WO2004065626A2 - Procede permettant de pronostiquer une sepsie - Google Patents

Procede permettant de pronostiquer une sepsie Download PDF

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WO2004065626A2
WO2004065626A2 PCT/DK2004/000027 DK2004000027W WO2004065626A2 WO 2004065626 A2 WO2004065626 A2 WO 2004065626A2 DK 2004000027 W DK2004000027 W DK 2004000027W WO 2004065626 A2 WO2004065626 A2 WO 2004065626A2
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seq
mbl
primer
individual
sepsis
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PCT/DK2004/000027
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WO2004065626A3 (fr
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Peter Garred
Hans Ole Madsen
Jens STRØM
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Rigshospitalet
Københavns Amt
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Publication of WO2004065626A2 publication Critical patent/WO2004065626A2/fr
Publication of WO2004065626A3 publication Critical patent/WO2004065626A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • 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
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention relates to a method for prognostic purposes of estimating whether an individual will develop sepsis when said individual suffers from systemic inflammatory response syndrome (SIRS).
  • SIRS systemic inflammatory response syndrome
  • SIRS systemic inflammatory response syndrome
  • ICU intensive care units
  • Mannose-binding lectin is a circulating liver synthesised serum protein of importance for innate immune defence and is one of the recognition molecules in the lectin complement activation pathway.
  • MBL deficiency is associated with increased risk of infections during early childhood, especially in ages between 6 and 18 months and also in patients with a concomitant immunodeficiency.
  • MBL variant alleles may be weakly associated with autoimmune conditions such as systemic lupus erythematosus as well as progression of rheumatoid arthritis.
  • autoimmune conditions such as systemic lupus erythematosus as well as progression of rheumatoid arthritis.
  • MBL susceptibility and modifying factor for development of sepsis is unknown.
  • WO 02/05833 in general relates to the use of sub-units and oligomers of MBL in prophylactic and/or curative treatment of infections in an individual receiving TNF- inhibiting treatment.
  • the description further describes an aspect of using a measurement of the MBL level as a prognostic marker for the risk of the individual receiving TNF- inhibiting treatment of acquiring an infection, and thereby an indication of the need for treatment. All patients are selected among individuals that have received treatment with either etanercept or infliximab and presented clinically significant infections.
  • WO 00/70043 relates to a process of producing a recombinant human MBL composition, a recombinant human MBL composition, a gene expression construct encoding a recombinant MBL polypeptide and to use of a MBL composition for the production of a pharmaceutical composition for the treatment, in an individual, of clinical conditions selected from infections, MBL deficiency, cancer, disorders associated with chemotherapy, miscarriages, disorders associated with neutropenia, and human immunodeficiency virus (HIV).
  • US 2001/0014449 relates to methods for the analysis and determination of the nature of single nucleic acid polymorphism's (SNPs) in a genetic target.
  • SNPs single nucleic acid polymorphism's
  • the nature of the SNPs is determined by the steps of providing a plurality of hybridisation complexes arrayed on a plurality of test sites on an electronically bioactive microchip.
  • the MBL gene locus is mentioned as a useful target since there are multiple closely spaced SNPs at a single genetic locus.
  • MBL gene locus is also described as correlating with susceptibility to sepsis in leukopenic patients.
  • US 2001/0014449 does not relate to the susceptibility to sepsis in SIRS patients.
  • MBL genotypes and serum concentrations have previously been determined in unrelated ethnic populations and that high frequencies of two different variant alleles are associated with low MBL concentrations (Madsen H.O. et al. 1998).
  • MBL gene polymorphism's are associated with major infection following allogenic hemopoietic stem cell transplantation (Mulligahn CG. et al.)
  • Steffensen describes the distribution of some variants of the MBL gene in a population of healthy unrelated Danes and correlates the genotype with the plasma MBL concentrations (Steffensen R. et al.) and others suggest the plasma level of MBL as predictor of sepsis after chemotherapy (Theil et al. 2000), thus limiting these conclusions to such subjects.
  • the importance of MBL in particular as susceptibility and modifying factor for development of sepsis in a patient having SIRS is unknown.
  • the present inventors have found a method of predicting whether and to what extend there is a risk of an individual having SIRS will develop sepsis based on the genotype and/or phenotype of said individual, and/or on the MBL concentration, in a biological sample of said individual.
  • the estimation of the sepsis risk of an individual can involve the comparison of the number and/or kind of polymorphic sequences of an individual and/or the MBL protein concentration in a biological sample of an individual with a predetermined sepsis risk profile. Such a profile can be based on statistical data obtained for a relevant reference group of individuals.
  • the present invention concerns a method of predicting whether an individual having SIRS will develop sepsis, said method comprising the steps of:
  • MBL mannose-binding lectin
  • step c) predicting the risk of said individual of developing sepsis by correlating the MBL genotype of step b) with a predefined risk value associated with said particular MBL genotype.
  • the invention relates to a method of predicting whether an individual wil develop sepsis comprising the steps of:
  • step c) predicting the risk of said individual of developing sepsis by correlating the MBL concentration of step b) with a predefined risk value associated with said particular MBL concentration.
  • the invention also describes a kit for predicting whether an individual will develop sepsis. Detailed description of the invention
  • the present invention discloses the connection between the MBL genotype of an individual having SIRS and the risk of developing sepsis, severe sepsis or septic shock.
  • the invention discloses how decreased levels of MBL and lack of functional MBL is crucial to the development of sepsis and septic shock in an individual having SIRS. Steps prior to an actual diagnosis of SIRS in a patient are furthermore an object of embodiments of the present invention, and the skilled addressee would easily recognise that employing the present invention on patients with diseases which are likely to evolve to SIRS, such as but not limited to infections, especially bacterial infections, traumas, tissue damages and burns; evaluating the genotype and/or concentration of MBL; and applying, for instance, MBL to said patient can prevent fatal outcome for those patients having high risk of later developing sepsis, as described by the present invention.
