Glossary
  • lung injury refers to any disorder, disease, condition, syndrome or combination of manifestations or symptoms recognized or diagnosed as a disorder of the lung .
  • the term includes acute and chronic lung injury.
  • ARDS acute respiratory distress syndrome
  • ARDS is an inflammatory disorder characterized by the accumulation of neutrophils in the lung and the development of non-cardiogenic pulmonary edema
  • Acute lung injury may result from many causes including but not limited to bacterial sepsis, hemorrhagic shock, toxic inhalation, and bleomycin and other drug-induced lung injury.
  • the term also includes fibrosis in epithelial organs, such as lung, liver, kidney, bladder, and esophagus.
  • the term includes injury due to mechanical ventilation [ventilator-induced lung injury (VILI)] which can worsen pre-existing lung injury, and contribute to multiple organ failure.
  • VILI mechanical ventilation
  • the term includes a variety of conditions or diseases associated with lung injury.
  • sample biological sample
  • biological sample means a material known or suspected of expressing or containing one or more LI Polynucleotides and/or one or more LI Markers.
  • a test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample.
  • the sample can be derived from any biological source, such as tissues, extracts, or cell cultures, including cells, cell lysates, and physiological fluids, such as, for example, whole blood, plasma, serum, saliva, ocular lens fluid, cerebral spinal fluid, sputum, aspirate or lavage from the lung, bronchoalveolar lavage, pulmonary aspirate, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, lavage fluid, and the like.
  • tissue, extracts, or cell cultures including cells, cell lysates, and physiological fluids, such as, for example, whole blood, plasma, serum, saliva, ocular lens fluid, cerebral spinal fluid, sputum, aspirate or lavage from the lung, bronchoalveolar lavage, pulmonary aspirate, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, lavage fluid, and the like.
  • the sample can be obtained from animals, preferably mammals, most preferably humans.
  • the sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, extraction, concentration, inactivation of interfering components, the addition of reagents, and the like.
  • the sample is a mammalian tissue sample. In a particular embodiment, the tissue is lung tissue.
  • subject refers to a warm-blooded animal such as a mammal. In particular, the terms refer to a human.
  • a subject, individual or patient may be afflicted with or suspected of having or being pre-disposed to lung injury or a condition as described herein.
  • the present invention may be particularly useful for determining ARDS development potential in at-risk patients suffering from particular ARDS predisposing conditions, including sepsis, severe pancreatitis, recent hypertransfusion, recent aspiration, severe abdominal trauma, severe chest trauma, and multiple fractures.
  • LI Marker or "Lung Injury Markers” includes a marker associated with lung injury.
  • the term includes native-sequence polypeptides isoforms, chimeric polypeptides, complexes, all homologs, fragments, precursors, and modified forms and derivatives of the markers.
  • the marker may be associated with a stage or phase of lung pathology.
  • the term includes a marker associated with lung injury identified using a method of the invention, in particular a marker listed in Table 1, neuropeptide Y, IFN- ⁇ - inducible protein -10, hemoglobin beta chain, beta-globin, glutathione S-transferase pi-1, and ferritin light chain 1.
  • the lung injury marker is neuropeptide Y (NPY).
  • neuropeptide Y includes human neuropeptide Y (NPY), in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, precursors, complexes, and modified forms and derivatives of human neuropeptide Y (NPY).
  • the amino acid sequence for native human neuropeptide Y (NPY) includes the sequences of protein ID NP_000896 (NCBI) shown as SEQ ID NO. 1.
  • the lung injury marker is IFN- ⁇ -inducible protein- 10 (IP-10, CXCLlO).
  • IFN- ⁇ -inducible protein -10 IP-10
  • IP-10 human IFN- ⁇ - inducible protein- 10
  • the amino acid sequence for native human CXCLlO includes the sequences of protein ID NP_001556 (NCBI) shown as SEQ ID NO.3.
  • the lung injury marker is hemoglobin beta-chain.
  • the term includes human hemoglobin beta chain, in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, precursors, complexes, and modified fo ⁇ ns and derivatives of human hemoglobin beta-chain.
  • the amino acid sequence for native hemoglobin beta-chain includes the sequences of the Protein ID Accession No. P02023 shown as SEQ ID NO. 7.
  • the lung injury marker is glutathione S-transferase pi-1 (GSTPl).
  • Glutathione S-transferase pi-1 includes human GSTPl, in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, precursors, complexes, and modified forms and derivatives of human GSTP 1 and rat Gstp 1 and Gstp2, and homologs of rat Gstpl and Gstp2.
  • the amino acid sequence for native human Gstpl includes the sequences of protein ID NP_000843 (NCBI) shown as SEQ ID NO. 9.
  • the lung injury marker is ferritin light chain 1 (e.g. human FTL and rat FtI 1 ) .
  • ferritin light chain 1 includes ferritin light chain 1 , in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, precursors, complexes, and modified forms and derivatives of human FTL and rat FtIl, and homologes of rat FtIl.
  • the amino acid sequence for native human FTL includes the sequences of protein ID NP_000137 (NCBI) shown as SEQ ID NO. 11.
  • a “native-sequence polypeptide” comprises a polypeptide having the same amino acid sequence of a polypeptide derived from nature. Such native-sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term specifically encompasses naturally occurring truncated or secreted forms of a polypeptide, polypeptide variants including naturally occurring variant forms (e.g. alternatively spliced forms or splice variants), and naturally occurring allelic variants.
  • polypeptide variant means a polypeptide having at least about 70-80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95% amino acid sequence identity with a native-sequence polypeptide.
  • Particular polypeptide variants have at least 70-80%, 85%, 90%, 95% amino acid sequence identity to the sequences identified in Table 1.
  • Such variants include, for instance, polypeptides wherein one or more amino acid residues are added to, or deleted from, the N- or C-terminus of the full-length or mature sequences of the polypeptide, including variants from other species, but excludes a native-sequence polypeptide.
  • allelic variant may also be created by introducing substitutions, additions, or deletions into a polynucleotide encoding a native polypeptide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations may be introduced by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. In an embodiment, conservative substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain. Amino acids with similar side chains are known in the art and include amino acids with basic side chains (e.g. Lys, Arg, His), acidic side chains (e.g.
  • Mutations can also be introduced randomly along part or all of the native sequence, for example, by saturation mutagenesis. Following mutagenesis the variant polypeptide can be recombinantly expressed and the activity of the polypeptide may be determined.
  • Polypeptide variants include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a native polypeptide which include fewer amino acids than the full length polypeptides.
  • a portion of a polypeptide can be a polypeptide which is for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids in length.
  • Portions in which regions of a polypeptide are deleted can be prepared by recombinant techniques and can be evaluated for one or more functional activities such as the ability to form antibodies specific for a polypeptide.
  • a naturally occurring allelic variant may contain conservative amino acid substitutions from the native polypeptide sequence or it may contain a substitution of an amino acid from a corresponding position in a polypeptide homolog, for example, a murine or rat polypeptide.
  • the invention also includes polypeptides that are substantially identical to the sequences of a LI Marker, in particular a lung injury marker, more particularly a marker listed in Table 1 (e.g. at least about 45%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity), and in particular polypeptides that retain the immunogenic activity of the corresponding native-sequence polypeptide.
  • a LI Marker in particular a lung injury marker, more particularly a marker listed in Table 1 (e.g. at least about 45%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity)
  • polypeptides that retain the immunogenic activity of the corresponding native-sequence polypeptide.
  • Percent identity of two amino acid sequences, or of two nucleic acid sequences is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues in a polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various conventional ways, for instance, using publicly available computer software including the GCG program package (Devereux J. et al., Nucleic Acids Research 12(1): 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S.F. et al.
  • BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J. MoI. Biol. 215: 403-410, 1990). Skilled artisans can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Methods to determine identity and similarity are codified in publicly available computer programs.
  • LI Markers include chimeric or fusion proteins.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably biologically active) of a LI Marker operably linked to a heterologous polypeptide (i.e., a polypeptide other than a LI Marker).
  • a heterologous polypeptide i.e., a polypeptide other than a LI Marker.
  • the term "operably linked” is intended to indicate that a LI Marker and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of a LI Marker.
  • a useful fusion protein is a GST fusion protein in which a LI Marker is fused to the C-terminus of GST sequences.
  • Another example of a fusion protein is an immunoglobulin fusion protein in which all or part of a LI
  • Marker is fused to sequences derived from a member of the immunoglobulin protein family.
  • Chimeric and fusion proteins can be produced by standard recombinant DNA techniques.
  • a modified form of a polypeptide referenced herein includes modified forms of the polypeptides and derivatives of the polypeptides, including but not limited to glycosylated, phosphorylated, acetylated, methylated or lapidated forms of the polypeptides.
  • modified forms of the polypeptides and derivatives of the polypeptides including but not limited to glycosylated, phosphorylated, acetylated, methylated or lapidated forms of the polypeptides.
  • an N-terminal methionine may be cleaved from a polypeptide, and a new N-terminal residue may or may not be acetylated.
  • LI Markers may be prepared by recombinant or synthetic methods, or isolated from a variety of sources, or by any combination of these and similar techniques.
  • Lung Injury Polynucleotides refers to polynucleotides associated with lung injury and/or encoding LI Markers including native-sequence polypeptides, polypeptide variants including a portion of a polypeptide, an isoform, precursor, complex, a chimeric polypeptide, or modified forms and derivatives of the polypeptides.
  • a polynucleotide encoding a native polypeptide employed in the present invention the LI Polynucleotides listed in Table 1.
  • a polynucleotide of the invention encodes neuropeptide
  • NPY polynucleotide sequence
  • a polynucleotide of the invention encodes IFN- ⁇ - inducible protein -10 (IP-IO), more particularly a polynucleotide sequence comprises the sequence for human CXCLlO which includes the sequences of LocusID 3627 (Org Hs) shown as SEQ ID NO. 5, or a fragment thereof.
  • the corresponding rat sequence is shown as
  • a polynucleotide of the invention encodes beta-globin, more particularly a polynucleotide sequence of GenBank Accession No. M94919 (Locus ID 3043) [SEQ ID NO 8], or a fragment thereof.
  • a polynucleotide of the invention encodes glutathione S- transferase pi-1 (GSTPl), more particularly a polynucleotide sequence comprises the sequence for human GSTPl which includes the sequences of LocusID 2950 (Org Hs) NM_000852 shown as SEQ ID NO. 10, or a fragment thereof.
  • GSTPl glutathione S- transferase pi-1
  • a polynucleotide of the invention encodes ferritin light chain 1, more particularly a polynucleotide sequence comprises the sequence for human FTL which includes the sequences of LocusID 2512 (Org Hs) NMJ)OO 146 shown as SEQ ID NO. 12, or a fragment thereof.
  • LI Polynucleotides include complementary nucleic acid sequences, and nucleic acids that are substantially identical to these sequences (e.g. at least about 45%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity).
  • LI Polynucleotides also include sequences that differ from a native sequence due to degeneracy in the genetic code.
  • DNA sequence polymorphisms within the nucleotide sequence of a LI Polynucleotide may result in silent mutations that do not affect the amino acid sequence.
  • Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation.
  • DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a polypeptide.
  • Polynucleotides also include nucleic acids that hybridize under stringent conditions, preferably high stringency conditions to a LI Polynucleotide. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-
  • 6.3.6 For example, 6.Ox sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50 0 C may be employed.
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, at about 65 0 C.
  • LI Polynucleotides also include truncated nucleic acids or nucleic acid fragments and variant forms of the nucleic acids that arise by alternative splicing of an mRNA corresponding to a DNA.
  • LI Polynucleotide markers are intended to include DNA and RNA (e.g. mRNA) and can be either double stranded or single stranded.
  • a polynucleotide may, but need not, include additional coding or non-coding sequences, or it may, but need not, be linked to other molecules and/or carrier or support materials.
  • the polynucleotides for use in the methods of the invention may be of any length suitable for a particular method. In certain applications the term refers to antisense polynucleotides (e.g. mRNA or DNA strand in the reverse orientation to sense polynucleotide markers).
  • Statistically different levels may represent levels that are higher or lower than the standard error of the detection assay.
  • the levels may be 1.5, 2, 3, 4, 5, or 6 times higher or lower than the control or standard.
  • Binding agent refers to a substance such as a polypeptide or antibody that specifically binds to one or more LI Marker.
  • a substance “specifically binds" to one or more LI Marker if is reacts at a detectable level with one or more LI Marker, and does not react detectably with peptides containing an unrelated or different sequence. Binding properties may be assessed using an ELISA, which may be readily performed by those skilled in the art (see for example, Newton et al , Develop. Dynamics 197: 1-13, 1993).
  • a binding agent may be a ribosome, with or without a peptide component, an aptamer, an RNA molecule, or a polypeptide.
  • a binding agent may be a polypeptide that comprises one or more LI Marker sequence, a peptide variant thereof, or a non-peptide mimetic of such a sequence.
  • an IFN- ⁇ -inducible protein -10 (IP-IO) sequence may be a peptide portion of an IP-IO that is capable of modulating a function mediated by IP-10.
  • An aptamer includes a DNA or RNA molecule that binds to nucleic acids and proteins.
  • An aptamer that binds to a protein (or binding domain) or a LI Polynucleotide can be produced using conventional techniques, without undue experimentation.
  • Klug et al. MoI. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol.4:427-429 (1994); Lato et al., Chem. Biol.2:291-303 (1995); Conrad et al., MoI. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996)].
  • Antibodies for use in the present invention include but are not limited to monoclonal or polyclonal antibodies, immunologically active fragments (e.g. a Fab or (Fab) 2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain F v molecules (Ladner et al, U.S. Pat. No. 4,946,778), chimeric antibodies, for example, antibodies which contain the binding specificity of murine antibodies, but in which the remaining portions are of human origin, or derivatives, such as enzyme conjugates or labeled derivatives.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras may be prepared using methods known to those skilled in the art.
  • Isolated native or recombinant LI Markers may be utilized to prepare antibodies. See, for example, Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) MoI Cell Biol 62: 109- 120 for the preparation of monoclonal antibodies; Huse etal.
  • the invention relates to a method of characterizing a sample of lung tissue by detecting or quantitating in the sample one or more polynucleotides extracted from the sample that are characteristic of a lung injury the method comprising assaying for differential expression of polynucleotides in the sample. Differential expression of the polynucleotides can be determined by microarray or by amplification of the extracted polynucleotides.
  • the invention relates to a method for identifying markers associated with lung injury, in particular associated with overdistension and/or derecruitment comprising:
  • a profile of nucleic acids is produced by a microarray or by amplification of the nucleic acids (e.g. using PCR).
  • the invention provides a method of characterizing a sample of lung tissue by detecting or quantitating in the sample one or more polynucleotides extracted from the sample that are characteristic of a lung injury the method comprising assaying for differential expression of polynucleotides in the sample by microarray of polynucleotides extracted from the sample.
  • the invention also relates to a method of characterizing a sample of lung tissue by detecting or quantitating in the sample one or more polypeptides extracted from the sample that are characteristic of a lung injury the method comprising assaying for differential expression of polypeptides in the sample. Differential expression of polypeptides can be assayed by mass spectroscopy of polypeptides extracted from the sample.
