MIRNAS AS BIOMARKERS FOR A SYSTEMIC INFLAMMATORY RESPONSE SYNDROME

The present invention relates to a method of diagnosing an individual as having/suffering from a systemic

inflammatory response syndrome (SIRS) or not. Further, the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual. Furthermore, the present invention relates to a method of prognosing a systemic inflammatory response syndrome (SIRS) in an individual. In addition, the present invention relates to a kit for conducting the above-mentioned methods . BACKGROUND OF THE INVENTION

The reaction of the human body on a certain insult may result in severe consequences. Especially if the reaction is systemic, it may cause death of patients in short time periods. The detection of systemic reactions of the human body to the external stimulus is the key for improved patient care .

A common form is the systemic inflammatory response syndrome (SIRS) . It is an inflammatory state affecting the whole body and frequently a response of the immune system to infection, but not necessarily so. Although the definition of SIRS refers to it as an "inflammatory" response, it actually has pro- and anti-inflammatory components. If signs of an infection are present, the syndrome is termed sepsis. Severe sepsis is defined as sepsis plus sepsis-induced organ

dysfunction or tissue hypoperfusion .

Sepsis is one of the oldest and most pressing problems in medicine . Even with treatment in modern intensive care units ( ICUs ) , mortality rates of patients with sepsis are high . In Germany, 150.000 patients a year suffer from sepsis , over 50.000 patients die from the disease, making sepsis to one of the most common causes of death in Germany almost reaching heart failure as most common cause of death . For other developed countries the numbers are similar, the incidence of sepsis in the United States is reaching e.g. 0.3% per year .

Importantly, immediate diagnosis followed by treatment is essential for surviving a sepsis . Within the first three hours of a suspected sepsis, diagnostic studies include counting white blood cells (WBCs) , measuring serum lactate and obtaining appropriate cultures before starting

antibiotics, so long as this does not delay their use by more than 45 minutes. To identify the causative organisms, blood cultures are measured. Importantly, bacteria are present in the blood in only about 30% of the cases, while for 70% of the cases no bacteria are found. Another possible method of detection is by polymerase chain reaction (PCR) . If other sources of infection are suspected, cultures of these

sources, such as urine, cerebrospinal fluid, wounds, or respiratory secretions, are also obtained, as long as this does not delay the use of antibiotics. Within six hours, if blood pressure remains low, despite initial fluid

resuscitation central venous pressure and central venous oxygen saturation are measured. Lactate is re-measured if the initial lactate was elevated. Within twelve hours, it is essential to diagnose or exclude any source of infection that would require emergent source control, such as necrotizing soft tissue infection, infection causing inflammation of the abdominal cavity lining, infection of the bile duct, or intestinal infarction. A pierced internal organ (free air on abdominal x-ray or CT scan) ; an abnormal chest x-ray

consistent with pneumonia (with focal opacification) ; or petechiae, purpura, or purpura fulminans can also be evident for an infection.

The current criteria used in clinical care are rather less ambiguous and the current examination procedures are very time consuming. Support by other techniques, in particular molecular techniques, is, therefore, highly desirable. Biomarker might help to diagnose and/or predict SIRS, in particular sepsis, fast, flexible and reliable.

However, the known molecular techniques are rather un- specific, provide not enough sensitivity and/or specificity and their dynamics remain unknown. The use of such markers in clinical care would potentially not add much to the clinical value . For example, Wacker C. et al . ("Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis", Lancet Infect Dis 2013, 13: 426-435) describe the

determination of the procalcitonin level in order to

distinguish sepsis from non-infectious causes of SIRS. The sensitivity of the test is 77% and the specificity is 79%. Wacker C. et al . suggest that the procalcitonin level may serve as a helpful diagnostic marker for sepsis, but at the same time caution that its level alone could not definitively make the diagnosis. Backes Y. et al . ("Usefulness of suPAR as a biological marker in patients with systemic inflammation or infection: a systematic review", Intensive Care Med, 2012, 38: 1418-1428) further describe that the soluble urokinase- type plasminogen activator receptor (SuPAR) is a nonspecific marker of inflammation and does not accurately diagnose sepsis.

Thus, there is still a need for new biomarkers allowing the diagnosis of SIRS, in particular sepsis, fast, flexible and reliable. In addition, there is a need for new biomarkers allowing the prediction of SIRS, in particular sepsis.