  • diseases which are likely to evolve to SIRS such as but not limited to infections, especially bacterial infections, traumas, tissue damages and burns
  • evaluating the genotype and/or concentration of MBL and applying, for instance, MBL to said patient can prevent fatal outcome for those patients having high risk of later developing sepsis, as described by the present invention.
  • MBL Mannose-binding lectin
  • SIRS systemic inflammatory response syndrome
  • the comparison shows that presence of MBL variant alleles is associated with development of sepsis, severe sepsis and septic shock.
  • the present invention relates to a method of predicting whether an individual having Systemic Inflammatory Response Syndrome (SIRS) will develop sepsis comprising the steps of:
  • MBL mannose-binding lectin
  • step c) predicting the risk of said individual of developing sepsis by correlating the MBL genotype determined in step b) with a predefined risk value associated with said particular MBL genotype.
  • a predefined risk value associated with said particular MBL genotype in the present context can be divided in a high risk and a low risk for developing sepsis in said individual having SIRS.
  • a high risk relates to a MBL genotype that is characterised in having at least one variant structural allele of the MBL gene and/or having two low-expression regulatory alleles of the MBL gene in said samples.
  • SIRS Systemic Inflammatory Response Syndrome
  • SIRS systemic inflammation response syndrome
  • any temperature above or equal to 38°C relates to temperatures from 38.0°C to any temperature by which the individual is capable of retain life, such as 38.1°C, 38.2°C, 38.3°C, 38.4°C, 38.5°C, 38.6°C, 38.7°C, 38.8°C, 38.9°C, 39.0°C, 39.1°C, 39.2°C, 39.3°C, 39.4°C, 39.5°C, 39.6°C, 39.7°C, 39.8°C, 39.9°C, 40.0°C, 40.1°C, 40.2°C, 40.3°C, 40.4°C, 40.5°C, 40.6°C, 40.7°C, 40.8°C, 40.9°C, 41.0°C, 41.1°C, 41.2°C, 41.3°C, 41.4°C, 41.5°C, 41.6°C, 41.7°C, 41.8°C, 41.9°C, or 4
  • any temperature below or equal to 36°C relates to temperatures from 36.0°C to any temperature by which the individual is capable of retain life, such as 35.9°C, 35.8°C, 35.7°C, 35.6°C, 35.5°C, 35.0°C, 34.5°C, 34.0°C, 33.5°C, 33.0°C, 32.5°C, 32.0°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, or 10°C.
  • a heart rate above 90 beats/mm relates to a heart rate from 91 beats/mm to 240 such as but not limited to a heart beat of 91 beats/mm, 92 beats/mm, 93 beats/mm of 94 beats/mm, 95 beats/min, 100 beats/mm, of 105 beats/mm, 110 beats/mm, 120 beats/mm, 125 beats/mm, 130 beats/mm, 135 beats/mm, 140 beats/mm of 145 beats/min, 150 beats/min, 155 beats/mm, of 160 beats/mm, 165 beats/min, 170 beats/mm, 180 beats/min, 185 beats/mm, 190 beats/mm, 195 beats/mm of 200 beats/mm, 205 beats/mm, 210 beats/mm, 215 beats/mm, or 220 beats/min.
  • a respiratory rate of more than or equal to 20 breaths/mm relates to a respiratory rate of 20 breaths/mm, 21 breaths/mm, 25 breaths/mm, 30 breaths/mm, 35 breaths/mm, 40 breaths/mm, 50 breaths/mm, 60 breaths/mm, 70 breaths/mm, 100 breaths/mm, 120 breaths/mm, 150 breaths/mm, 175 breaths/mm, 200 breaths/mm, 250 breaths/min, 275 breaths/min, 300 breaths/mm, 350 breaths/min, 375 breaths/min or 400 breath/mm.
  • a respiratory rate with a PaC02 ratio of below or equal to 4.3 kPa includes PaC02 ratios of 4.3 kPa, 4.2 kPa, 4.1 kPa, 4.0 kPa, 3.9 kPa, 3.8 kPa, 3.7 kPa, 3.6 kPa, 3.5 kPa, 3.4 kPa, 3.3 kPa, 3.2 kPa, 3.1 kPa, 3.0 kPa, 2.9 kPa, 2.8 kPa, 2.7 kPa, 2.6 kPa, 2.5 kPa, 2.4 kPa, 2.3 kPa, 2.2 kPa, 2.1 kPa, 2.0 kPa, 1 9 kPa, 1.8 kPa, 1.7 kPa, 1.6 kPa, 1.5 kPa, 1.4 kPa, 1.3
  • a white blood cell count of above or equal to 5 12.0xl0 9 cells/1 or less than or equal to 4.0xl0 9 cells/1 relates to white blood cell counts of 12-OxlO 9 cells/1, 12.1xl0 9 cells/I, 12.2xl0 9 cells/1, 12.3xl0 9 cells/I, 12.4xl0 9 cells/1, 12.5x10 s cells/1, 13.0xl0 9 cells/I, 13.5xl0 9 cells/1, 14.0xl0 9 cells/1, 14.5xl0 9 cells/1, 15.0x10 s cells/I, 16.0xl0 9 cells/1, 17.0xl0 9 cells/1, 18.0xl0 9 cells/1 or above, or 4.0xl0 9 cells/I, 3.9 xlO 9 cells/1, 3.8 xlO 9 cells/1, 3.7 xlO 9 cells/1, 3.6xl0 9 cells/I, 3.5 xlO 9 cells/I, 3.4 10 xlO 9 cells/
  • the invention also relates a method according to the present invention, wherein said individual has a white blood cell count of above or equal to
  • immature neutrophils As the skilled addressee would recognise a differential count showing above 10% immature neutrophils relates to 10.5% immature neutrophils, 11% immature neutrophils, 25 12% immature neutrophils, 13% immature neutrophils, 14% immature neutrophils, 15% immature neutrophils, 20% immature neutrophils or above.
  • the sepsis criteria in the present context is SIRS with a documented infection or a clinically suspected infection as in postoperative intra-abdominal sepsis in which the intestinal tract 30 had either been perforated or required partial resection for ischemia, or intestinal content appeared intra-abdominally due to leak from prior gut anastomosis; or radiographic evidence of pneumonia in association with purulent sputum.
