[go: up one dir, main page]

WO2017037477A1 - Marqueur biologique de sepsis - Google Patents

Marqueur biologique de sepsis Download PDF

Info

Publication number
WO2017037477A1
WO2017037477A1 PCT/GB2016/052724 GB2016052724W WO2017037477A1 WO 2017037477 A1 WO2017037477 A1 WO 2017037477A1 GB 2016052724 W GB2016052724 W GB 2016052724W WO 2017037477 A1 WO2017037477 A1 WO 2017037477A1
Authority
WO
WIPO (PCT)
Prior art keywords
mirna
concentration
sepsis
sirs
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2016/052724
Other languages
English (en)
Inventor
Stefano Caserta
Martin LLEWELYN
Sarah NEWBURY
Florian Kern
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Sussex
Original Assignee
University of Sussex
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Sussex filed Critical University of Sussex
Publication of WO2017037477A1 publication Critical patent/WO2017037477A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to biological markers for distinguishing between sepsis and SIRS, and in particular to the use of micro RNAs as diagnostic markers that may be used to distinguish between sepsis and SIRS.
  • the invention extends to methods and kit which detect for such diagnostic micro RNAs for distinguishing between sepsis and SIRS.
  • Sepsis is defined as the systemic inflammatory response syndrome (SIRS) initiated by infection ⁇ ]. Severe sepsis (sepsis accompanied by acute organ dysfunction) is a leading cause of death worldwide ( ⁇ 19 million deaths/year) and the most common cause of death (30% mortality rate) among patients on Intensive Care Units (ICUs)[2]. Research over the past three decades has focused primarily on the inflammatory responses that underlie sepsis. Biomarkers of inflammation ⁇ ] have been identified but investigational treatments which attempt to 'switch-off inflammation in sepsis have uniformly failed to improve patient outcomes. More recently there has been a growing recognition that anti-inflammatory, regulatory mechanisms accompany sepsis [4]. These are
  • MicroRNAs are small ( ⁇ 22nt) regulatory RNAs that function as post- transcriptional gene regulators[6,2]- In 2003, only 255 human miRNAs were predicted to exist[8,s], but the number of known human miRNAs had risen dramatically to 2588 by 2014 (miRBase.org[io]). In 2008, it was demonstrated for the first time that miRNAs could be identified circulating in blood[ii-i3]. It is now thought 100-200 miRNAs may be detectable in the The impact of disease on circulating miRNAs has been assessed principally in the context of cancer[ 11,16,12]. A handful of studies have measured miRNAs present in blood of sepsis patients, and reported conflicting findings [18 ⁇ 25].
  • a method for distinguishing between sepsis and SIRS in a subject comprising analysing the concentration of one or more type of microRNA molecule in a bodily sample from a test subject and comparing this concentration with:-
  • concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference suggests that the subject is suffering from SIRS, and wherein if there is no difference in the concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference then this suggests that the subject is suffering from sepsis; and/ or
  • concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference suggests that the subject is suffering from sepsis, and wherein if there is no difference in the
  • concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference then this suggests that the subject is suffering from SIRS.
  • kits for distinguishing between sepsis and SIRS in a subject comprising: -
  • RNA molecule in a sample from a test subject RNA molecule in a sample from a test subject
  • the kit is used to identify a difference in the concentration of the one or more type of microRNA molecule in the sample from the test subject compared to the reference concentration, thereby suggesting that the test subject suffers from SIRS, or wherein the kit is used to determine that there is no difference in the concentration of the one or more type of micro RNA molecule in the sample from the test subject compared to the reference concentration, thereby suggesting that the test subject suffers from sepsis, and/or a reference for the concentration of the one or more type of microRNA molecule in a sample from an individual who suffers from SIRS, wherein the kit is used to identify a difference in the
  • the kit is used to determine that there is no difference in the concentration of the one or more type of microRNA molecule in the sample from the test subject compared to the reference concentration, thereby suggesting that the test subject suffers from SIRS.
  • a method of treating an individual suffering from sepsis comprising the steps of:
  • a method of treating an individual suffering from SIRS comprising the steps of:
  • the inventors have identified six CIR- miRNAs that that are highly discriminatory for sepsis from SIRS having AUCs by ROC analysis comparable or better than clinical biomarkers, C-reactive protein (CRP) and procalcitonin (PCT). Notably, they found that CIR-miRNA levels correlate inversely with pro-inflammatory biomarkers. More importantly, the inventors have found that the levels of some CIR-miRNAs are differentially affected in sepsis and non-infective SIRS.
  • CRP C-reactive protein
  • PCT procalcitonin
  • CIR-miRNAs inversely correlate with the plasma levels of key pro-inflammatory mediators such as IL-i, IL-6, IL-8 and C-reactive protein (CRP), which have previously identified as increased in systemic inflammation and sepsis [3,34].
  • the method according to the first aspect and the kit according to the second aspect are useful for enabling a clinician to make decisions with regards to the best course of treatment for a subject who is currently or who may suffer from either sepsis or SIRS in the future. It is preferred that the method of the first aspect or the kit according to the second aspect is useful for enabling a clinician to decide how to treat a subject who is suffering from sepsis or SIRS, according to the methods of the third and fourth aspects. The methods and the kit are therefore very useful for guiding a SIRS or sepsis treatment regime for the clinician.
  • the clinician may use the kit of the invention in conjunction with existing diagnostic tests to improve the accuracy of diagnosis.
  • Micro RNA molecules are non-coding, post-transcriptional regulators that normally bind to complementary sequences in the 3' untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing.
  • miRNAs are short ribonucleic acid (RNA) molecules, on average only about 22 nucleotides long.
  • the miRNA detected in the methods and kit of the invention may be about 15 to 30 nucleotides long, or about 18 to 25 nucleotides long, or about 21 to 23 nucleotides long.
  • the methods and kit of the invention may comprise analysing the concentration of any of the known miRNAs, and which can be found on the miRBase website (http://www.mirbase.org/).