  • the invention relates to a method for identifying LI Markers associated with lung injury comprising:
  • Polypeptides may be extracted from the samples in a manner known in the art.
  • polypeptides may be extracted by first digesting or disrupting cell membranes by standard methods such as detergents or homogenization in an isotonic sucrose solution, followed by ultra-centrifugation or other standard techniques.
  • the separated polypeptides may be digested into peptides, in particular using proteolytic enzymes such as trypsin, pepsin, subtilisin, and proteinase.
  • proteolytic enzymes such as trypsin, pepsin, subtilisin, and proteinase.
  • polypeptides may be treated with trypsin which cleaves at the sites of lysine and arginine, to provide doubly-charged peptides with a length of from about 5 to 50 amino acids.
  • Such peptides may be particularly appropriate for mass spectrometry analysis, especially electrospray ionization mass spectrometry.
  • Chemical reagents including cyanogens bromide may also be utilized to digest proteins.
  • Mass spectrometers that may be used to analyze the peptides or polypeptides include a Matrix-Assisted Laser Desorptioon/Ioniation Time-of-Flight Mass Spectrometer (“MALDI-TOF”) (e.g. from PerSeptive Biosystems, Framingham, Mass.); an Electrospray Ionization (“ESI”) ion trap spectrometer, (e.g. from Finnigan MAT, San Jose, Calif.), an ESI quadrupole mass spectrometer (e.g.
  • MALDI-TOF Matrix-Assisted Laser Desorptioon/Ioniation Time-of-Flight Mass Spectrometer
  • ESI Electrospray Ionization
  • ESI Electrospray Ionization
  • Finnigan MAT MAT
  • San Jose, Calif. an ESI quadrupole mass spectrometer
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of lung injury or status involving one or more LI Markers and LI Polynucleotides, and the identification of subjects with a predisposition to lung injury.
  • Such methods may, for example, utilize LI Polynucleotides, and fragments thereof, and binding agents (e.g. antibodies) against one or more LI Markers, including peptide fragments.
  • the polynucleotides and antibodies may be used, for example, for (1) the detection of the presence of LI Polynucleotide mutations, or the detection of either an over- or under- expression of LI Polynucleotide mRNA relative to a non-injury state, or the qualitative or quantitative detection of alternatively spliced forms of LI Polynucleotide transcripts which may correlate with certain conditions or susceptibility toward lung injury; and (2) the detection of either an over- or an under-abundance of one or more LI Markers relative to a non-injury state or a different stage or type of injury or the presence of a modified (e.g., less than full length) LI Marker which correlates with an injury state or a progression toward lung injury, or a particular type or stage of lung injury.
  • a modified (e.g., less than full length) LI Marker which correlates with an injury state or a progression toward lung injury, or a particular type or stage of lung injury.
  • the invention contemplates a method for detecting the stage or type of lung injury, comprising producing a profile of levels of one or more LI Marker and/or LI Polynucleotides, and optionally other markers associated with lung injury in a sample (e.g. cells) from a patient, and comparing the profile with a reference to identify a profile for the patient indicative of the stage or type of lung injury.
  • a sample e.g. cells
  • the invention also contemplates a method for detecting a lung injury comprising producing a profile of levels of one or more LI Marker and/or LI polynucleotides, and other markers associated with lung injury in a sample (e.g. cells) from a patient, and comparing the profile with a reference to identify a profile for the patient indicative of lung injury.
  • the methods described herein may be used to evaluate the probability of the presence of lung injury, for example, in a sample (e.g group of cells) freshly removed from a host. Such methods can be used to detect lung injury and help in the diagnosis and prognosis of disease.
  • the methods can be used to detect the presence of lung injury and to monitor lung injury or a therapy.
  • the methods described herein can be adapted for diagnosing and monitoring a lung injury by detecting one or more LI Markers or LI Polynucleotides in biological samples from a subject. These applications require that the amount of LI Markers or LI Polynucleotides quantitated in a sample from a subject being tested be compared to a predetermined standard or cut-off value.
  • the standard may correspond to levels quantitated for another sample or an earlier sample from the subject, or levels quantitated for a control sample.
  • Levels for control samples from healthy subjects, different stages or types of lung injury may be established by prospective and/or retrospective statistical studies. Healthy subjects who have no clinically evident lung injury or abnormalities may be selected for statistical studies.
  • Diagnosis may be made by a finding of statistically different levels of detected LI Markers associated with disease or LI Polynucleotides, compared to a control sample or previous levels quantitated for the same subject.
  • the methods described herein may also use multiple markers for lung injury.
  • the invention contemplates a method for analyzing a biological sample for the presence of one or more LI Markers and LI Polynucleotides, and other markers that are specific indicators of a lung injury.
  • the methods described herein may be modified by including reagents to detect the additional markers.
  • a lung injury or stage or type of same may be detected based on the level of LI Polynucleotides in a sample.
  • Techniques for detecting polynucleotides such as polymerase chain reaction (PCR) and hybridization assays are well known in the art.
  • Probes may be used in hybridization techniques to detect polynucleotide markers.
  • the technique generally involves contacting and incubating polynucleotides (e.g. recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe under conditions favourable for the specific annealing of the probes to complementary sequences in the polynucleotides. After incubation, the non- annealed nucleic acids are removed, and the presence of polynucleotides that have hybridized to the probe if any are detected.
  • polynucleotides e.g. recombinant DNA molecules, cloned genes
  • Nucleotide probes for use in the detection of nucleic acid sequences in samples may be constructed using conventional methods known in the art. Suitable probes may be based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of a LI Polynucleotide, preferably they comprise 15 to 40 nucleotides.
  • a nucleotide probe may be labeled with a detectable substance such as a radioactive label that provides for an adequate signal and has sufficient half-life such as 32 P, 3 H, 14 C or the like.
  • Other detectable substances that may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization.
  • Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleic acid probes may be used to detect LI Polynucleotides, preferably in human cells.
  • the nucleotide probes may also be useful in the diagnosis of a lung injury involving one or more LI Polynucleotides; in monitoring the progression of such disorder; or monitoring a therapeutic treatment.
  • the detection of LI Polynucleotides may involve the amplification of specific gene sequences using an amplification method such as polymerase chain reaction (PCR), followed by the analysis of the amplified molecules using techniques known to those skilled in the art.
  • PCR polymerase chain reaction
  • Suitable primers can be routinely designed by one of skill in the art.
  • at least two oligonucleotide primers may be employed in a PCR based assay to amplify a portion of a polynculeotide encoding one or more LI Marker derived from a sample, wherein at least one of the oligonucleotide primers is specific for (i.e. hybridizes to) a LI Polynucleotide.
  • primers and probes employed in the methods of the invention generally have at least about 60%, preferably at least about 75%, and more preferably at least about 90% identity to a portion of a LI Polynucleotide; that is, they are at least 10 nucleotides, and preferably at least 20 nucleotides in length. In an embodiment the primers and probes are at least about 10-40 nucleotides in length.
  • Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of LI Polynucleotide expression.
  • RNA may be isolated from a cell type or tissue known to express a LI Polynucleotide and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques referred to herein.
  • the primers and probes may be used in the above-described methods in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
  • a method employing reverse transcriptase- polymerase chain reaction (RT-PCR), in which PCR is applied in combination with reverse transcription.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • RNA is extracted from a sample tissue using standard techniques (for example, guanidine isothiocyanate extraction as described by Chomcynski and Sacchi, Anal. Biochem. 162:156-159, 1987) and is reverse transcribed to produce cDNA.
  • the cDNA is used as a template for a polymerase chain reaction.
  • the cDNA is hybridized to a set of primers, at least one of which is specifically designed against an LI Marker sequence.
  • a DNA polymerase is employed to extend from the primer, to synthesize a copy of the template.