  • Severe sepsis is defined as sepsis and either hypotension or evidence of hypoperfusion and organ dysfunction developing within 24 hour of study enrolment. Individual would have to meet at least one of the following criteria to be defined as having organ dysfunction;
  • Septic shock was defined as sepsis with hypotension in combination with one of the other criteria of acute organ dysfunction. Patients were excluded from participation if any of the following conditions were present: neutrophil count of less than 1.0 x 10 9 /l prior to the onset of sepsis; infections associated with burns; documented or suspected recent acute myocardial infarction; or lack of commitment to full life-support measures by the primary physician.
  • MOF Multiple organ failure
  • the method of the present invention determines the presence of both variant and normal polymorphism of the MBL gene in said sample.
  • MBL single nucleotide polymorphism's in form of the structural variants named B (codon 54), C (codon 57), and D (codon 52) as well as the regulatory variants named H/L (-550), X/Y (-221), and P/Q (+4) were in the present invention typed by PCR using sequence specific priming (PCR-SSP).
  • PCR-SSP sequence specific priming
  • the present inventors included a PCR covering exon 4 of the mbl2 gene as shown in the examples below.
  • Figure 1 shows seven patterns necessary for covering all combinations of each of the six complementary reactions.
  • the typing system was validated by automated sequencing (ABI 3100 platform) of PCR products from the seven control samples covering all of the polymorphic positions and in addition, by comparing the typing system with the previously used typing techniques.
  • All three structural variant alleles ( ⁇ , C, and D) have a considerable effect on MBL concentrations and to avoid small groups, the three alleles were grouped in one category called allele "O" for statistical analyses. Likewise, for statistical analyses described below, the present inventors only included the X/Y promoter variation at position -221.
  • the X variant is always found on a functional haplotype (LXPA) and has been shown to have a down regulating effect on MBL expression.
  • LXPA functional haplotype
  • the A/A group two normal structural alleles with high-expression promoter activity in position -221 (YA/YA) or one high-expression promoter and one low-expression promoter (YA/XA) or two low-expression promoters (XA/XA);
  • the A/0 group one variant structural allele (i.e. defective allele) and one normal structural allele regulated by a high-expression promoter (YA/O) or a low- expression promoter (XA/O), and the
  • the present invention relates to a method according to the present invention, wherein the MBL genotype has at least one variant structural allele of the MBL gene.
  • the present invention relates to a method according to the present invention, wherein the MBL has two low-expression regulatory alleles of the MBL gene.
  • the regulatory allele may be situated in the promoter region of the MBL gene.
  • the present invention relates to a method according the present invention, wherein the polymorphism lies in position 602, which generates a low-expression regulatory allele when position 602 has a base C substitution (SEQ ID NO: 26 (GeneBank, ID No. Y16580), or generates a high-expression regulatory allele when position 602 has a base G substitution (SEQ ID NO: 24 (GeneBank, ID No. Y16581).
  • the present invention relates to a method according to the present invention, wherein the polymorphism lies in codon 52 in position 1045, which generates a variant structural allele, when position 1045 has a base T (SEQ ID NO: 25 (GeneBank, ID No. Y16582)) or generates a normal structural allele, when position 1045 has a base C (SEQ ID NO: 24 (GeneBank, ID No. Y16581)).
  • the present invention relates to a method according to the present invention, wherein the polymorphism lies in codon 54 in position 1052, which generates a variant structural allele, when position 1052 has a base A (SEQ ID NO: 23 (GeneBank, ID No. Y16579)) or generates a normal structural allele, when position 1052 has a base G (SEQ ID NO: 24 (GeneBank, ID No. Y16581)).
  • the present invention relates to a method according to the present invention, wherein the polymorphism lies in codon 57 in position 1055, which generates a variant structural allele, when position 1055 has a base A (SEQ ID NO: 22 (GeneBank, ID No. Y16576)) or generates a normal structural allele, when position 1055 has a base G (SEQ ID NO: 20 (GeneBank, ID No. Y16576)).
  • the allele may be normal (see for example SEQ ID NO: 20, 21, 22 and 26), or it may be a variant, such as a structural variant or a non-structural variant.
  • structural allele is meant a polymorphic position situated in the protein coding part of the gene.
  • variant structural allele is meant a structural allele that differs from the wild-type structural allele by a substitution and/or a deletion and/or an insertion of a nucleotide or more nucleotides.
  • a wild type gene relates to a gene a functionally normal protein, whereas a variant gene encodes a variant protein with a function and/or structure that differs from that of a wild type protein.
  • structural variant is meant a polymorphism in the part of the MBL gene responsible for encoding the protein.
  • non-structural variant is meant an allele not associated with part of the MBL gene responsible for encoding the protein, such as a regulatory allele.
  • MBL melatonin-like growth factor-like cell sorting
  • An analogue is a compound (or molecule) that is a (chemical) structural derivative of MBL. It is also used to describe a molecule which may be structurally similar (but not identical) to another, and which exhibits many or some of the same biological functions of MBL.
  • An analogue is to be understood as being any peptide sequence capable of the same biological functions as wild-type MBL, including recombinant MBL.
  • the MBL genotype of an individual is determined according to at least two alleles by determining the presence of at least one structural allele of the MBL gene and/or the presence of at least one non-structural allele of the MBL gene of said individual.
  • the MBL gene is MBL2 (see SEQ ID NO: 20, 21, 22, 26).
  • Non-structural variant According to the invention in one aspect the non-structural variant is in the promoter region of the MBL gene.
  • non-structural variant is in the regulatory region of the MBL gene.
  • the non-structural allele may be selected among the following sequences:
  • the non-structural allele has a base C substitution in position 602 of SEQ ID NO: 26 (GeneBank, ID No. Y16580) or a base G substitution in position 602 of SEQ ID NO: 21 (GeneBank, ID No. Y16577).
  • the non-structural allele has a base C substitution in position 273 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base G substitution in position 273 of SEQ ID NO: 25 (GeneBank, ID No. Y16581).