Specifically, it would be essential to know prior to an intervention such as a surgery whether the patient has a predisposition to develop SIRS, in particular sepsis. For example, in case that a surgery is not in an acute setting, it may be shifted for several days if the patient is

identified as having a risk of developing SIRS. The present inventors studied the role of miRNAs as key modulators of the immune system. They found that miRNAs measured in biological samples, preferably in body fluids, more preferably in blood cells, reflect early and very specific immune responses against human pathologies. They considered this observation as an indication to further study the role of miRNAs in SIRS, e.g. sepsis. In particular, they analysed miRNA expression profiles of patients prior to surgical interventions and followed up the respective

patients over time. Afterwards, they classified said patients in patients not developing SIRS, in particular SIRS without infection as well as SIRS with infection (sepsis) (=

controls) , patients developing SIRS without infection, and patients developing SIRS with infection (sepsis) .

Subsequently, they performed a pairwise analysis of the groups: (i) controls pre-surgical intervention/post-surgical intervention, (ii) patients having SIRS without infection pre-surgical intervention/post-surgical intervention, and (iii) patients having SIRS with infection (sepsis) pre- surgical intervention/post-surgical intervention and

identified miRNAs that allow to diagnose SIRS, in particular sepsis, and to distinguish between SIRS without infection and SIRS with infection (sepsis) which high diagnostic power. The diagnosis can be performed following a severe intervention (e.g. surgery) or if first symptoms are present (e.g. in an acute care setting) . In contrast to former studies , the present inventors have taken into account in their

measurements and data analysis that also in controls , i.e. in patients that did not develop SIRS, the miRNA level slightly changed pre and post surgical intervention . This might be caused by the fact that the immune system is specifically challenged and stressed during surgical intervention, which is also apparent on the miRNA level . Thus , having regard to these circumstances , the inventors of the present invention determined miRNAs as biomarkers that allow to determine SIRS, in particular sepsis , with high reliability . In addition, the present inventors identified miRNAs that are already dysregulated prior to surgical intervention. These miRNAs are suitable as predictive biomarkers of SIRS, in particular sepsis. Such biomarkers are not known yet.

Moreover, the present inventors identified a specific blood cell sample type, preferably a leukocyte sample type, more preferably a lymphocyte sample type, as a special source of miRNAs having high diagnostic power.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the step of:

determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual.

In a second aspect, the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual (having SIRS) comprising the step of:

determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual.

In a third aspect, the present invention relates to a method of prognosing a systemic inflammatory response

syndrome (SIRS) in an individual comprising the step of:

determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32 and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual. In a fourth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual (suspected of having SIRS) for diagnosing the individual as

having/suffering from a systemic inflammatory response syndrome (SIRS) or not,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25.

In a fifth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual (having SIRS) for determining the severity of a systemic inflammatory response syndrome (SIRS) in the individual,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25.

In a sixth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual for prognosing a systemic inflammatory response syndrome (SIRS) in the individual ,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32.

In a seventh aspect, the present invention relates to a kit comprising:

(i) means for determining the level of at least one miRNA selected from the group consisting of

(a) SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80% sequence identity thereto, (b) SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto, and/or

(c) SEQ ID NO: 26 to SEQ ID NO: 32, and a sequence

having at least 80% sequence identity thereto in a biological sample isolated from an individual, and

(ii) optionally at least one reference.

This summary of the invention does not necessarily describe all features of the present invention. Other

embodiments will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Definitions Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be

understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as

described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds . (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) . Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank

Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present

specification, the text of the present specification takes precedence . The term "comprise" or variations such as "comprises" or

"comprising" according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term "consisting essentially of" according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term "consisting of" or variations such as "consists of" according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The terms "microRNA" or "miRNA", as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked

together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences

(e.g. biotin stretches). The miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs) . The genes encoding miRNAs are longer than the processed mature miRNA molecules. The miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70 nucleotide stem- loop structures known as pre-miRNAs in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) . When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5' end. The remaining strand, known as the miRNA*, anti-guide (anti-strand), or passenger strand, is degraded as a RISC substrate. Therefore, the miRNA*s are derived from the same hairpin structure like the "normal" miRNAs. So if the "normal" miRNA is then later called the "mature miRNA" or "guide strand", the miRNA* is the "anti- guide strand" or "passenger strand".

The terms "microRNA*" or "miRNA*", as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked

together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 18 to 23 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences (e.g. biotin stretches) . The "miRNA*s", also known as the "anti-guide strands" or "passenger strands", are mostly complementary to the "mature miRNAs" or "guide strands", but have usually single-stranded overhangs on each end. There are usually one or more mispairs and there are sometimes extra or missing bases causing single-stranded "bubbles". The miRNA*s are likely to act in a regulatory fashion as the miRNAs (see also above) . In the context of the present invention, the terms "miRNA" and "miRNA*" are interchangeable used. The term "miRBase", as used herein, refers to a well established repository of validated miRNAs. The miRBase

(www . mirbase . org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database) , with information on the location and sequence of the mature miRNA sequence

(termed miR) . Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download. The

sequences of the miRNAs described herein are based on miRBase version 21.