  • the miRBase (release 21) currently includes 2588 mature human miRNAs, all of which are processed from longer precursors and differ from each other in nucleotide sequence.
  • the current understanding is that each miRNA is expressed in one or more human tissues and binds to one or more target RNA sequences expressed in particular tissues.
  • the methods according to the invention may comprise analysing the concentration of one or more type of microRNA molecule selected from the group of miRNA molecules consisting of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p, and the kit according to the invention may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • microRNA as a biomarker for distinguishing between sepsis and SIRS in a subject
  • the one or more type of microRNA molecule is selected from the group of miRNA molecules consisting of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p.
  • the inventors have found that three of the miRNA molecules (miRNA-3od-5p, miRNA- 30a-5p and miRNA-i92-5p) described herein act as particularly robust biomarkers for distinguishing between sepsis and SIRS, and form preferred embodiments of the invention.
  • miRNA-3od-5p as a biomarker for distinguishing between sepsis and SIRS in a subject, optionally wherein one or more additional microRNA molecule is used and is selected from the group of miRNA molecules consisting of miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a- 3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p.
  • miRNA-3oa-5p as a biomarker for distinguishing between sepsis and SIRS in a subject, optionally wherein one or more additional micro RNA molecule is used and is selected from the group of miRNA molecules consisting of miRNA-3od-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a- 3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p.
  • miRNA-i92-5p as a biomarker for distinguishing between sepsis and SIRS in a subject, optionally wherein one or more additional micro RNA molecule is used and is selected from the group of miRNA molecules consisting of miRNA-30a-5p, miRNA-3od-5p, miRNA-26a-5p, miRNA- 23a- 3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p and miRNA-i9i-5p, and optionally at least one of miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p, and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, and optionally at least one of miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-101- 3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- 15 ia-3p, miRNA- I46a-5p and let-7f-5p, , and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-3od-5p, and optionally at least one of miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p, and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-30a-5p, and optionally at least one of miRNA-3od-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA-i5ia-3p, miRNA-i46a-5p and let-7f-5p, and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-i92-5p, and optionally at least one of miRNA-3od-5p, miRNA-30a-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA-i5ia-3p, miRNA-i46a-5p and let-7f-5p, and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • the best normalizer for the dataset was a combination of miR320a and miR486-5p.
  • the methods and uses of the invention may further comprise analysing the concentration of miR320a and/or miR486-5p and then adjusting the concentration of the one or more type of microRNA molecule being detected in the bodily sample before comparing the detected concentration against the reference value
  • the kit according to the invention may comprise a means for normalising the concentration of the one or more type of microRNA molecule in a test sample with respect to miR320a and/or miR486-5p.
  • miRNA-3od-5p miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA-i5ia-3p, miRNA-i46a-5p and let-7f-5p provide a very robust and reliable diagnosis when distinguishing between SIRS and sepsis.
  • the methods and uses of the invention may comprise analysing the concentration of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA-i5ia-3p, miRNA-i46a-5p and let-7f-5p, and the kit may comprise a means for analysing the concentration of the one or more type of microRNA molecule.
  • these miRNAs are very useful for generating a circulating inflammation-related miRNAs (CIR-miRNA) score.
  • CIR-miRNA circulating inflammation-related miRNAs
  • Xi -6 are the measurements of the top 6 miRNAs in a specific individual and the variables, a -e, and the constant, k, are the coefficients returned by the binary logistic regression model.
  • the miRNA being detected is miRNA-30a-5p.
  • the sequence of miRNA-30a-5p is 22 nucleotides long, and is referred to herein as SEQ ID No.i, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.i, or the complementary sequence thereof, or a variant or fragment thereof.
  • the miRNA being detected is miRNA-3od-5p.
  • the sequence of miRNA"3od is, 22 nucleotides long, and is referred to herein as SEQ ID No.2, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.2or a variant or fragment thereof.
  • the miRNA being detected is miRNA-i92-5p.
  • the sequence of miRNA-192 is 21 nucleotides long, and is referred to herein as SEQ ID N0.3, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID N0.3, or a variant or fragment thereof.
  • the miRNA being detected is miRNA-26a-5p.
  • the sequence of miRNA-26a-5p is 22 nucleotides long, and is referred to herein as SEQ ID N0.4, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID N0.4 or a variant, or fragment thereof.
  • the miRNA being detected is miRNA-23a.
  • the sequence of miRNA-23a-3p is 21 nucleotides long, and is referred to herein as SEQ ID N0.5, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID N0.5, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA-i9i-5p.
  • the sequence of miRNA-i9i-5p is 23 nucleotides long, and is referred to herein as SEQ ID No.6, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.6, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA-ioi-3p.
  • the sequence of miRNA-ioi-3p is 21 nucleotides long, and is referred to herein as SEQ ID N0.7, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.7, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA-i22-5p.
  • the sequence of miRNA-i22-5p is 22 nucleotides long, and is referred to herein as SEQ ID No.8, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.8, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA-378a-3p.
  • the sequence of miRNA-378a-3p is 21 nucleotides long, and is referred to herein as SEQ ID N0.9, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.9, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA- I5ia-3p.
  • the sequence of miRNA- I5ia-3p is 21 nucleotides long, and is referred to herein as SEQ ID No.10, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.10, or a variant, or fragment thereof.
  • the miRNA being detected is miRNA- I46a-5p.
  • the sequence of miRNA- I46a-5p is 22 nucleotides long, and is referred to herein as SEQ ID No.11, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.11, or a variant, or fragment thereof.