  • the DNA strands are denatured, and the procedure is repeated many times until sufficient DNA is generated to allow visualization by ethidium bromide staining and agarose gel electrophoresis.
  • Amplification may be performed on samples obtained from a subject with a suspected lung injury and an individual who is not afflicted with a lung injury.
  • the reaction may be performed on several dilutions of cDNA spanning at least two orders of magnitude. A statistically significant difference in expression in several dilutions of the subject sample as compared to the same dilutions of the normal sample may be considered positive for the presence of a lung injury.
  • the invention provides methods for determining the presence or absence of a lung injury in a subject comprising (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to one or more LI Polynucleotides; and (b) detecting in the sample a level of nucleic acids that hybridize to the polynucleotides relative to a predetermined cut-off value, and therefrom determining the presence or absence of a lung injury in the subject.
  • the invention provides a method wherein an LI Polynucleotide which is mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more LI Polynucleotides, to produce amplification products; (d) analyzing the amplification products to detect amounts of mRNA encoding LI Polynucleotides; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal tissue derived using similar nucleic acid primers.
  • LI Marker-positive samples or alternatively higher levels in patients compared to a control may be indicative of advanced injury, and/or that the patient is not responsive to or tolerant of a therapy.
  • negative samples or lower levels compared to a control e.g. normal samples or negative samples may also be indicative of progressive injury.
  • the invention provides methods for determining the presence or absence of lung injury in a subject comprising (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to one or more LI Polynucleotides; and (b) detecting in the sample levels of polynucleotides that hybridize to the LI Polynucleotides relative to a predetermined cut-off value, and therefrom determining the presence or absence of lung injury in the subject.
  • the LI Polynucleotides encode one or more polypeptides listed in Table 1.
  • the invention provides a method wherein NPY mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to a NPY polynucleotide, to produce amplification products; (d) analyzing the amplification products to detect an amount of NPY mRNA; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal tissue derived using similar nucleic acid primers.
  • Marker-positive samples or alternatively higher levels, in particular significantly higher levels of NPY polynucleotides in patients compared to a control (e.g. normal) are indicative of lung injury, in particular overdistension.
  • the levels may also be indicative of progressive injury and poor prognosis.
  • the invention provides a method wherein IP-10 mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to an IP- 10 polynucleotide, to produce amplification products; (d) analyzing the amplification products to detect an amount of TP- 10 mRNA; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal tissue derived using similar nucleic acid primers.
  • Marker-positive samples or alternatively higher levels are indicative of lung injury, in particular overdistension and/or derecruitment. The levels may also be indicative of progressive injury and poor prognosis. Marker-positive samples or alternatively higher levels, in particular significantly higher levels of M94919, glutathione S-transferase 2, and/or ferritin light chain 1 in patients compared to a control (e.g. normal) are indicative of lung injury, in particular VILI.
  • Oligonucleotides or longer fragments derived from an LI Polynucleotides may be used as targets in a microarray.
  • the microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms.
  • the information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • the invention also includes an array comprising one or more LI Polynucleotidess (in particular the markers listed in Table 1), and optionally other markers.
  • the array can be used to assay expression of LI Polynucleotides in the array.
  • the invention allows the quantitation of expression of one or more LI Polynucleotides.
  • the array can be used to monitor the time course of expression of one or more LI Polynucleotides in the array. This can occur in various biological contexts such as progression of lung injury.
  • the array is also useful for ascertaining differential expression patterns of LI Polynucleotides, and optionally other markers, in normal and abnormal cells. This may provide a battery of nucleic acids that could serve as molecular targets for diagnosis or therapeutic intervention. Protein Methods
  • Binding agents may be used for a variety of diagnostic and assay applications. There are a variety of assay formats known to the skilled artisan for using a binding agent to detect a target molecule in a sample. (For example, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of a lung injury or stage or type of lung injury in a subject may be determined by (a) contacting a sample from the subject with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined standard or cut-off value.
  • the binding agent is an antibody.
  • Antibodies specifically reactive with one or more LI Marker, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect one or more LI Marker in various samples (e.g. biological materials). They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of expression of one or more LI Marker.
  • Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on lung injury involving one or more LI Markers, and other conditions. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies.
  • the invention provides a diagnostic method for monitoring or diagnosing a lung injury in a subject by quantitating one or more LI Markers in a biological sample from the subject comprising reacting the sample with antibodies specific for one or more LI Markers, which are directly or indirectly labeled with detectable substances and detecting the detectable substances.
  • LI Markers are quantitated or measured.
  • a method for detecting a lung injury comprising:
  • the invention contemplates a method for monitoring the progression of a lung injury in an individual, comprising:
  • step (d) comparing the result of step (b) with the result of step (c), wherein a difference in the amount of complex formation is indicative of lung injury, or stage or type of injury in said individual.
  • the amount of complexes may also be compared to a value representative of the amount of the complexes from an individual not at risk of, or afflicted with, a lung injury at different stages. A significant difference in complex formation may be indicative of advanced lung injury, or an unfavourable prognosis.
  • NPY is detected in samples and higher levels, in particular significantly higher levels compared to a control (normal) is indicative of lung injury, in particular overdistension.
  • IP-IO is detected in samples and higher levels, in particular significantly higher levels compared to a control (normal) is indicative of lung injury, in particular overdistension and derecruitment.
  • hemoglobin beta-chain, glutathione S-transferase pi-1, and/or ferritin light chain 1 is detected in samples and higher levels, in particular significantly higher levels compared to a control (normal) is indicative of lung injury, in particular VILI.
  • Antibodies may be used in any known immunoassays that rely on the binding interaction between antigenic determinants of one or more LI Marker and the antibodies.
  • Immunoassay procedures for in vitro detection of antigens in fluid samples are also well known in the art. [See for example, Paterson et al., Int. J. Can.37:659 (1986) and Burchell et al., Int. J. Can. 34:763 (1984) for a general description of immunoassay procedures].
  • Qualitative and/or quantitative determinations of one or more LI Marker in a sample may be accomplished by competitive or non-competitive immunoassay procedures in either a direct or indirect format.
  • Detection of one or more LI Marker using antibodies can be done utilizing immunoassays which are run in either the forward, reverse or simultaneous modes.
  • immunoassays are radioimmunoassays (RIA), enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, histochemical tests, and sandwich (immunometric) assays. These terms are well understood by those skilled in the art. A person skilled in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • an immunoassay for detecting more than one LI Marker in a biological sample comprises contacting binding agents that specifically bind to LI Markers in the sample under conditions that allow the formation of first complexes comprising a binding agent and LI Markers and determining the presence or amount of the complexes as a measure of the amount of LI Markers contained in the sample.
  • the binding agents are labeled differently or are capable of binding to different labels.
  • Binding agents e.g. antibodies
  • Binding agents may be used in immunohistochemical analyses, for example, at the cellular and sub-subcellular level, to detect one or more LI Markers, to localize them to particular lung cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
  • Immunohistochemical methods for the detection of antigens in tissue samples are well known in the art. For example, immunohistochemical methods are described in Taylor, Arch. Pathol. Lab. Med. 102:112 (1978). Briefly, in the context of the present invention, a tissue sample obtained from a subject suspected of having a lung injury is contacted with antibodies, preferably monoclonal antibodies recognizing one or more LI Markers. The site at which the antibodies are bound is determined by selective staining of the sample by standard immunohistochemical procedures. The same procedure may be repeated on the same sample using other antibodies that recognize one or more LI Markers.
  • a sample may be contacted with antibodies against one or more LI Markers simultaneously, provided that the antibodies are labeled differently or are able to bind to a different label.
  • the tissue sample may be normal lung tissue.
  • Antibodies specific for one or more LI Marker may be labelled with a detectable substance and localised in biological samples based upon the presence of the detectable substance.
  • detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35 S, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached via spacer arms of various lengths to reduce potential ster
  • detectably labeled is to link it directly to an enzyme.
  • the enzyme when later exposed to its substrate will produce a product that can be detected.
  • detectable substances that are enzymes are horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase, malate dehydrogenase, ribonuclease, urease, catalase, glucose-6-phosphate, staphylococcal nuclease, delta-5-steriod isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, triose phosphate isomerase, asparaginase, glucose oxidase, and acetylcholine esterase.
  • a bioluminescent compound may also be used as a detectable substance.
  • Bioluminescence is a type of chemiluminescence found in biological systems where a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent molecule is determined by detecting the presence of luminescence.
  • bioluminescent detectable substances examples include luciferin, luciferase and aequorin.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against one or more LI Markers.
  • a second antibody having specificity for the antibody reactive against one or more LI Markers.
  • the antibody having specificity against one or more LI Markers is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect one or more LI Markers.
  • antibodies may be labeled with detectable substances and one or more LI Markers may be localised in tissues and cells based upon the presence of the detectable substances.
  • the sample, binding agents e.g. antibodies specific for one or more LI Markers
  • one or more LI Markers may be immobilized on a carrier or support.
  • suitable carriers or supports are agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros, filter paper, magnetite, ion-exchange resin, plastic film, plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the support material may have any possible configuration including spherical (e.g.
  • the carrier may be in the shape of, for example, a tube, test plate, well, beads, disc, sphere, etc.
  • the immobilized antibody may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • An antibody may be indirectly immobilized using a second antibody specific for the antibody.
  • mouse antibody specific for an LI Marker may be immobilized using sheep anti-mouse IgG Fc fragment specific antibody coated on the carrier or support.
  • one or more LI Marker may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • One or more LI Marker antibodies may also be indirectly labelled with an enzyme using ligand binding pairs.
  • the antibodies may be conjugated to one partner of a ligand binding pair, and the enzyme may be coupled to the other partner of the ligand binding pair.
  • Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein.
  • the antibodies are biotinylated, and the enzyme is coupled to streptavidin.
  • an antibody specific for LI Marker antibody is labeled with an enzyme.
  • Computer readable media comprising one or more LI Markers, and/or LI Polynucleotides, and optionally other markers (e.g. markers of lung injury) is also provided.
  • Computer readable media refers to any medium that can be read and accessed directly by a computer, including but not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • the invention contemplates computer readable medium having recorded thereon markers identified for patients and controls.
  • Recorded refers to a process for storing information on computer readable medium.
  • the skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising information on one or more LI Markers, and optionally other markers.
  • a variety of data processor programs and formats can be used to store information on one or more LI Markers, and/or LI Polynucleotides, and other markers on computer readable medium.
  • the information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • Any number of dataprocessor structuring formats (e.g., text file or database) may be adapted in order to obtain computer readable medium having recorded thereon the marker information.
  • marker information in computer readable form
  • one skilled in the art can use the information in computer readable form to compare marker information obtained during or following therapy with the information stored within the data storage means.
  • the invention also provides in an electronic system and/or in a network, a method for determining whether a subject has a lung injury or a pre-disposition to a lung injury, comprising determining the presence or absence of one or more LI Markers, and/or LI Polynucleotides, and optionally other markers, and based on the presence or absence of the one or more LI Markers, and/or LI Polynucleotides, and optionally other markers, determining whether the subject has a lung injury, or a pre-disposition to a lung injury, and optionally recommending a procedure or treatment.
  • the invention further provides in a network, a method for determining whether a subject has a lung injury or a pre-disposition to a lung injury comprising: (a) receiving phenotypic information on the subject and information on one or more LI Markers, and/or LI Polynucleotides, and optionally other markers associated with samples from the subject; (b) acquiring information from the network corresponding to the one or more LI Markers, and/or LI Polynucleotides, and optionally other markers; and (c) based on the phenotypic information and information on the one or more LI Markers, and/or LI Polynucleotides, and optionally other markers, determining whether the subject has a lung injury or a pre ⁇ disposition to a lung injury; and (d) optionally recommending a procedure or treatment.
  • a system of the invention generally comprises a digital computer; a database server coupled to the computer; a database coupled to the database server having data stored therein, the data comprising records of data comprising one or more LI Markers, and/or LI polynucleotides, and optionally other markers, and a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records which match the desired selection criteria.
  • Markers and/or LI polynucleotides, and optionally other markers of lung injury;
  • step (c) using a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records of step (a) which provide a match of the desired selection criteria of the database of step (b) the presence of a match being a positive indication that the markers of step (a) have been isolated from cells or tissue that are injured.
  • the invention contemplates a business method for determining whether a subject has a lung injury or a pre-disposition to lung injury comprising: (a) receiving phenotypic information on the subject and information on one or more LI Markers, and/or LI
  • Polynucleotides, and optionally other markers, associated with samples from the subject (b) acquiring information from a network corresponding to one or more LI Markers, and/or LI
  • Polynucleotides, and optionally other markers and (c) based on the phenotypic information, information on one or more LI Markers, and/or LI Polynucleotides encoding the markers, and optionally other markers, and acquired information, determining whether the subject has a lung injury or a pre-disposition to a lung injury; and (d) optionally recommending a procedure or treatment.
  • the computer systems, components, and methods described herein are used to monitor lung injury or determine the stage or type of a lung injury.
  • Binding agents in particular antibodies, specific for one or more LI Markers may also be used in imaging methodologies in the management of a lung injury.
  • the invention provides a method for imaging injured lung tissue or cells associated with one or more LI Markers.
  • the invention also contemplates imaging methods described herein using multiple markers for a lung injury or stage or type of lung injury.
  • each agent is labeled so that it can be distinguished during the imaging.
  • the method is an in vivo method and a subject or patient is administered one or more agents that carry an imaging label and that are capable of targeting or binding to one or more LI Markers.
  • the agent is allowed to incubate in vivo and bind to the LI Markers associated with an injured cell or tissue.
  • the presence of the label is localized to the injured cell or tissue, and the localized label is detected using imaging devices known to those skilled in the art.
  • the agent may be an antibody or chemical entity that recognizes the LI Markers.
  • the agent is a polyclonal antibody or monoclonal antibody, or fragments thereof, or constructs thereof including but not limited to, single chain antibodies, bifunctional antibodies, molecular recognition units, and peptides or entities that mimic peptides.
  • the antibodies specific for the LI Markers used in the methods of the invention may be obtained from scientific or commercial sources, or isolated native LI Markers or recombinant LI Markers may be utilized to prepare antibodies etc. as described herein.
  • An agent may be a peptide that mimics the epitope for an antibody specific for an LI Marker and binds to the marker.
  • the peptide may be produced on a commercial synthesizer using conventional solid phase chemistry.
  • a peptide may be prepared that includes either tyrosine, lysine, or phenylalanine to which N 2 S 2 chelate is complexed (See U.S. Patent No. 4,897,255).
  • a marker peptide conjugate is then combined with a radiolabel (e.g. sodium 99m Tc pertechnetate or sodium 188 Re perrhenate) and it may be used to locate an LI Marker producing cell or tissue.
  • the agent carries a label to image the LI Markers.
  • the agent may be labelled for use in radionuclide imaging.
  • the agent may be directly or indirectly labelled with a radioisotope.