  • the non-structural allele has a base A substitution in position 396 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base C substitution in position 396 of SEQ ID NO: 20 (GeneBank, ID No. Y16576).
  • the non-structural allele has a base A substitution in position 474 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base G substitution in position 474 of SEQ ID NO: 20 (GeneBank, ID No. Y16576).
  • the non-structural allele has a base A substitution in position 487 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base G substitution in position 487 of SEQ ID NO: 20 (GeneBank, Seq ID No. Y16576).
  • the non-structural allele has a deletion in base sequence AAAGAG in position 495-500 of SEQ ID NO: 21 (GeneBank, ID No. Y16577).
  • the non-structural allele has a base C substitution in position 753 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base T substitution in position 747 10 of SEQ ID NO: 20 (GeneBank, Seq ID No. Y16576).
  • the non-structural allele has a base C substitution in position 826 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or a base T substitution in position 820 of SEQ ID NO: 20 (GeneBank, ID No. Y16576).
  • At least one structural allele has a substitution in codon 54 in base G in position 1052 of SEQ ID NO: 21 (GeneBank, ID No. Y16577) or in base A in position 1052 of SEQ ID NO: 20 23 (GeneBank, ID No. Y16579).
  • the at least one structural allele has a substitution in codon 57 in base G in position 1055 of SEQ ID NO: 20 (GeneBank, ID No. Y16576) or in base A in position 1055 of SEQ ID NO: 22 (GeneBank, ID No. Y16578). 25
  • the at least one structural allele has a substitution in codon 52 in base C in position 1045 of SEQ ID NO: 24 (GeneBank, ID No. Y16581) or in base T in position 1045 of SEQ ID NO: 25 (GeneBank, ID No. Y16582).
  • the variety of the genotypes may be used in the determination of the risk factor of an individual of developing sepsis, severe sepsis, or septic shock.
  • the determination of the risk factor of an individual of 35 developing sepsis, severe sepsis, or septic shock may be applied to both healthy and sick individuals. It is envisioned that the above risk factor of an individual may be registered in a file, for example in a hospital for use in situations where an individual has been injured. The risk information would provide valuable information, which may be used in determining a treatment strategy of said individual.
  • the genotype comprises two normal structural alleles regulated by two high-expression promoters.
  • the genotype comprises two normal structural alleles regulated by two low-expression promoters.
  • high-expression promoters By the term “high-expression promoters” is meant promoter alleles associated with a high MBL protein expression/concentration.
  • low-expression promoters is meant promoter alleles associated with a low MBL protein expression/concentration.
  • the genotype comprises two normal structural alleles regulated by one low-expression promoter and one high-expression promoter.
  • the genotype comprises one normal structural allele regulated by one high-expression promoter and one variant structural allele.
  • the genotype comprises one normal structural allele regulated by one low-expression promoter and one variant structural allele.
  • the genotype comprises two variant structural alleles.
  • the nucleic acid sequences and/or the MBL concentration according to the present invention makes it possible to estimate sepsis risk in an individual.
  • the risk estimation is based on sequence polymorphism originating from specific regions of the MBL gene.
  • the MBL concentration may be determined using conventional methods mentioned further below.
  • sequence polymorphism is understood any single nucleotide, tandem repeat, insert, deletion or block polymorphism, which varies among humans, whether it is of biological importance or not.
  • the estimation of the risk of sepsis has a number of important applications: Companies, hospitals or other institutions can with the methods of the present invention offer a service to help individuals determine whether they belong to a risk group and if so provide treatment accordingly.
  • One possible application could be in an emergency situation, such as in an ambulance after an accident where emergency staff may determine the risk factor of the injured individual and provide a treatment accordingly.
  • the present invention concerns a method of determining the risk factor of a person of developing sepsis, severe sepsis or septic shock.
  • risk factor is expressed as an arbitrary risk value, which has been defined prior to determining the actual risk (likelihood) of an individual developing sepsis, severe sepsis, or septic shock.
  • One embodiment relates to various risk values associated with particular MBL genotypes.
  • the particular MBL genotype associated with a low risk value comprises two normal MBL structural alleles regulated by two high-expression promoters.
  • the particular MBL genotype associated with a medium-low risk value comprises two normal MBL structural alleles regulated by one low-expression promoter and one high-expression promoter.
  • the particular MBL genotype associated with a medium risk value comprises two normal MBL structural alleles regulated by two low-expression promoters.
  • the particular MBL genotype associated with a medium-high risk value comprises one normal structural MBL allele regulated by a high- expression promoter and one variant structural allele.
  • the particular MBL genotype associated with a high-risk value comprises one normal structural allele regulated by a low-expression promoter and one variant structural allele.
  • the particular MBL genotype associated with an ultra-high risk value comprises two MBL variant structural alleles.
  • nucleic acid from any nucleated cell can be used as the starting point for such assay techniques and may be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art.
  • allelic variation requires an amplification reaction and, a signal generation system and a mutation discrimination technique.
  • Table A lists a number of mutation detection techniques, some are based on the PCR technique. These may be used in combination with a number of signal generation systems a selection of which is listed in Table B. Further amplification techniques are listed in Table C.
  • variant alleles of the present invention may be subjected to amplification, thus a presently preferred embodiment relates to a method according to the present invention, wherein the at least one structural allele or the at least one regulatory allele is subjected to amplification.
  • the amplification may be performed by an amplification method selected from the group consisting of polymerase chain reaction (PCR), Ligase Chain Reaction (LCR), Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification, rolling circle amplification, and T7-polymerase amplification.
  • PCR polymerase chain reaction
  • LCR Ligase Chain Reaction
  • NASBA Nucleic Acid Sequence-Based Amplification
  • strand displacement amplification strand displacement amplification
  • rolling circle amplification rolling circle amplification
  • T7-polymerase amplification T7-polymerase amplification.
  • the amplification is performed by PCR.
  • the amplification is carried out by means of sequence-specific primers (SSP).
  • SSP sequence-specific primers
  • Table B illustrates various mutation detection techniques capable of being used for SNP detection.