  • the miRNA being detected is let-7f-5p.
  • the sequence of let-7f-5p is 22 nucleotides long, and is referred to herein as SEQ ID No.12, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.12, or a variant or fragment thereof.
  • sequence of miR32oa is 22 nucleotides long, and is referred to herein as SEQ ID No.13, as follows:
  • sequence of miR486-5p is 22 nucleotides long, and is referred to herein as SEQ ID No.14, as follows:
  • miRNAs of the invention have a hairpin loop structure.
  • the nucleotide sequence of certain micro RNAs according to the invention are located 5' (-5p) of the hairpin loop (i.e. miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA-i9i-5p, miRNA-i22-5p, miRNA- I46a-5p, let- 7f-5P and miR486-5p), whereas the nucleotide sequence of the remaining microRNAs is located 3' (-3p) of the hairpin loop (i.e. miRNA-23a-3p, miRNA-378a-3p and miRNA-i5ia-3p).
  • the methods uses of the invention may comprise determining the concentration of one or more type of microRNA molecule selected from the group of miRNA molecules comprising a nucleotide sequence substantially as set out in any or SEQ ID No.i to 12, or variants or fragments thereof, and the kit according to the invention may comprise a means for determining the concentration of the one or more type of microRNA molecule.
  • the methods and uses of the invention may comprise determining the concentration of miRNA320a (SEQ ID No:i3) and/or miR486-5p (SEQ ID No:i4) in a bodily sample and then adjusting the concentration of the one or more type of microRNA molecule being detected in the bodily sample with respect to the level of expression of miR320a and/or miR486-5p before comparing the detected
  • miRNA-3od-5p The pattern of expression of miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and/ or let-7f-5p are found at either significantly higher or significantly lower levels in a bodily sample compared to a test subject (e.g. plasma or serum from peripheral blood) maybe termed the "miRNA signature".
  • a test subject e.g. plasma or serum from peripheral blood
  • miR320a SEQ ID No:i3
  • miR486-5p SEQ ID No:i4
  • the biomarker is one or more microRNAs selected from miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p.
  • Variants and fragments of any of the miRNA molecules that may be detected may include truncations or additions of nucleotides of the miRNA molecule, for example SEQ ID No.1-12.
  • a truncation may comprise the miRNA molecule having been reduced in size by the removal of at least one nucleotide from the 5' and/or 3' end of the miRNA, or by deletion of one of more nucleotides from within the core or centre of the miRNA.
  • the truncation may comprise deletion of at least 2, 3, 4 or 5 nucleotides from the miRNA molecule.
  • An addition may comprise the miRNA molecule having been increased in size by the addition of at least one nucleotide to the 5' and/or 3' end of the miRNA, or by the introduction of one of more nucleotides into the core or centre of the miRNA.
  • the addition may comprise addition of at least 2, 3, 4, 5, or up to 10
  • nucleotides to the miRNA molecule are nucleotides to the miRNA molecule.
  • the concentration of the at least one type of miRNA molecule may act as a diagnostic and/or prognostic marker for sepsis or SIRS.
  • the inventors investigated the expression levels of a large number of miRNA molecules in sepsis and SIRS patients, and were surprised to observe that a number of miRNAs (i.e.
  • miRNA-3od-5p miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-i9i-5p, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p) exhibited increased levels in SIRS than in sepsis patients.
  • This pattern of increased expression can be used to form a miRNA signature.
  • the inventors therefore realised that these miRNA molecules, which together form a miRNA signature, represents a useful and robust physiological marker for distinguishing between a patient suffering from sepsis and a patient suffering from SIRS.
  • each of these biomarkers can be robustly used for prognostic and diagnostic purposes.
  • the inventors have established that circulating levels of miRNAs in a test subject is highly suggestive of whether the subject suffers from either sepsis or SIRS, and is sufficiently sensitive to detect the disorder at an early stage. Accordingly, the kit and methods the invention provides a very reliable prognostic marker for monitoring conditions, both before and after treatment. Accordingly, assaying for miRNA molecules is a substantial improvement over assaying for other markers, because it is more sensitive and also provides enhanced specificity. In addition, assaying for miRNA molecules also provides far more information to the clinician, and will help stratify the disease, be that either sepsis or SIRS.
  • detecting one particular type of miRNA molecule may be of use by itself as a biomarker for distinguishing between sepsis and SIRS. Further, detecting more than one type of miRNA molecule, may provide a more robust diagnosis or prognosis of the disease.
  • the biomarker may also be used in combination with an assay of another biological marker indicative of sepsis or SIRS. Hence, assaying for one or more miRNA molecules may be used to complement the use of another marker to provide even more information to the clinician.
  • the subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc. However, it is preferred that the subject is a mammal, such as a human, either male or female.
  • a sample is taken from the subject, and the concentration of the one or more type of miRNA molecule may be measured.
  • the kit of the second aspect may comprise sample extraction means for obtaining the sample from the test subject.
  • the sample extraction means may comprise a needle or syringe or the like.
  • the kit comprises one or more microRNAs for normalising the expression of levels of the biomarker in the sample. More preferably, the micro RNA for normalising the expression levels of the biomarker in the sample is miRNA320a and/or miR486-5p.
  • the sample may be any bodily sample into which miRNA molecules are secreted, e.g. it maybe lymph or interstitial fluid.
  • the sample maybe a urine sample or a blood sample. It is preferred that the miRNA molecule is measured or assayed in a blood sample.
  • the blood sample may be venous or arterial.
  • the kit may comprise a sample collection container for receiving the extracted sample.
  • Blood samples may be assayed for miRNA molecule levels immediately.
  • the blood may be stored at low temperatures, for example in a fridge or even frozen before the miRNA assay is conducted. Measurement of miRNA may be made on whole blood.
  • the blood maybe further processed before the assay is performed.
  • an anticoagulant such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added.
  • the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.
  • the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which maybe used for analysis.