  • radioisotopes that may be used in the present invention are the following: 277 Ac, 211 At, 128 Ba, 131 Ba, 7 Be, 204 Bi, 205 Bi, 206 Bi, 76 Br, 77 Br, 82 Br, 109 Cd, 47 Ca, 11 C, 14 C, 36 Cl, 48 Cr, 51 Cr, 62 Cu, 64 Cu, 67 Cu, 165 Dy, 155 Eu, 18 F, 153 Gd, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 198 Au, 3 H, 166 Ho, 111 In, 113m In, 115m In, 123 1, 125 1, 131 1, 189 Ir, 191m Ir, 192 Ir, 194 Ir, 52 Fe, 55 Fe, 59 Fe, 177 Lu, 15 0, 191m"191 Os, 109 Pd, 32 P, 33 P
  • the radioisotope is 131 I, 125 I, 123 I, 111 I, 99m Tc, 90 Y, 186 Re, 188 Re, 32 P, 153 Sm, 67 Ga, 201 Tl 77 Br, or 18 F, and is imaged with a photoscanning device.
  • Procedures for labeling biological agents with the radioactive isotopes are generally known in the art.
  • U.S. Pat. No.4,302,438 describes tritium labeling procedures.
  • Procedures for iodinating, tritium labeling, and 35 S labeling especially adapted for murine monoclonal antibodies are described by Goding, J. W. (supra, pp 124-126) and the references cited therein.
  • Other procedures for iodinating biological agents, such as antibodies, binding portions thereof, probes, or ligands are described in the scientific literature (see Hunter and Greenwood, Nature 144:945 (1962), David et al., Biochemistry 13:1014-1021 (1974), and U.S. Pat. Nos.
  • the agent may be administered to the patient, it is localized to the cell or tissue having an LI Marker with which the agent binds, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography.
  • radionuclear scanning e.g., a gamma camera or emission tomography.
  • a positron emission transaxial tomography scanner such as designated Pet VI located at Brookhaven National Laboratory, can also be used where the radiolabel emits positrons (e.g., 11 C, I8 F, 15 O, and 13 N).
  • An agent may also be labeled with a paramagnetic isotope for purposes of an in vivo method of the invention.
  • the paramagnetic compound may comprise a monocrystalline nanoparticle, e.g., a nanoparticle comprising a lanthanide (e.g., Gd) or iron oxide; or, a metal ion comprising a lanthanide.
  • “Lanthanides” refers to elements of atomic numbers 58 to 70, a transition metal of atomic numbers 21 to 29, 42 or 44, a Gd(III), a Mn(II), or an element comprising an Fe element.
  • Paramagnetic compounds can also comprise a neodymium iron oxide (NdFeO 3 ) or a dysprosium iron oxide (DyFeO 3 ).
  • Examples of elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof. (See, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986) Magn. Reson. Med.
  • An imaging agent may carry a bioluminescent or chemiluminescent label.
  • Such labels include polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or, that act as enzymes on a specific substrate (reagent), or can generate a fluorescent, bioluminescent or chemiluminescent molecule.
  • bioluminescent or chemiluminescent labels include luciferases, aequorin, obelin, mnemiopsin, berovin, a phenanthridinium ester, and variations thereof and combinations thereof.
  • a substrate for the bioluminescent or chemiluminescent polypeptide may also be utilized in a method of the invention.
  • the chemiluminescent polypeptide can be luciferase and the reagent luciferin.
  • a substrate for a bioluminescent or chemiluminescent label can be administered before, at the same time (e.g., in the same formulation), or after administration of the agent.
  • An image can be generated in a method of the invention by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BBI bioluminescence imaging
  • Test agents and compounds include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab) 2 , and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules.
  • the agents or compounds may be endogenous physiological compounds or natural or synthetic compounds.
  • the invention provides a method for assessing the potential efficacy of a test agent for inhibiting a lung injury in a patient, the method comprising comparing:
  • the first and second samples may be portions of a single sample obtained from a patient or portions of pooled samples obtained from a patient.
  • the invention provides a method of selecting an agent for inhibiting a lung injury in a patient comprising: (a) obtaining a sample from the patient;
  • Still another aspect of the present invention provides a method of conducting a drug discovery business comprising: (a) providing one or more methods or assay systems for identifying agents that inhibit, prevent or reduce a lung injury or affect a stage or type of lung injury in a patient; (b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.
  • the subject method can also include a step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
  • the invention also contemplates a method of assessing the potential of a test compound to contribute to a lung injury comprising:
  • Kits may typically comprise two or more components required for performing a diagnostic assay.
  • Components include but are not limited to compounds, reagents, containers, and/or equipment.
  • the methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising one or more specific LI Marker polynucleotide or binding agent (e.g. antibody) described herein, which may be conveniently used, e.g., in clinical settings to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a lung injury.
  • diagnostic kits comprising one or more specific LI Marker polynucleotide or binding agent (e.g. antibody) described herein, which may be conveniently used, e.g., in clinical settings to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a lung injury.
  • a container with a kit comprises a binding agent as described herein.
  • the kit may contain antibodies or antibody fragments which bind specifically to epitopes of one or more LI Markers and optionally other markers, antibodies against the antibodies labelled with an enzyme; and a substrate for the enzyme.
  • the kit may also contain microtiter plate wells, standards, assay diluent, wash buffer, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit.
  • the kit includes antibodies or fragments of antibodies which bind specifically to an epitope of one or more markers listed in Table 1, and means for detecting binding of the antibodies to their epitope associated with lung injury, either as concentrates (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages.
  • the kits are intended for in vivo use, single dosages may be provided in sterilized containers, having the desired amount and concentration of agents. Containers that provide a formulation for direct use, usually do not require other reagents, as for example, where the kit contains a radiolabeled antibody preparation for in vivo imaging.
  • a kit may be designed to detect the level of polynucleotides encoding one or more LI
  • kits generally comprise at least one oligonucleotide probe or primer, as described herein, that hybridizes to a LI Polynucleotide.
  • oligonucleotide may be used, for example, within a PCR or hybridization procedure.
  • Additional components that may be present within the kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate detection of a polynucleotide encoding one or more LI Markers.
  • reagents suitable for applying the screening methods of the invention to evaluate compounds may be packaged into convenient kits described herein providing the necessary materials packaged into suitable containers.
  • the invention relates to a kit for assessing the suitability of each of a plurality of test compounds for inhibiting a lung injury in a patient.
  • the kit comprises reagents for assessing one or more LI Markers or LI polynucleotides, and optionally a plurality of test agents or compounds.
  • the invention contemplates a kit for assessing the presence of injured lung cells, wherein the kit comprises antibodies specific for one or more LI Markers, or primers or probes for LI Polynucleotides, and optionally probes, primers or antibodies specific for other markers associated with a lung injury (e.g. ). Additionally the invention provides a kit for assessing the potential of a test compound to contribute to a lung injury.
  • the kit comprises injured lung cells and reagents for assessing one or more LI Markers, LI Polynucleotides, and optionally other markers associated with a lung injury.
  • Therapeutic Applications One or more LI Markers may be targets for immunotherapy. Immunotherapeutic methods include the use of antibody therapy.
  • the invention provides one or more LI Marker antibodies that may be used to treat a lung injury associated with the marker.
  • the invention provides a method of preventing, inhibiting or reducing lung injury, comprising administering to a patient an antibody which binds specifically to one or more LI Markers in an amount effective to prevent, inhibit, or reduce lung injury.
  • the methods of the invention contemplate the administration of single LI Marker antibodies as well as combinations, or "cocktails", of different individual antibodies such as those recognizing different epitopes of other markers. Such cocktails may have certain advantages inasmuch as they contain antibodies that bind to different epitopes of LI Markers and/or exploit different effector mechanisms. Such antibodies in combination may exhibit synergistic therapeutic effects.
  • the administration of one or more LI Marker specif ⁇ c antibodies may be combined with other therapeutic agents.
  • the LI Marker specific antibodies may be administered in their "naked” or unconjugated form, or may have therapeutic agents conjugated to them.
  • the LI Marker specific antibodies used in the methods of the invention may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.