The term "nucleotides", as used herein, refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals:

adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. The term "nucleosides" refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH) , producing nucleotides, which are the molecular building blocks of DNA and RNA. The term "polynucleotide", as used herein, means a molecule of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally spacer elements and/or elongation elements described below. The depiction of a single strand of a polynucleotide also defines the sequence of the complementary strand. Polynucleotides may be single stranded or double stranded, or may contain

portions of both double stranded and single stranded

sequences. The term "polynucleotide" means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. In detail, the polynucleotide may be DNA, both cDNA and genomic DNA, RNA, cRNA or a hybrid, where the polynucleotide sequence may contain combinations of deoxyribonucleotide or

ribonucleotide bases, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.

Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods .

In the context of the present invention, a polynucleotide as a single polynucleotide strand provides a probe (e.g. miRNA capture probe) that is capable of binding to, hybridizing with, or detecting a target of complementary sequence, such as a nucleotide sequence of a miRNA or miRNA*, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.

Polynucleotides in their function as probes may bind target sequences, such as nucleotide sequences of miRNAs or miRNAs*, lacking complete complementarity with the polynucleotide sequences depending upon the stringency of the hybridization condition. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence, such as a nucleotide sequence of a miRNA or miRNA*, and the single stranded polynucleotide described herein.

However, if the number of mutations is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequences are no complementary sequences. The polynucleotide variants including

polynucleotide fragments or polynucleotide mutants and the miRNA variants including miRNA fragments or miRNA mutants are further defined below. Described herein are polynucleotides in form of single polynucleotide strands as probes for binding to, hybridizing with or detecting complementary sequences of miRNAs (targets) , which are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32. The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind. The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may also be modified, e.g. may comprise an

elongation (EL) element. For use in a RAKE or MPEA assay, a polynucleotide with an elongation element may be used as a probe. The elongation element comprises a nucleotide sequence with 1 to 30 nucleotides chosen on the basis of showing low complementarity to potential target sequences, such as nucleotide sequences of miRNAs or miRNAs*, therefore

resulting in not to low degree of cross-hybridization to a target mixture. Preferred is a homomeric sequence stretch N _n with n = 1 to 30, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and N = A or C, or T or G. Particularly preferred is a homomeric sequence stretch N _n with n = 1 to 12, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and N = A or C, or T or G. The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may be present in form of a tandem, i.e. in form of a polynucleotide hybrid of two different or identical polynucleotides, both in the same orientation, i.e. 5' to 3' or 3' to 5', or in different orientation, i.e. 5' to 3' and 3' to 5' . Said polynucleotide hybrid/tandem may comprise a spacer element. For use in a tandem hybridization assay, the polynucleotide hybrid/tandem as a probe may comprise a spacer (SP) element. The spacer element represents a nucleotide sequence with n = 0 to 12, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, nucleotides chosen on the basis of showing low

complementarity to potential target sequences, such as nucleotide sequences of miRNAs or anti-miRNAs, therefore resulting in not to low degree of cross-hybridization to a target mixture. It is preferred that n is 0, i.e. that there is no spacer between the two miRNA sequence stretches.

The term "label", as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA) , biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids at any position, e.g. at the 3' or 5' end or internally. The polynucleotide for detecting a miRNA

(polynucleotide probe) and/or the miRNA itself may be

labeled .

The term "stringent hybridization conditions", as used herein, means conditions under which a first nucleic acid sequence (e.g. polynucleotide in its function as a probe for detecting a miRNA or miRNA*) will hybridize to a second nucleic acid sequence (e.g. target sequence such as

nucleotide sequence of a miRNA or miRNA*), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at

equilibrium) . Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 tol.O M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20°C for short probes (e.g., about 10-35 nucleotides) and up to 60°C for long probes (e.g., greater than about 50

nucleotides) . Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x

SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C; or 6x SSPE, 10 % formamide, 0.01 %,Tween 20, 0.1 x TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42°C with wash in 05x SSPE and 6x SSPE at 45°C.

The term "antisense", as used herein, refers to

nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand.

Residues in two or more polynucleotide s are said to "correspond" to each other if the residues occupy an

analogous position in the polynucleotide structures. It is well known in the art that analogous positions in two or more polynucleotides can be determined by aligning the

polynucleotide sequences based on nucleic acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW (see www .ebi.ac.uk/clustalw) or Align (see

http : //www .ebi.ac.uk/emboss/align/index . html ) using standard settings, preferably for Align EMBOSS :: needle, Matrix:

Blosum62, Gap Open 10.0, Gap Extend 0.5.