  • the miRNA is analysed or assayed in a blood plasma or a blood serum sample. It is preferred that miRNA concentration is measured in vitro from a blood serum sample or a plasma sample taken from the subject.
  • freshness samples may be analysed immediately after they have been taken from a subject.
  • the serum or plasma samples may be frozen and stored. The sample may then be de-frosted and analysed at a later date.
  • the inventors monitored the concentration of various miRNAs in numerous patients who suffered from either sepsis or SIRS, and compared them to the concentration of the same miRNAs in individuals who did not suffer from either condition. They demonstrated that there was a statistically significant increase or decrease in the concentration of certain miRNA molecules described herein in the patients suffering from sepsis or SIRS. Thus, the difference in concentration maybe an increase or a decrease compared to the reference taken from individuals who do not suffer from either condition. It will be appreciated that the concentration of a certain miRNA molecule in sepsis or SIRS patients is highly dependent on a number of factors, for example how far the disease has progressed, and the age and gender of the subject.
  • the concentration of miRNAs in individuals who do not suffer from sepsis or SIRS may fluctuate to some degree, but that on average over a given period of time, the concentration tends to be substantially constant.
  • the concentration of miRNA in one group of individuals who do not suffer from, for example, sepsis maybe different to the concentration of those miRNAs in another group of individuals who do not suffer sepsis.
  • the skilled technician will know how to determine the average concentration of certain miRNAs in individuals who suffer from either sepsis or SIRS, and this is referred to as the 'normal' concentration of miRNA for the disease.
  • the normal concentration corresponds to the reference values discussed above in the first to third aspects.
  • the miRNAs may be extracted from the bodily sample by a variety of techniques.
  • these may comprise addition of a protein denaturant (such as Trizol or guanidine thiocyanate) to the sample, centrifugation to remove protein debris, addition of DNasel to remove DNA, and extraction of RNA using a suitable column.
  • RNA samples maybe further concentrated by ethanol/isopropanol precipitation and/or centrifugal concentration.
  • the preferred extraction kit is supplied by Ambion, but other extraction kits could be used, depending on availability and/ or suitability in subsequent downstream reactions.
  • PCR may be used to amplify the one or more type of miRNA molecule.
  • the PCR technology may be selected from the group consisting of real-time PCR, reverse transcriptase PCR, multiplex PCR or molecular beacon PCR. It will be appreciated that PCR involves the use of two primers which are substantially complementary to the miRNA molecule being assayed in the sample.
  • the kit according to the second aspect comprises means for determining the concentration of one or more type of miRNA molecule in a sample from a test subject.
  • the kit may comprise a container in which the means for determining the concentration of one or more type of miRNA molecule in a sample from a test subject maybe contained.
  • the kit may also comprise instructions for use.
  • the kit may comprise detection means for determining the concentration of the one or more type of miRNA in the sample once this has been obtained from the subject.
  • the detection means may comprise one or more primer, for use in a PCR method for amplifying the miRNA.
  • detection of the one or more type of miRNA molecule may be achieved by TaqMan quantitative RT-PCR using primer and probe sets specific for particular human miRNAs, as described on the Applied Biosystems website
  • detection may be achieved using an Exiqon micro RNA detection kit. (http://www.exiqon.com/ls).
  • Exiqon micro RNA detection kit http://www.exiqon.com/ls.
  • other PCR-based and microarray- based detection methods are also applicable to this invention.
  • the primers may comprise at least partial sequence identity with the miRNA molecule being detected, for example, miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-191-5, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- 151a- 3p, miRNA- I46a-5p and/or let-7f-5p.
  • miRNA-3od-5p miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-191-5, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- 151a- 3p, miRNA- I46a-5p and/or let-7f-5p.
  • the Reverse Transcriptase and PCR reactions may comprise the procedure as set out in Examples.
  • the reference values may be obtained by assaying a statistically significant number of control samples (i.e. samples from subjects who suffer from sepsis but do not suffer from SIRS or vice versa). Accordingly, the reference (ii) according to the kit of the second aspect of the invention maybe a control sample (for assaying).
  • the kit may comprise a positive control (preferably provided in a container), which corresponds to total RNA extracted from a sample (e.g. the plasma) of a subject having, for example, sepsis where it has been established that the relevant miRNAs (for example, miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-191-5, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- 151a- 3p, miRNA- I46a-5p and let-7f-5p) are present at statistically higher or lower levels than those present in a subject suffering from SIRS.
  • a positive control preferably provided in a container
  • the positive control maybe total RNA extracted from a sample of a subject having SIRS, where it has been established that the relevant miRNAs (for example, miRNA-3od-5p, miRNA-30a-5p, miRNA-i92-5p, miRNA-26a-5p, miRNA- 23a-3p, miRNA-191-5, miRNA-ioi-3p, miRNA-i22-5p, miRNA-378a-3p, miRNA- I5ia-3p, miRNA- I46a-5p and let-7f-5p) are present at statistically higher or lower levels than those present in a subject suffering from sepsis.
  • the positive control miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.1-12, or a variant, or fragment thereof.
  • the kit may comprise a negative control (preferably provided in a container), which corresponds to total RNA extracted from a sample (e.g. the plasma) of a subject without sepsis or SIRS where it has previously been established that the above miRNAs are detectable at significantly lower or higher levels.
  • a negative control preferably provided in a container
  • the kit may comprise the reference, a positive control and a negative control.
  • the kit will also comprise further controls, as necessary, such as “spike-in” controls to provide a reference for concentration, and further positive controls for each of the "signature" micro RNAs.
  • the blood plasma concentration of the signature miRNA in sepsis patient may not be detectable, whereas the concentration of certain signature miRNAs in a patient with SIRS may be at least 1.5-, 5-, 10, 15- or 20-fold higher (or vice versa, in terms of sepsis and SIRS).