  • Hybridisation Based techniques Solid phase hybridisation: Dot blot hybridization, MASDA, Reverse dot blot hybridization, Oligonucleotide arrays (DNA Chips)
  • Fluorescence Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation— United Kingdom Patent No. 2228998 (Zeneca Limited)
  • Preferred mutation detection techniques include ARMS.TM' or PCR-SSP, ALEX.TM., COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.
  • Particularly preferred methods include FRET, Taqman, ARMS.TM, or PCR-SSP and RFLP based methods.
  • the presence of the at least one variant allele is determined by hybridising a primer to a target nucleic acid sequence comprising at least one structural allele or at least one non-structural allele or at least one regulatory allele, or hybridising to any complementary sequence of said alleles.
  • the amplification is carried out by means of sequence- specific primers (SSP).
  • SSP sequence- specific primers
  • SIRS necessitate a fast and reliable overview of said individuals genetype in order to select and initiate the proper treatment in due time, thus it is an object of preferred embodiments of the present invention to provide the results as quickly as possible.
  • the invention relates to a method according to present invention, wherein said method is carried out within 168 hours of the initiation of the indications of SIRS, such as but not limited to 150 hours of the initiation of the indications of SIRS, 140 hours of the initiation of the indications of SIRS, 130 hours of the initiation of the indications of SIRS, 120 hours of the initiation of the indications of SIRS, 110 hours of the initiation of the indications of SIRS, 100 hours of the initiation of the indications of SIRS, 90 hours of the initiation of the indications of SIRS, 80 hours of the initiation of the indications of SIRS, 70 hours of the initiation of the indications of SIRS, 60 hours of the initiation of the indications of SIRS, 50 hours of the initiation of the indications of SIRS,48 hours of the initiation of the indications of SIRS, 36 hours of the initiation of the indications of SIRS, 24 hours of the initiation of the indications of SIRS, 20 hours of the initiation of the indications
  • the typing of the polymorphism is performed within at the most 5 hours, such as within at the most 4.5 hours, within at the most 4 hours, within at the most 3.5 hours, within at the most 3 hours, within at the most 2.5 hours, within at the most 2 hours, within at the most 1.5 hours, within at the most 60 minutes, within at the most 50 minutes, for example within at the most 40 minutes, such as within at the most 30 minutes, for example within at the most 20 minutes, such as at the most 10 minutes.
  • the PCR-SSP technique is used.
  • the mutation detection technique used is without an amplification step.
  • mutations or polymorphism's can be detected by using a microassay of nucleic acid sequences immobilized to a substrate or "gene chip” (see, e.g. Cronin, et al., 1996, Human Mutation 7:244-255).
  • nucleic acid reagents such as those described above may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).
  • the present invention may combine the result of MBL2 gene polymorphism within the codons 52, 54 and 57 with gene polymorphism's outside these codons in order to increase the probability of the significance of the correlation between MBL polymorphism's and sepsis risk.
  • the risk assessment of the invention may be conducted by means of at least one nucleic acid primer or probe, such as a primer or probe of DNA, RNA or a nucleic acid analogue such as peptide nucleic acid (PNA) or locked nucleic acid (LNA).
  • the nucleotide primer or probe is preferably capable of hybridising to a subsequence of the region corresponding to any of the MBL haplotype sequences, or a part thereof, or a complementary region thereof.
  • Such probes will be apparent to the molecular biologist of ordinary skill.
  • Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 bases in length.
  • such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the MBL gene.
  • one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected.
  • the probes of the invention may carry one or more labels to facilitate detection.
  • the amplification employs the following sequence-specific primers: 5' primer: AGTCGACCCAGATTGTAGGACAGAG (SEQ ID NO: l) 3' primer: CCTGGTTCCCCCTTTTCTC (SEQ ID NO: 5)
  • the amplification employs the following sequence-specific primers: 5' primer: AGTCGACCCAGATTGTAGGACAGAG (SEQ ID NO: l)
  • the amplification employs the following sequence-specific primers: 5' primer: AGTCGACCCAGATTGTAGGACAGAG (SEQ ID NO: l) 3' primer: TCCCTTGGTGCCATCACG (SEQ ID NO: 7)
  • the invention concerns in one aspect the amplification employing the following sequence-specific primers:
  • the invention discloses the amplification employing the following sequence-specific primers:
  • the amplification employs the following sequence-specific primers:
  • the amplification employs the following sequence-specific primers:
  • the amplification employs the following sequence-specific primers:
  • the amplification employs the following sequence-specific primers: 5' primer: TAGGACAGAGGGCATGCTC(SEQ ID NO: 15) 3' primer: AGGATCCAGGCAGTTTCCTCTGGAAGG (SEQ ID NO: 16)
  • the amplification employs the following sequence-specific primers: 5' primer: TAGGACAGAGGGCATGCTT (SEQ ID NO: 17)
  • the amplification employs the following sequence-specific primers: 5' primer: GAGTTTCACCCAC I I I I I CACA (SEQ ID NO: 18)
  • SEQ ID NO: 18 and SEQ ID NO: 19 are control primers.
  • a presently preferred embodiment of the present invention relates to a method according to the present invention, wherein the amplification employs the following sequence-specific primer pairs selected form the group consisting of at least one of:
  • One aspect of the invention concerns a method of predicting whether an individual will develop sepsis, severe sepsis, or septic shock comprising determining the concentration of MBL in a biological sample from an individual, such as a blood sample, for example a serum sample. The determination may in one aspect be performed directly in the blood stream of said individual. In a further aspect the MBL concentration is determined in a blood sample collected from said individual.
  • step c) predicting the risk of said individual of developing sepsis by correlating the MBL concentration of step b) with a predefined risk value associated with said particular MBL concentration.