  • Suitable carriers include any material which when combined with the antibodies retains the function of the antibody and is non-reactive with the subject's immune systems. Examples include any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th Edition, A. Osal., Ed., 1980).
  • One or more LI Marker specific antibody formulations may be administered via any route capable of delivering the antibodies to the site or injury.
  • Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intradermal, into the lung and the like.
  • the route of administration is by intravenous injection or intra-tracheally.
  • Antibody preparations may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, beri2yl alcohol preservative, or in sterile water prior to injection.
  • Treatment will generally involve the repeated administration of the antibody preparation via an acceptable route of administration at an effective dose. Dosages will depend upon various factors generally appreciated by those of skill in the art, including the type of injury and the severity, stage of the injury, the binding affinity and half life of the antibodies used, the degree of LI Marker expression in the patient, the desired steady-state antibody concentration level, frequency of treatment, and the influence of any therapeutic agents used in combination with the treatment method of the invention.
  • a determining factor in defining the appropriate dose is the amount of a particular antibody necessary to be therapeutically effective in a particular context. Repeated administrations may be required to achieve a desired effect. Direct administration of one or more LI Marker antibodies is also possible and may have advantages in certain situations.
  • Patients may be evaluated for markers in order to assist in the determination of the most effective dosing regimen and related factors.
  • the assay methods described herein, or similar assays may be used for quantitating LI Marker levels in patients prior to treatment. Such assays may also be used for monitoring throughout therapy, and may be useful to gauge therapeutic success in combination with evaluating other parameters such as levels of LI Markers.
  • LI Polynucleotides can be turned off by transfecting a cell or tissue with vectors that express high levels of a desired LI Polynucleotide. Such constructs can inundate cells with untranslatable sense or antisense sequences. Even in the absence of integration into the
  • DNA such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases.
  • Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used to deliver LI Polynucleotides to a targeted organ, tissue, or cell population. Methods well known to those skilled in the art may be used to construct recombinant vectors that will express antisense LI Polynucleotides (See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra)).
  • Methods for introducing vectors into cells or tissues include those methods discussed herein and which are suitable for in vivo, in vitro and ex vivo therapy.
  • delivery by transfection and by liposome are well known in the art.
  • Modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA or PNA, to the regulatory regions of a LI Polynucleotide, i.e., the promoters, enhancers, and introns.
  • oligonucleotides are derived from the transcription initiation site, e.g. between -10 and +10 regions of the leader sequence.
  • the antisense molecules may also be designed so that they block translation of mRNA by preventing the transcript from binding to ribosomes. Inhibition may also be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Therapeutic advances using triplex DNA were reviewed by Gee J E et al (In: Huber B E and B I Carr ( 1994) Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N. Y.).
  • Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. The invention therefore contemplates engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of LI Polynucleotides. Specif ⁇ c ribozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be determined by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • One or more LI Markers and LI Polynucleotides, and fragments thereof, and compounds or agents identified using a method of the invention may be used to prevent, treat, or reduce a lung injury in a subject.
  • the markers or polynucleotides may be formulated into compositions for administration to subjects suffering from a lung injury. Therefore, the present invention also relates to a composition comprising one or more LI Markers or LI Polynucleotides, or a fragment thereof, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method for treating or preventing a lung injury in a subject comprising administering to a patient in need thereof, one or more LI Markers or LI Polynucleotides, an agent or compound identified using a method of the invention, or a composition of the invention.
  • the invention further provides a method of preventing, inhibiting, or reducing a lung injury in a patient comprising:
  • test agents which alters the levels of the LI Markers, and/or LI Polynucleotides in the aliquot containing that test agent, relative to the other test agents.
  • An active therapeutic substance described herein may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, intra-tracheally, inhalation, transdermal application, or rectal administration.
  • the active substance may be coated in a material to protect the substance from the action of enzymes, acids and other natural conditions that may inactivate the substance.
  • Solutions of an active compound as a free base or pharmaceutically acceptable salt can be prepared in an appropriate solvent with a suitable surfactant.
  • Dispersions may be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the compositions include, albeit not exclusively, solutions of the active substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment.
  • the compositions of the invention may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.
  • compositions and agents/compounds identified using a method of the invention may be evaluated in vivo using a suitable animal model.
  • Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a lung injury.
  • patients have ARDS or VILI.
  • suitable subjects for treatment in accordance with the invention include persons that are susceptible to, suffering from or that have ARDS or VILI.
  • the methods and compositions described herein are used prophylactically to prevent development of lung injury, for example, prior to the start of mechanical ventilation or after a subject develops a risk factor predisposing them to develop ARDS or VILI (e.g. major trauma, pancreatitis etc.).
  • ARDS or VILI e.g. major trauma, pancreatitis etc.
  • RNA was isolated from lung tissue (n 5 per group) (RNEasy; Qiagen, Valencia, CA). Pooled RNA was hybridized to Affymetrix (Santa Clara, CA) rat A34 GeneChips in duplicate. Data were analyzed using Microarray Suite 3.1 software (Affymetrix). Expression analysis files were transferred to a database (Microsoft Access) and analyzed by JMP software (cluster analysis; SAS Institute, Cary, NC) and the SAM program (12).
  • RNA (10 ⁇ g) from lung tissue (n 5 per group) was reverse transcribed using random hexamers (Applied Biosystems), and the resulting templates quantified by real time PCR (ABI Prism 7700) using TaqMan PCR reactions. Histology and immunohistochemistry
  • the lung tissue fixed in formalin were processed for histologic analysis (H&E) and immunohistochemistry for NPY, CGRP and IP-IO.
  • ELISA histologic analysis
  • Frozen lung tissues were homogenized in cell lysis buffer, and supernatants were assayed using specific ELISA kits for rat Neuropeptide Y (Phoenix pharmaceuticals, Belmont, CA) and rat IL-6, murine IL-6, and murine IP-IO (R&D systems, Minneapolis, MN).
  • mice C57BL/6 mice (21-26g; Jackson Laboratories) received either 50 ⁇ g of anti-mouse IP-10 (R&D systems) or control antibodies i.p. 30 min before surgical procedures.
  • right lungs were snap frozen for ELISA.
  • BAL was performed ( 1 ml of PBS x 3) in left lung for the total and differential cell counts.
  • RNA was extracted from lung tissues after 3 h of ventilation in 3 acid treated groups (b ⁇ l, b l, and b>l); from animals without any interventions except anesthesia (normal group); and from animals after anesthesia and surgical preparation (sham group).
  • neuropeptide Y neuropeptide Y
  • galanin chemokines
  • IP-IO chemokines
  • MIP-3 ⁇ MIP-3 ⁇
  • MIP- l ⁇ adhesion molecules
  • E-selectin adhesion molecules
  • PAI-I clotting related genes
  • u-PAR u-PAR
  • t-PA clotting related genes
  • RT-PCR analysis largely confirmed the microarray data attests to the usefulness of the SAM procedure for gene selection and suggests that most of the unconfirmed microarray data for these genes are likely to be valid as well.
  • genes include the transcription factors c-fos, Fra-1, CREM, Tcf21 and nerve growth factor inducible-B (NGFI-B, rat homolog of human NR4A1) the cytokines IL-6 and platelet factor 4, the heme-related proteins haptoglobin, ⁇ -globin (gene highly similar to human HBAl) and aminolevulinate synthase 2 ⁇ as well as a variety of other genes including MCA-32, Tapl, Cish and P- selectin.
  • NPY neuropeptides NPY and galanin in overdistended, but not derecruited lungs suggesting that overdistension specifically enhances expression of certain neuropeptides and activates a neural- inflammatory axis.
  • NPY is a neurotransmitter that is released from excited sympathetic nerves and widely distributed throughout the central and peripheral nervous system (13, 14).