  • the decrease in concentration of certain signature miRNAs in sepsis maybe at least 1.5- 5-, 10, 15- or 20-fold lower than a SIRS patient (or vice versa, in terms of sepsis and SIRS).
  • concentration and therefore infer whether that subject is suffering from sepsis or SIRS.
  • statistical significance is found at 10%.
  • the preferred statistical significance value is 5%.
  • the increase in concentration of miRNA compared to the 'sepsis or SIRS' concentration may be at least 1.5-, 5-, 10-, 15- or 20-fold higher than the 'normal' or reference concentration.
  • the decrease in concentration of miRNA compared to the 'normal' concentration may be at least 1.5-, 5-, 10-, 15- or 20-fold lower than the 'normal' or reference concentration.
  • concentration infer that the test subject is suffering from either SIRS or sepsis is suffering from either SIRS or sepsis.
  • a clinician would be able to make a decision as to the preferred course of treatment required, for example the type and dosage of the therapeutic agent according to the third aspect to be administered.
  • nucleic acid or variant, derivative or analogue thereof which comprises substantially the nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • substantially the nucleotide sequence can be a sequence that has at least 40% sequence identity with the nucleotide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleotide identified as SEQ ID No: i (i.e. miRNA-30a) or SEQ ID No.2 (i.e. miRNA-3od), and so on, for all of the miRNAs described herein.
  • nucleotide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the nucleotide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two nucleotide sequences.
  • an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • the pair-score matrix e.g. BLOSUM62, PAM250, Gonnet etc.
  • gap-penalty e.g. functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs.
  • percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process.
  • ClustalW The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention.
  • calculation of percentage identities between two nucleotide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1-12, or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/ sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences described herein. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • Figure 1 is a series of 12 graphs showing the results of patients whose plasma was tested for miRNAs in Illumina next generation sequencing (NGS).
  • Plasma total RNA was extracted from 10 pools (representative of 89 ICU patients, as in Table 1) using the miRVana PARIS technology and then human miRNAs were sequenced using the Illumina next generation sequencing (NGS) platform.
  • A. Representative plots show the number of blood miRNAs (x-axis, sorted based on their abundance in the first duplicate of SIRS) and relative NGS counts (y-axis), in SIRS, sepsis and no-SIRS patients. Many miRNA were expressed below 1/10 5 NGS counts (orange shadowed areas) consistently across all pools and were excluded from further analysis.
  • B Representative plots show the number of blood miRNAs (x-axis, sorted based on their abundance in the first duplicate of SIRS) and relative NGS counts (y-axis), in SIRS, sepsis and no-SIRS patients. Many miRNA were expressed below
  • C miRNA counts in 2 identical replicates are shown in scatter plots for SIRS, sepsis and no-SIRS patients. Reproducible results were obtained for miRNAs with NGS counts>io/io 5 (red lines) and miRNA in the grey area were excluded.
  • FIG. 2 is two graphs that show the results of shortlisted internal normalizers in NGS and miRNA Q-PCR arrays.
  • A. Among the finally shortlisted miRNAs (miR320a, miR92-3p and miR486-5p), the fold-differences (fd) of average NGS counts seen in severe and non-severe sepsis and SIRS groups (8 pools representative of 73 individuals) relative to no-SIRS controls (2 duplicate pools, n 16) are shown.
  • fd fold-differences
  • FIG. 3 show the results of shortlisted CIR-miRNAs measured with Exiqon miRNA qPCR arrays.
  • Cp of a single miRNA is compared to the mean Cp of 2 normalizers (as from Figure 2) to give delta- Cp (dCp).
  • A. Volcano plot shows fold changes (log2, D/A) relative to p values (-logio) in each miRNA assay.
  • FIG 4 is a series of 14 graphs which show that CIR-miRNAs are good-to-excellent biomarkers of sepsis.
  • miRNA qPCR arrays data was analyzed as in Figure 3 and the top-6 differentially expressed miRNA in sepsis compared to SIRS (after the Benjamini- Hochberg correction) are shown.
  • the relative receiver operator curve (ROC, right) is shown with the Area Under the Curve (AUC).
  • Each of the top 6 significant CIR-miRNAs is a good-to-excellent biomarker and CIR-miRNAs were mostly downregulated in Sepsis compared to SIRS in Exiqon miRNA qPCR arrays.
  • B. A model combines the top-6 significant CIR-miRNAs to maximize distinction between SIRS and sepsis. The CIR- miRNA score is directly related to the odds of having SIRS or sepsis given the measurements of the 6 top miRNAs (see Material and Methods for further details).
  • Left dot plot shows the model interpolation of the experimental cohort: SIRS patients -that have high CIR-miRNA levels (in A)- tend to score>o, whilst sepsis patients tend to score ⁇ o.
  • ROC and AUC, right shows that the 6 CIR-miRNAs combined outperformed single miRNAs.
  • Figure 5 are a series of eight graphs which show the correlation of the model scores with pathology scores and plasma levels of immune mediators relevant in sepsis and SIRS.
  • the model scores that combine the top-6 CIR-miRNA measurements in Severe SIRS and Severe Sepsis patients were plotted against the pathology score (SOFA, sequential organ failure assessment); markers of disease and inflammation such as Hb (free hemoglobin), CRP (C-reactive protein), and PSP (pancreatic soluble protein); and markers of immune cell activation: soluble CD25 (SCD25), IL-6 IL-8 and IL-i.
  • SOFA sequential organ failure assessment
  • markers of disease and inflammation such as Hb (free hemoglobin), CRP (C-reactive protein), and PSP (pancreatic soluble protein)
  • markers of immune cell activation markers of immune cell activation: soluble CD25 (SCD25), IL-6 IL-8 and IL-i.
  • Figure 6 shows the proposed model for CIR-miRNA and inflammatory mediator plasma levels.
  • the triangular shapes represent plasma levels of CIR-miRNA
  • FIG. 7 is two graphs that show the average hemoglobin levels in an experimental cohort in which of hemolyzed samples have been excluded.
  • Hb levels are shown in any experimental group used in NGS and miRNA Q-PCR array, after the exclusion of outliers. Importantly, average Hb did not differ significantly across groups, suggesting that RBC lysis is equally represented across the experimental groups prior to the NGS analysis. RBCs may be responsible for miRNA presence in the blood.
  • FIG. 8 is a graph that showing an independent validation of hemolysis levels in miRNA qPCR arrays.
  • Figure 7 the qPCR platform confirmed similar levels of hemolysis across the groups and only 1 patient sample in the severe sepsis group was deemed to be excluded from further analysis.