  • any MBL concentration below 1.5 mg/l relates to a higher risk for said individual having SIRS developing sepsis, such as but not limited to below 1.45 mg/l, below 1.40 mg/l, below 1.35 mg/l, below 1.30 mg/l, below 1.25 mg/l, below 1.20 mg/l, below 1.15 mg/l, below 1.10 mg/l, below 1.05 mg/l, below 1.00 mg/l, below 0.95 mg/l, below 0.90 mg/l, below 0.85 mg/l, below 0.80 mg/l, below 0.75 mg/l, below 0.70 mg/l, below 0.65 mg/l, below 0.60 mg/l, below 0.50 mg/l, below 0.40 mg/l, below 0.35 mg/l, below 0.30 mg/l, below 0.25 mg/l, below 0.20 mg/l, below 0.15 mg/l, 0.10 mg/l or no detectable MBL concentration.
  • the invention relates to a method of predicting whether an individual will develop sepsis, severe sepsis, or septic shock comprising the steps of:
  • MBL mannose-binding lectin
  • step b) predicting the risk of said individual of developing sepsis, severe sepsis or septic shock by correlating the MBL genotype or the MBL concentration of step b) with a predefined risk value associated with said particular MBL genotype or said particular MBL concentration.
  • MBL genotype of an individual will be reflected in the level of MBL protein concentration in for example blood.
  • the MBL concentration is determined by the means of for example, but not exclusively, ELISA, RIA or TRIFMA.
  • the MBL concentration is determined by determining the activity of MASP (MBL associated serine proteases).
  • the biological sample is provided from an individual having at least one indication of systemic inflammation response syndrome (SIRS).
  • SIRS systemic inflammation response syndrome
  • the biological sample is provided from an individual having at least one indication of systemic inflammation response syndrome (SIRS), sepsis or severe sepsis.
  • SIRS systemic inflammation response syndrome
  • sepsis sepsis or severe sepsis.
  • SIRS criteria a core temperature of ⁇ 38°C or ⁇ 36°C; a heart rate of >90 beats/min; a respiratory rate of >20 breaths/min or a PaC0 2 of ⁇ 4.3 kPa (32 mm Hg) or a need of mechanical ventilation; a white blood cell count of >12.0 10 9 /l or ⁇ 4.0 10 9 /l or a differential count showing >10 percent immature neutrophils.
  • Sepsis criteria are SIRS with a documented infection or a clinically suspected infection as in postoperative intra- abdominal sepsis in which the intestinal tract is either perforated or required partial resection for ischemia, or intestinal content appeared intra-abdominally due to leak from prior gut anastomosis; or radiographic evidence of pneumonia in association with purulent sputum. Infection is considered documented by a positive culture or convincing gram stain no more than 3 days prior to admittance to intensive care. Severe sepsis is defined as sepsis and either hypotension or evidence of hypoperfusion and organ dysfunction developing within 24 hour of study enrolment.
  • MBL seems to be crucial in controlling systemic dissemination of different infectious agents in individuals with acute medical and surgical stress. This observation is also in agreement with the inventor's finding that an increased proportion of individuals carrying MBL variant alleles have a positive bacterial blood culture.
  • MBL may suppress the release of proinflammatory cytokines.
  • MBL may have both a direct antimicrobial role and may also have a modulating effect on the inflammatory response.
  • MBL has been shown to function as scavenger molecule towards cells undergoing apoptosis and necrosis.
  • the present invention provides a method for genetically determining the differences in the mbl2 gene and thereby develop a prognostic method useful in for example hospitals.
  • a rapid determination of a patient's MBL genotype is important in identifying individuals at risk of developing sepsis, severe sepsis or septic shock.
  • MBL substitution therapy now is possible is within the scope of the present invention to use MBL in prophylaxis and treatment of the sepsis syndrome.
  • One aspect of the invention relates to a method of predicting whether an individual having SIRS will develop sepsis comprising combining the two methods described in the present application.
  • the biological sample is body fluid, such as blood salvia, urine, faeces, cerebrospinal fluid, plasma and/or serum, for example the biological sample used in the present invention may be any suitable biological sample capable of providing the genetic material for use in the method.
  • the body fluid is blood.
  • the biological sample is a tissue sample, a sample of secretion, semen, ovum, a washing of a body surface (e.g. a buccal swap), a clipping of a body surface (hairs, or nails), such as wherein the cell is selected from white blood cells and tumour tissue.
  • the biological test sample may equally well be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR before use in the analysis of MBL variation.
  • the MBL variation analysis and the PCR are performed simultaneously, for example by using the PCR-SSP (ARMS.TM technique).
  • the present method comprises an additional step d) of assessing the risk of said individual of developing sepsis, severe sepsis or septic shock and accordingly determining a treatment profile for said individual.
  • At least one 5' primer selected from the group comprising the nucleic acid sequences as defined in SEQ ID NO. : l, SEQ ID NO. :3, SEQ ID NO. :9, SEQ ID NO. : ll, and SEQ ID NO. : 18.
  • At least one 3' primer selected from the group comprising the nucleic acid sequences as defined in SEQ ID NO. :2, SEQ ID NO. :4, SEQ ID NO. : 5, SEQ ID NO. :6, SEQ ID NO. :7, SEQ ID NO. :8, SEQ ID NO. -.10 and SEQ ID NO. : 19 for amplification of variant alleles of the MBL gene.
  • a prognostic kit comprising at least one prognostic primer of the invention and/or at least one allele-specific oligonucleotide primer or probe of the invention.
  • the prognostic kit may comprise appropriate packaging and instructions for use in the methods of the invention.
  • Such kit may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example Taq DNA polymerase.
  • a preferred kit may comprise means for amplifying the relevant sequence such as primers, polymerase, deoxynucleotides, buffer, metal ions; and/or means for discriminating the polymorphism, such as one or a set of primers or probes hybridising to the polymorphic site, a sequence reaction covering the polymorphic site, an enzyme or an antibody; and/or a secondary amplification system, such as enzyme-conjugated antibodies, or fluorescent antibodies.
  • the kit-of-parts preferably also comprises a detection system, such as a fluorometer, a film, an enzyme reagent or another highly sensitive detection device.
  • An antibody-based kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and optionally (2) a second different antibody which binds to either the polypeptide or to the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second different antibody which binds to either the polypeptide or to the first antibody and is conjugated to a detectable agent.