  • Figure 3a protein levels of NPY were significantly higher in lungs ventilated with b>l.
  • NPY neuropeptide binds to a number of G protein-coupled receptors (Yl -Y6; the putative Y3 receptor has not yet been cloned) [15, 16]. Yl receptors have NF- ⁇ B response elements in their promoter regions and are expressed in the lung [17]. The role of IP-IO in VILI
  • IP-10 (CXCL-IO, in the rat also called mob-1) is a highly inducible, primary response gene that belongs to the CXC chemokine superfamily. Further studies were performed on IP-10 because (i) it was one of the few genes that were significantly upregulated by both overdistension and derecruitment; (ii) it is strongly upregulated in many types of inflammation, but its function is largely unknown; and (iii) it has been associated with acute lung injury induced by the SARS virus (19). Protein levels of IP-10 were significantly higher in animals ventilated with both
  • NPY and IP-10 Plasma levels were measured in ARDS patients who had been randomized to either conventional ventilation with relatively high tidal volumes, or a lung protective strategy in the course of a previous clinical trial [20]. Plasma levels of NPY ( Figure 5a) and IP-lO/CXCL-10 ( Figure 5b) were significantly higher in the conventionally ventilated group than in the lung protective group.
  • ARDS lungs are heterogeneously injured, and the current experiments were designed to mimic recruitment/de-recruitment ([b ⁇ l] strategy) or overdistension ([b ⁇ l] strategy) that occurs in the lungs of patients with acute lung injury.
  • NPY meets several criteria for a transmitter involved in neuro- immune interactions [14]: (1) it is synthesized and stored in postganglionic nerves terminating in the lung [25-28]; (2) it is released into lymphatic organs [29-31]; (3) it modulates the activity of immune cells including PMN [32, 33] and macrophages [29]; (4) it alters the levels of inflammatory cytokines [34-36]; (5) it increases vascular permeability in the lung and may contribute to neurogenic edema [37].
  • the fact that NPY was induced in both nerve endings and alveolar macrophages is pronounced of the distribution of neurokinins, which also contribute to VILI and show a similar anatomical distribution [38].
  • IP-lO/CXCL-10 showed a strong induction by both overdistension and derecruitment.
  • IP-10 was expressed in alveolar macrophages in rat acute lung injury elicited by IL-2 or LPS/platelet activating factor (PAF) [39].
  • the data described herein demonstrate that blockade of IP-10 attenuated the infiltration of neutrophils in the lung, supporting a chemokine-like function of IP- 10 on neutrophils. This is consistent with the observation that administration of IP-10 causes neutrophil infiltration [39] and that blockade of CXCR3, a receptor of IP-10, reduced the leukocyte recruitment and severity of idiopathic pneumonia in mice [40].
  • the results may have clinical relevance for the development of novel therapies for the treatment of patients with ARDS. Due to the tremendous spatial heterogeneity of the lung injury in many patients with ARDS, it will not be possible to develop ventilatory strategies that are completely protective, especially in patients with the most severe underlying injury - a strategy that limits injury in some regions, such as the use of high positive end-expiratory pressure to mitigate recruitment/de-recruitment, may lead to injury in other regions due to overdistension. For these patients in particular, other non-ventilatory approaches are necessary. In this study a number of therapeutic targets were identified that may be useful in this context.
  • antibody is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • NPY a newly discovered peptide is present in the mammalian respiratory tract.
  • Neuropeptides by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype. Proc.Natl.Acad.Sci. U.S.A 95, 12544-12549 (1998). 36. Kawamura, N. et al. Differential effects of neuropeptides on cytokine production by mouse helper T cell subsets. Neuroimmunomodiilation. 5, 9-15 (1998).
  • NA Neuropeptide Y (human NPY) Locus ID 4852 (Org Hs) ; NM_000905 (human)
  • IFN- ⁇ -inducible protein -10 (rat CxcllO) Protein ID NP_620789 (Rattus norwegicus)
  • NA IFN- ⁇ -inducible protein -10 (CXCLlO) Locus ID 3627 (Org Hs); NM 001565 (human)
  • NA IFN- ⁇ -inducible protein-10 Locus ID 245920 (Org Rn) ; NM_139089 (rat)
  • HBB Hemoglobin beta-chain
  • GenBank Accession No. AA M94919 (Rattus norvegicus (Norway rat))
  • Protein ID No. NP_000843 [microsomal GST-II [Homo sapiens]]
  • NA Glutathione S-transferase pi human GSTPl
  • Locus ID 2950 Org Hs
  • NA light polypeptide human FTL
  • Locus ID 2512 Org Hs

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Abstract

La présente invention a trait à de nouveaux marqueurs de la lésion pulmonaire, à des procédés pour l'évaluation de l'état du tissu pulmonaire, et à des procédés pour le diagnostic et le traitement de lésions pulmonaires.
PCT/CA2005/001603 2004-10-15 2005-10-17 Marqueurs de la lesion pulmonaire WO2006039819A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2013170735A1 (fr) * 2012-05-15 2013-11-21 中国医学科学院基础医学研究所 Méthode de traitement et/ou de prévention d'une lésion pulmonaire
WO2014125164A1 (fr) * 2013-02-14 2014-08-21 Faron Pharmaceuticals Oy Procédé pour déterminer des biomarqueurs liés au syndrome de détresse respiratoire aiguë (sdra), procédé pour suivre le développement et le traitement du sdra chez un patient
WO2017220988A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps multispécifiques pour l'immuno-oncologie
WO2021189015A1 (fr) * 2020-03-20 2021-09-23 FORSYTH DENTAL INFIRMARY FOR CHILDREN, d/b/a THE FORSYTH INSTITUTE Procédés de détection et de traitement de réponses immunitaires associées à une infection virale

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CA2331934A1 (fr) * 2000-12-04 2002-06-04 Ruddy Wattiez Proteine et ses sequences de nucleotides de codage pour le diagnostic, la prevention et le traitement des lesions et des troubles pulmonaires
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HILDEBRANT G.C. ET AL.: "Blockade of CXCR3 Receptor: Ligand Interactions Reduces Leukocyte Recruitment to the Lung and the Severity of Experimental Idiopathic Pneumonia Syndrome", J. IMMUNOL., vol. 173, no. 3, August 2004 (2004-08-01), pages 2050 - 2059 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013170735A1 (fr) * 2012-05-15 2013-11-21 中国医学科学院基础医学研究所 Méthode de traitement et/ou de prévention d'une lésion pulmonaire
WO2014125164A1 (fr) * 2013-02-14 2014-08-21 Faron Pharmaceuticals Oy Procédé pour déterminer des biomarqueurs liés au syndrome de détresse respiratoire aiguë (sdra), procédé pour suivre le développement et le traitement du sdra chez un patient
JP2016513253A (ja) * 2013-02-14 2016-05-12 ファロン ファーマシューティカルズ オサケ ユキチュア 急性呼吸促迫症候群（ａｒｄｓ）関連バイオマーカーを測定する方法、患者におけるａｒｄｓの進行および治療をモニターする方法
US10247730B2 (en) 2013-02-14 2019-04-02 Faron Pharmaceuticals Oy Method for determining acute respiratory distress syndrome (ARDS) related biomarkers, a method to monitor the development and treatment of ARDS in a patient
WO2017220988A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps multispécifiques pour l'immuno-oncologie
WO2017220990A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps anti-pd-l1
WO2017220989A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 et cytokines il-2
WO2021189015A1 (fr) * 2020-03-20 2021-09-23 FORSYTH DENTAL INFIRMARY FOR CHILDREN, d/b/a THE FORSYTH INSTITUTE Procédés de détection et de traitement de réponses immunitaires associées à une infection virale

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