  • the inventors set out to measure miRNAs present in blood of patients with critical illness categorized on the basis of having sepsis or non-infective systemic SIRS, in comparison with control patients having critical illness without a systemic
  • Example 1 the different patient populations were established.
  • Example 2 next generation sequencing (NGSQ33 . ]) was used to identify normalizer miRNAs (present at consistent levels between patient groups) and then to identify a long-list of candidate miRNAs differentially present in the blood of patients with sepsis, non-infective SIRS and without SIRS.
  • Example 3 miRNAs stably expressed in sepsis, SIRS and normal individuals were identified.
  • the inventors used miRNA RT-qPCR to validate the most differentiating miRNAs and explore their performance in distinguishing sepsis from non-infective SIRS used singly and in combination.
  • the patients comprised unselected adult admissions to the intensive or high-dependency care units at an English acute hospital (Brighton and Wales University Hospitals NHS Trust).
  • SOFA Sequential Organ Failure Assessment
  • Study blood samples were collected in Na-citrate tubes from patients within 6 hours of ICU admission and centrifuged. Plasma was stored at -8o°C until the day of analysis, thawed on ice and kept at 4°C until the RNA extraction.
  • Red blood cell (RBC) lysis during sample handling has the potential to bias micro RNA content in The concentration of free hemoglobin ([Hb]) in plasma reflects the degree of any hemolysis [33].
  • Free [Hb] in patient samples was assessed by the Harboe spectrophotometric method[40, i] and samples with [Hb]>o.6g/L were excluded from further analysis ⁇ 42 ⁇ . Briefly, the total [Hb] in a freshly prepared Hb standard was validated using SysMex SLS-technology[43] to detect any Hb form in the human blood. Standard dilutions and plasma samples (1:10) were tested in triplicate to determine the A415, A380, and A450 and the Harboe [oxy-Hb] [3 .
  • each sample was denatured and processed according to manufacturer's instructions to extract RNA with Acid-Phenol :CHC1 3 ; the recovered aqueous phase was mixed with ethanol (molecular biology grade; SIGMA; 1:1.25) and loaded onto replicate columns to bind RNA. After multiple column washes, RNA was eluted in 95°C DEPC-treated H 2 o (Life Technologies) from replicate columns, pooled and quantified using a Nanodrop spectrophotometer. Typically, 679 ⁇ i65 pg RNA/ ⁇ of plasma (mean ⁇ SD) was recovered.
  • RNA input of 849 ⁇ 2o6 ng was created for technical duplicates of NGS and stored at -8o°C.
  • RNA preparations were validated for the presence of miRNA using a Taqman miRNA assay (Life Technologies) for human miR-16.
  • NGS cDNA libraries were prepared and validated from plasma RNA by ARK Genomics (University of Edinburgh, UK), following manufacturer instructions, with specific barcodes for each cDNA library (Illumina TruSeq Small RNA sample protocol). Briefly, samples were ligated with an adapter (3' end) and a primer (5' end) before being reversely transcribed.
  • the cDNA obtained was used as a template for PCR to add sample specific barcodes and extend adapters. Thereafter, samples were purified by electrophoresis (6% polyacrylamide gels) and bands corresponding to ⁇ 22 nucleotides in the original sample were size-selected (correct insert size: i46bp) after band staining and visualization under UV-light. The amplified size selected DNA was extracted from the gel by overnight soaking (H 2 o) and concentrated. The final preparation was checked for size and potential adapter-dimer contamination by electrophoresis.
  • the libraries were finally eluted from gels and run on the High Sensitivity DiK ScreenTape (Agilent Technologies) to determine size and purity prior to final quantification by qPCR and sequenced on a HiSeqTM 2500 Illumina instrument by loading duplicate libraries on separate lanes.
  • ⁇ io 8 NGS reads were acquired and, after filtering and sorting by library barcodes, sequences in any sample were mapped to the miRBase (release 20) database.
  • the resulting mapped reads (called counts) were arbitrarily normalized as miRNA counts/ 10 5 .
  • RNA (2 ⁇ ) was reverse transcribed using the miRCURY LNATM Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon).
  • cDNA (1:50) was assayed in qPCR as by the miRCURY LNATM Universal RT microRNA PCR protocol.
  • Each microRNA was assayed once by qPCR (on the microRNA Ready-to-Use PCR, Pick-&-Mix using ExiLENT SYBR® Green master mix) in 2 independent technical repeat experiments including negative controls (no-template from the reverse transcription reaction). In each experimental group, ⁇ 8 biological replicates were included.
  • the amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche) in 384-well plates.
  • the amplification curves were analyzed using the Roche LC software, both for determination of Cq (2nd derivative method) and for melting curve analysis. Amplification efficiency was calculated using a linear regression method. All assays were inspected for distinct melting curves and the Tm was confirmed to be within known specifications for the assay. Assays returning 3 Cq less than the negative control and Cq ⁇ 37 were accepted and sample runs not matching these criteria were omitted from further analysis (e.g., miR-92b-3p). The stability values of candidate normalizers were assessed using the 'NormFinder' software ⁇ ].
  • Cytokine levels (IL-6, IL-8, IL- ⁇ ) were measured on a Luminex LX200 using
  • Data describing demographics, severity of illness and key inflammatory biomarkers are shown in Table 1.
  • the median age of the patients was 66 years (IQR 54-75 years), 38 and 51 patients (43% and 57%) were male and female, respectively.
  • next generation sequencing NGS
  • Plasma pools were preferred to individual samples because they decrease the impact of individual outliers on the analysis.
  • the inventors compiled pools in such a way that average levels of hemolysis were comparable (Fig. 7B and Table 1).
  • Total RNA was then extracted from equal volumes of plasma pools and technical duplicates of cDNA libraries for Illumina NGS created. Results from 10 pools representative of 89 individuals are shown in Figure 1.
  • CIR- miRNAs circulating inflammation-related miRNAs
  • Example - Identification o fnormalizer miRNAs To allow for robust comparison of miRNA levels in blood between individual samples, endogenous miRNA normalizers must be established (i.e. similar to "housekeeping" miRNAs in blood). Previous studies have used different and inconsistent approaches to miRNA normalization in blood[i8,2i,42]- The inventors first used NGS to identify potential normalizers present at consistent levels across the four other pools
  • the optimal normalizer by NormFinder stability value ⁇ P was the mean Cp of miR320a and miR486-5p, which performed better than any other single miRNA detected and with levels consistent across 89 patients (Fig. 2B).