  • kits for predicting whether an individual will develop sepsis, severe sepsis or septic shock comprises:
  • At least one 5' primer selected from the group comprising the nucleic acid sequences as defined in SEQ ID NO. : l, SEQ ID NO.:3, SEQ ID NO. :9, SEQ ID NO. : ll, SEQ ID NO. : 12, SEQ ID NO. : 15, SEQ ID NO. : 17, and SEQ ID NO. : 18.
  • At least one 3' primer selected from the group comprising the nucleic acid sequences as defined in SEQ ID NO.:2, SEQ ID NO. :4, SEQ ID NO.:5, SEQ ID NO. :6, SEQ ID NO.:7, SEQ ID NO. :8, SEQ ID NO. : 10, SEQ ID NO.: 13, SEQ ID NO. : 14, SEQ ID NO. : 16, and SEQ ID NO. : 19 for amplification of variant alleles of the MBL gene.
  • the at least one primer of the kit comprises a detectable label.
  • the detectable may be selected from TEX, TET, TAM, ROX, R6G, ORG, HEX, FLU, FAM, DABSYL, Cy7, Cy5, Cy3, BOFL, BOF, BO-X, BO-TRX, BO-TMR, JOE, 6JOE, VIC, 6FAM, LCRed640, LCRed705, TAMRA, Biotin, Digoxigenin, DuO-family, and Daq-family.
  • the present kit comprises one or more reagents/materials for use in establishing the variant alleles of the MBL gene.
  • the present kit comprises in one aspect primers selected from SEQ ID NO.: l, SEQ ID NO. :3, SEQ ID NO. :9, SEQ ID NO. : ll, SEQ ID NO. : 12, SEQ ID NO. : 15, SEQ ID NO.: 17, SEQ ID NO. : 18, or variants thereof, or primers selected from the complementary strings of the above sequences.
  • the primer of the kit is selected from SEQ ID NO. : 2, SEQ ID NO. :4, SEQ ID NO. :5, SEQ ID NO. :6, SEQ ID NO. :7, SEQ ID NO. :8, SEQ ID NO. : 10, SEQ ID NO. : 13, SEQ ID NO. : 14, SEQ ID NO. : 16, SEQ ID NO. : 19, or variants thereof, or primers selected from the complementary strings of the above sequences.
  • the above primers have the "3 position in the polymorphic base of the sequences or their complementary strands.
  • the primer is operably linked to at least one label, such as operably linked to two different labels.
  • operably linked in the present context means that the primers can be linked to at least one label without the primer being impaired.
  • the label may be selected from, but is not limited to TEX, TET, TAM, ROX, R6G, ORG, HEX, FLU, FAM, DABSYL, Cy7, Cy5, Cy3, BOFL, BOF, BO-X, BO-TRX, BO-TMR, JOE, 6JOE, VIC, 6FAM, LCRed640, LCRed705, TAMRA, Biotin, Digoxigenin, DuO- family, and Daq-family.
  • the primer is operably linked to a surface, such as the surface is the surface of microbeads or a DNA chip or plastic.
  • the present invention provides a method for identifying an individual as having an increased likelihood of responding positively to a sepsis treatment, comprising determining the allele genotype correlated with an increased likelihood of positive response to treatment, whereby the presence of the genotype identifies the subject as having an increased likelihood of responding to sepsis treatment.
  • the treatment mentioned herein may be any sepsis treatment, such as conventional sepsis treatment, for example antibodies.
  • Gene products of the MBL gene or peptide fragments thereof can be prepared for a variety of uses.
  • such gene products, or peptide fragments thereof can be used for the generation of antibodies in prognostic assays.
  • the invention discloses the use of MBL for the manufacture of a medicament for the prevention and/or treatment of sepsis, severe sepsis, or septic shock.
  • the pharmaceutical of the invention comprises an effective amount of MBL in combination with pharmaceutically acceptable additives.
  • Such pharmaceutical may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration.
  • Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are for example water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, which enhance the effectiveness or transportation of the preparation.
  • Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.
  • an MBL based pharmaceutical is intravenous.
  • the application is subcutaneous and/or intramuscular.
  • a localised or substantially localised application may be performed.
  • the preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect.
  • Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations.
  • traditional binders and carriers include polyalkylene glycols or triglycerides.
  • Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
  • the active compound may be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are of the order 1 mg to 10 mg active ingredient per administration with a preferred range of from about 3 mg to 7 mg. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dose would be in the interval 1.0 mg to 10 mg per 70 kg body weight.
  • MBL according to the invention may be advantageous to administer MBL according to the invention with other substances to obtain a synergistic effect.
  • other substances may be a growth factor, which can induce cell differentiation, or a hormone, or a transplant of cells, including a transplant of stem cells, or gene therapy, or immuno- therapy.
  • Administration may be a continuous infusion, such as intra-ventricular infusion or administration in more doses, such as more times a day, daily, more times a week, or weekly.
  • administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to sepsis.
  • the medicament may be administered to an individual prior to an operation to prevent said individual to develop sepsis following the operation.
  • the invention relates to a method of treating an individual suffering from sepsis, septic shock or severe sepsis by administering a pharmaceutical composition comprising MBL to said individual.
  • the invention concerns a method of treating an individual suffering from sepsis, severe sepsis, or septic shock by administering to said individual a pharmaceutical composition comprising MBL to said individual.
  • LYPB/LYQC heterozygotes give a weak signal in rxn #1
  • LYPB/HYPD heterozygotes give a weak signal in rxn #5 reactions #1 and #2, #3 and #4, etc. are complementary Table 2
  • Non-operative medical including patients with no surgery latest 7 days
  • postoperative surgical diagnoses surgery within last 7 days
  • COPD chronic obstructive lung disease
  • G-I gastrointestinal
  • CNS • central nervous system.
  • SIRS systemic inflammatory response syndrome
  • A indicates normal structural allele.
  • Table 4a MBL genotypes, structural alleles, comparisons of patients without sepsis with sepsis, severe sepsis and septic shock in patients with inflammatory response syndrome (SIRS)
  • SIRS systemic inflammatory response syndrome
  • SAPS II Simplified Acute Physiology Score
  • SIRS systemic inflammatory response syndrome
  • A indicates the normal structural allele.