  • CIR-miRNAs with high levels of detection in blood were selected (by excluding CIR-miRNAs with consistently less than 35/10 5 NGS counts in any group) and with fd ⁇ 0.66 or fd ⁇ i.5 (when comparing sepsis to SIRS), leaving a panel of 47 CIR-miRNAs to be validated in 89 individuals -including 3 potential normalizers (miR320a, miR92b-3p and miR486-5p) - in RT-qPCR miRNA arrays.
  • the top-12 significantly different CIR-miRNAs showed inverse patterns in sepsis and SIRS.
  • Principal component analysis demonstrated that a combination of the top 5 significantly different CIR-miRNAs (including miR3od-5p, miR30a-5p, miRi92-5p, miR26a-5p and miR23a-5p) was able to discriminate severe sepsis from SIRS, as patients with SIRS (Fig. 3C, blue dots) tended to group in a different quadrant from sepsis patients (Fig. 3C, green dots).
  • the inventors further created a model combining the top 6 CIR-miRNA levels into a score that maximized the distinction between SIRS and sepsis (Fig. 4B).
  • SIRS and sepsis patients tended to score respectively >o and ⁇ o; hence the higher the model score the more likely patients are to have non-infective SIRS rather than sepsis, as described by a
  • CIR-miRNA score concomitant increase of multiple CIR-miRNAs.
  • the ROC curve with AUC 0.917 for the model interpolation data shows that the top-6 significant CIR-miRNAs combined together outperformed any single miRNA.
  • CIR-miRNAs are excellent biomarkers to distinguish SIRS from sepsis.
  • Example - Correlations between inflammatory cytokines and CIR-miRNA scores The inventors obtained CIR-miRNA scores as a mathematical function of the plasma levels of 6 CIR-miRNAs found to be consistently reduced in sepsis (and preferentially leading to score ⁇ o). The CIR-miRNA scores were then correlated to plasma levels of pro-inflammatory mediators, and SOFA severity scores, across sepsis and SIRS patients (Figure 5). CIR-miRNA scores did not correlate with SOFA scores (Fig. 5).
  • CIR-miRNA scores negatively correlated with levels of pro-inflammatory mediators, suggesting that a marked increase of multiple CIR-miRNAs is significantly associated with low levels of pro-inflammatory cytokines (IL-i, IL-8 and IL-6, Fig. 5) and mediators (CRP and SCD25, Fig. 5).
  • CIR-miRNAs change in the opposite direction to pro-inflammatory mediators.
  • CIR-miRNAs circulating inflammation-related miRNAs
  • the inventors have found a general upregulation of circulating inflammation-related miRNAs (CIR-miRNAs) in both sepsis and non-infective SIRS patients when compared with no-SIRS controls.
  • CIR-miRNAs were higher in non-infective SIRS than in sepsis, indicating that CIR-miRNAs is differentially affected in systemic inflammatory conditions depending on etiology.
  • the inventors have identified six CIR-miRNAs that that are highly discriminatory for sepsis from SIRS having AUCs by ROC analysis comparable or better than clinical biomarkers, CRP and PCT. Notably, they found that CIR-miRNA levels correlate inversely with pro-inflammatory biomarkers.
  • the inventors undertook an experimentally robust evaluation of blood miRNAs during systemic inflammation. They recruited robustly, and prospectively, clinically characterized patient groups. The focus of infection and causative organism may influence the inflammatory response in sepsis[48] and thus they enrolled specifically patients with abdominal sepsis where infection will predominantly be caused by Gram negative enterobacteriaceae.
  • the sepsis and non- infective SIRS groups were strictly stratified and matched for severity of illness. They used critically ill patients without SIRS as their controls. This is a particularly important feature of the study, since it is the distinction of sepsis from non-infective SIRS among critically ill patients with is crucial in research and clinical practice.
  • the inventors used NGS to screen miRNA species using pooled samples representative of many individuals, hence minimizing inter-individual variability. They rigorously normalized blood miRNAs and accounted for variation in hemolysis.
  • the best normalizer for the dataset was a combination of miR320a and miR486-5p, while miR92b-3p was excluded because its levels fell below the detection limit of qPCR in many individuals.
  • the results highlight the importance of choosing plasma miRNAs (either as normalizers or biomarkers) that are expressed at detectable levels within a relatively large cohort of individuals rather than miRNA species (or other small RNAs, including nuclear RNAs[2i,42]), the presence of which had not been validated across all the individuals in the cohort in the blood[i8,2i].
  • CIR-miRNAs are up-regulated in sepsis (including miR-223), thus reconciling this study with previous literature. Furthermore, this study is compatible with a previous report[20] in which miR223 and miRi46a are both downregulated in sepsis compared to SIRS. Interestingly, 6/7 miRNAs investigated in the same study also showed a tendency to decrease in sepsis compared to SIRS.
  • CIR-miRNAs do not directly address the cellular origin of CIR-miRNAs, except for the exclusion of RBC as a source of differentially expressed CIR-miRNAs. Still, they revealed a vast change in CIR-miRNA levels in systemic inflammatory disease.
  • CIR-miRNAs may regulate ⁇ 30% of human genes ⁇ 55 ⁇ , yet it is unclear whether CIR-miRNAs are a means of intercellular communication[54,56,57.]. According to recent research, Blimp-i[s8], P53/MDM2[52] and ⁇ 60 ⁇ may be targets of the top 3 CIR-miRNAs downregulated in sepsis in our study. Interestingly, these are kinases or transcription factors important in immune-cell differentiation and regulation.
  • Pro-inflammatory protein biomarkers are predominantly acute phase reactants which are upregulated in sepsis [3da, ]-
  • the inventors have found that levels of CIR-miRNAs inversely correlate with levels of inflammatory cytokines that are typically elevated in sepsis such as IL-ib, IL-6, and IL-8, and CRP[3,6i].
  • CIR-miRNAs may be part of the anti-inflammatory response ⁇ ] suppressing immune cell activation in severe sepsis and inflammation (illustrated in Figure 6).
  • This hypothesis is compatible with the recent discovery that (murine) regulatory T cells which suppress inflammatory responses can secrete a number of miRNAs analogous to the human CIR-miRNAs found in this study[fyi].
  • Argonaute2 complexes carry a population of circulating micro RNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108: 5003-5008.
  • MicroRNA fingerprints identify miR- 150 as a plasma prognostic marker in patients with sepsis. PLoS One 4: 67405.
  • microRNAs identified as diagnostic biomarkers of sepsis. J Trauma Acute Care Surg 73: 850-854.
  • miR-i46a is critical for endotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY. J Biol Chem 284: 34590-34599 ⁇
  • PRDMi is directly targeted by miR-3oa-5p and modulates the Wnt/beta-catenin pathway in a Dkki-dependent manner during glioma growth. Cancer Lett 331: 211-219.
  • P53-inducible microRNAs 192, 194, and 215 impairs the P53/MDM2

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des marqueurs biologiques permettant de distinguer la sepsis du syndrome de réponse inflammatoire systémique (SIRS), et en particulier l'utilisation de micro-ARN à titre de marqueurs de diagnostic pouvant être utilisés pour distinguer la septicémie du SIRS. Des procédés et des kits de détection desdits micro-ARN permettant de distinguer la sepsis du SIRS, et des méthodes de traitement sont en outre décrits.
PCT/GB2016/052724 2015-09-04 2016-09-05 Marqueur biologique de sepsis Ceased WO2017037477A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1515724.1 2015-09-04
GBGB1515724.1A GB201515724D0 (en) 2015-09-04 2015-09-04 Sepsis and systemic inflammatory response syndrome

Publications (1)

Publication Number Publication Date
WO2017037477A1 true WO2017037477A1 (fr) 2017-03-09

Family

ID=54345797

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2016/052724 Ceased WO2017037477A1 (fr) 2015-09-04 2016-09-05 Marqueur biologique de sepsis

Country Status (2)

Country Link
GB (1) GB201515724D0 (fr)
WO (1) WO2017037477A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018219998A1 (fr) * 2017-05-30 2018-12-06 Siemens Aktiengesellschaft Miarn en tant que biomarqueurs pour un syndrome de réponse inflammatoire systémique
CN111455044A (zh) * 2020-06-10 2020-07-28 新疆农垦科学院 一种用于母羊早期妊娠诊断的外泌体miRNA标志物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090075258A1 (en) * 2007-09-14 2009-03-19 Latham Gary J Methods of Normalization in microRNA Detection Assays
WO2015117205A1 (fr) * 2014-02-06 2015-08-13 Immunexpress Pty Ltd Méthode de signature de biomarqueur, et appareil et kits associés

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090075258A1 (en) * 2007-09-14 2009-03-19 Latham Gary J Methods of Normalization in microRNA Detection Assays
WO2015117205A1 (fr) * 2014-02-06 2015-08-13 Immunexpress Pty Ltd Méthode de signature de biomarqueur, et appareil et kits associés

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHA CHEN: "Differential expression of plasma miR-146a in sepsis patients compared with non-sepsis-SIRS patients", EXPERIMENTAL AND THERAPEUTIC MEDICINE, 30 January 2013 (2013-01-30), GR, XP055316969, ISSN: 1792-0981, DOI: 10.3892/etm.2013.937 *
LIQIONG YAO ET AL: "Original Article Clinical evaluation of circulating microRNA-25 level change in sepsis and its potential relationship with oxidative stress", INT J CLIN EXP PATHOL, 1 January 2015 (2015-01-01), XP055316965, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555662/pdf/ijcep0008-7675.pdf> *
YUQIAN MA ET AL: "Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis", PLOS ONE, vol. 8, no. 10, 16 October 2013 (2013-10-16), pages e75918, XP055183318, DOI: 10.1371/journal.pone.0075918 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018219998A1 (fr) * 2017-05-30 2018-12-06 Siemens Aktiengesellschaft Miarn en tant que biomarqueurs pour un syndrome de réponse inflammatoire systémique
CN111455044A (zh) * 2020-06-10 2020-07-28 新疆农垦科学院 一种用于母羊早期妊娠诊断的外泌体miRNA标志物及其应用
CN111455044B (zh) * 2020-06-10 2023-05-23 新疆农垦科学院 一种用于母羊早期妊娠诊断的外泌体miRNA标志物及其应用

Also Published As

Publication number Publication date
GB201515724D0 (en) 2015-10-21

Similar Documents

Publication Publication Date Title
Wang et al. Evidence for serum miR-15a and miR-16 levels as biomarkers that distinguish sepsis from systemic inflammatory response syndrome in human subjects
Joilin et al. Identification of a potential non-coding RNA biomarker signature for amyotrophic lateral sclerosis
Giannella et al. Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients
Xie et al. An increased ratio of serum miR-21 to miR-181a levels is associated with the early pathogenic process of chronic obstructive pulmonary disease in asymptomatic heavy smokers
JP7390285B2 (ja) 老化細胞を検出するための新規バイオマーカー
Sanders et al. Next-generation sequencing reveals broad down-regulation of microRNAs in secondary progressive multiple sclerosis CD4+ T cells
CN102884206B (zh) 用于确定心血管疾病的风险的手段和方法
JP2024075761A (ja) 脳卒中の血液バイオマーカー
WO2015148919A2 (fr) Micro-arn circulants en tant que biomarqueurs pour l&#39;endométriose
EP3630999A1 (fr) Nouveaux biomarqueurs d&#39;arn circulaire pour l&#39;insuffisance cardiaque
Yang et al. Effect of miR-126 on intracranial aneurysms and its predictive value for rupture of aneurysms.
US20230014092A1 (en) Materials and methods for monitoring inflammation
Xu et al. MicroRNAs combined with the TLR4/TDAG8 mRNAs and proinflammatory cytokines are biomarkers for the rapid diagnosis of sepsis
EP4073510A1 (fr) Matériaux et procédés destinés à des marqueurs moléculaires inflammatoires
JP6827067B2 (ja) ループス腎炎の検出またはそのリスクを予測する方法およびその応用
WO2017037477A1 (fr) Marqueur biologique de sepsis
US20140194467A1 (en) Plasma cell disorders
US20120164653A1 (en) Methods for the diagnosis of multiple sclerosis based on its microrna expression profiling
TW201514311A (zh) 用於判定胰臟癌預後之方法
US10465250B2 (en) Method for determining the survival prognosis of a patient suffering from pancreatic cancer
Meiri et al. Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure
ES3040135T3 (en) In vitro method for the diagnosis of synucleinopathies
WO2021118349A1 (fr) Procédés de typage de tumeurs de cellules germinales
Dubey et al. Factors influencing circulating microRNAs as biomarkers for liver diseases
Zhao et al. Systematic and integrated analysis of tRNA-derived small RNAs reveals novel potential pathogenesis targets of sarcoidosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16762854

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16762854

Country of ref document: EP

Kind code of ref document: A1