  • O is the common designation of the variant alleles ( ⁇ , codon 54, C, codon 57 and D, codon 52). Mean values or numbers. Brackets indicate ⁇ SD or %. * Without surgery latest 7 days.
  • APS acute physiology score
  • FIG. 1 MBL genotyping pattern. MBL genotyping by PCR- Sequence Specific Priming (SSP). The numbers 1-12 indicate the PCR reactions listed in table.
  • M Molecular weight marker, pBR327/Hae III.
  • Figure 2 Relationship between MBL serum concentrations and MBL genotypes. MBL serum concentrations in 272 patients with systemic inflammatory response syndrome (SIRS) are shown in relation to MBL structural alleles (O) as well as the MBL promoter alleles in position -221 (X/Y). Ranges, quartiles and medians are indicated. The detection limit in the assay is 20 ⁇ g/L.
  • SIRS systemic inflammatory response syndrome
  • Table 1 show the primers and the MBL haplotypes.
  • Table 2 shows the clinical diagnosis of ICU patients with SIRS.
  • Table 3 shows the MBL genotypes of SIRS patients.
  • Table 4 shows microbiological species in cultures taken from SIRS patients.
  • Table 5 shows MBL genotypes in individuals with or without sepsis.
  • Table 6 shows MBL genotypes in individuals with or without SIRS.
  • Table 7 shows a SAPS II score for patients having SIRS and with MBL structural variant alleles.
  • Table 8 Demographic and clinical variables in survivors and non-survivors.
  • SAPS II is based on a large international sample of medical and surgical patients and provides an estimate of the risk of death without having to specify a diagnostic category. The parameters included are listed in table 7. The worst values within the first 24 h after admission to the intensive care unit were recorded.
  • MBL concentrations in serum were measured in a double enzyme immuno-assay as previously described (Garred et al. 1992).
  • MBL single nucleotide polymorphism's (SNPs) in form of the structural vari ants named B (codon 54), C (codon 57), and D (codon 52) as well as the regulatory vari ants named H/L (-550), X/Y (-221), and P/Q (+4) were typed by a PCR and sequence sped fie primers (PCR-SSP) using the 12 reactions listed in table 1.
  • PCR-SSP sequence sped fie primers
  • Figure 1 shows seven patterns necessary for covering all combinations of each of the six complementary reactions. Although the typing was performed as SNP-typing the results were combined in haplotypes, based on strong linkage equilibrium between the SNPs. All three structural alleles ( ⁇ , C, and D) have a considerable effect on MBL concentrations and to avoid small groups, the three alleles were grouped in one category (called allele "O") for statistical analyses. The normal allele is designated A.
  • the A/A group two normal structural alleles with high-expression promoter activity in position -221 (YA/YA) or one high-expression promoter and one low-expression promoter (YA/XA) or two low-expression promoters (XA/XA);
  • the A/0 group one variant structural allele (i.e. defective allele) and one normal structural allele regulated by a high-expression promoter (YA/0) or a low- expression promoter (XA/0) and the 0/0 group with two defective structural alleles.
  • MBL seems to be crucial in controlling systemic dissemination of different infectious agents in patients with acute medical and surgical stress. This observation is also in agreement with the finding that a significant increased proportion of patients carrying MBL variant alleles also had a positive bacterial blood culture.
  • MBL exert its largest effect during the vulnerable window of infancy between 6 and 18 months of age. Nevertheless, MBL deficiency has been associated with a number of infections, particular in patients with concomitant immunodeficiencies.
  • the initial SIRS insult creates a precondition rendering the patient partly immunocompromised. This increases the patient's susceptibility for infection, thereby exposing the clinical MBL phenotype.
  • the necessity that an accompanying condition has to be present before MBL deficiency becomes clinically important has recently been indicated in relation to pneumococcal pneumonia. In unselected patients a variable association is seen, while in selected patients with a concomitant disorder a clear association is revealed.
  • MBL may suppress the release of proinflammatory cytokines.
  • MBL may have both a direct antimicrobial role and it may also have a modulating effect on the inflammatory response.
  • MBL has been shown to function as scavenger molecule towards cells undergoing apoptosis and necrosis. Lack of functional MBL could also very well play a role in the development of sepsis severity.
  • Diallelic polymorphism may explain variations of blood concentration of mannan-binding protein in Eskimos, but not in black Africans. Ear J Immunogenetics. 1992;19:403-412.

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Abstract

La présente invention se rapporte à un procédé destiné à prédire si une personne atteinte du syndrome de réponse inflammatoire systémique (SRIS) contractera une sepsie, et ce par la mise en corrélation du génotype MBL avec une estimation du risque prédéfinie associée au génotype MBL en question. Dans le présent contexte, une estimation du risque prédéfinie associée au génotype MBL en question peut se diviser en un risque élevé et un risque faible que ladite personne atteinte du SRIS contracte une sepsie. En général, un génotype MBL présentant un risque élevé est caractérisé en ce qu'il possède au moins un allèle structurel variant du gène MBL et/ou possédant deux allèles régulateurs à faible expression du gène MBL dans les échantillons prélevés.
PCT/DK2004/000027 2003-01-17 2004-01-16 Procede permettant de pronostiquer une sepsie WO2004065626A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107377A2 (fr) * 2007-03-02 2008-09-12 Innogenetics N.V. Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2010041232A2 (fr) * 2008-10-09 2010-04-15 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Procédé d’estimation du risque de sepsie chez un individu ayant une infection

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107377A2 (fr) * 2007-03-02 2008-09-12 Innogenetics N.V. Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2008107377A3 (fr) * 2007-03-02 2008-11-27 Innogenetics Nv Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2010041232A2 (fr) * 2008-10-09 2010-04-15 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Procédé d’estimation du risque de sepsie chez un individu ayant une infection
WO2010041232A3 (fr) * 2008-10-09 2010-06-03 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Procédé d’estimation du risque de sepsie chez un individu ayant une infection

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