The term "systemic inflammatory response syndrome

(SIRS)", as used herein, refers to a systemic inflammatory response to a variety of severe clinical insults. The causes of SIRS are broadly classified as infectious or non ^¬ infectious. The causes of SIRS include, but are not limited to, infection, trauma, burns, pancreatitis, ischemia, hemorrhage, and others. The other causes of SIRS include, but are not limited to, complications of surgery, adrenal

insufficiency, pulmonary embolism, complicated aortic

aneurysm, cardiac tamponade, and anaphylaxis. The syndrome is manifested by more than one of the following conditions: (i) body temperature less than 36 C or greater than 38°C, (ii) heart rate greater than 90 beats per minute, (iii) tachypnea (high respiratory rate) , with greater than 20 breaths per minute; or, an arterial partial pressure of

carbon dioxide less than 4.3 kPa (32 mmHg) , and (iv) white blood cell count less than 4000 cells/mm ³ (4 x 10 ⁹ cells/1) or greater than 12,000 cells/mm ³ (12 x 10 ⁹ cells/1); or the presence of greater than 10% immature neutrophils (band forms) .

If signs of an infection are present, the syndrome is termed sepsis .

In this clinical circumstance, sepsis represents the systemic inflammatory response to the presence of infection. Sepsis has been well recognized as a systemic inflammatory response to an active infectious process in the host, e.g. in the human . Based on the above, SIRS may be designated as SIRS with infection (sepsis) or SIRS without infection. In other words, SIRS may be termed SIRS dependent on/caused by infection (sepsis) or SIRS independent of/not caused by infection.

The term "infection", as used herein, refers to a microbial phenomenon characterized by an inflammatory

response to the presence of microorganisms or the invasion of normally sterile host tissue by those organisms.

The infection may be caused by bacteria, fungi,

parasites, viruses, and other microorganism. The infection may alternatively be designated as bacteremia, viremia, fungemia, or parasitemia. The term "bacteremia", as used herein, refers to the presence of (viable) bacteria in the blood. The term "viremia", as used herein, refers to a condition where (viable) viruses enter the bloodstream and hence have access to the rest of the body. The term

"fungemia", as used herein, refers to the presence of

(viable) fungi or yeasts in the blood. The term

"parasitemia", as used herein, refers to a condition where (viable) parasites enter the bloodstream and hence have access to the rest of the body. As mentioned above, when SIRS is the result of an infectious process, it is termed sepsis. The term "sepsis", as used herein, refers to the systemic response to infection. In association with infection, manifestations of sepsis are the same as those described for SIRS, and include more than one of the following conditions:

(i) body temperature less than 36 C or greater than 38°C,

(ii) heart rate greater than 90 beats per minute, (iii)

tachypnea (high respiratory rate) , with greater than 20 breaths per minute; or, an arterial partial pressure of

carbon dioxide less than 4.3 kPa (32 mmHg) , and (iv) white blood cell count less than 4000 cells/mm ³ (4 x 10 ⁹ cells/1) or greater than 12,000 cells/mm ³ (12 x 10 ⁹ cells/1); or the presence of greater than 10% immature neutrophils (band forms) .

Preferably, sepsis is severe sepsis, more preferably septic shock .

The term "severe sepsis", as used herein, refers to sepsis associated with organ dysfunction, hypoperfusion abnormality, or sepsis-induced hypotension. Hypoperfusion abnormalities include, but are not limited to, lactic

acidosis, oliguria, or an acute alteration in mental status.

The term "sepsis-induced hypotension", as used herein, is defined by the presence of a systolic blood pressure of less than 90 mm Hg or its reduction by 40 mm Hg or more from baseline in the absence of other causes for hypotension (e.g. cardiogenic shock) .

The term "septic shock", as used herein, refers to a subset of severe sepsis and is defined as sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction. Perfusion abnormalities include, but are not limited to, lactic acidosis, oliguria, or an acute alternation in mental status. In this respect, it should be noted that patients who are receiving inotropic or

vasopressor agents may not be hypotensive at the time that perfusion abnormalities are measured.

The term "diagnosing an individual as having a systemic inflammatory response syndrome (SIRS) or not", as used herein, means determining whether an individual shows signs of or suffers from SIRS or not. SIRS may be SIRS without infection or SIRS with infection (sepsis) . Thus, the

individual may be diagnosed as suffering from SIRS without infection or SIRS with infection (sepsis) , or as not

suffering from both. The term "determining the severity of a systemic

inflammatory response syndrome (SIRS)", as used herein, means determining whether an individual has a serious form of SIRS. Preferably, the serious form of SIRS is sepsis. More

preferably, the sepsis is severe sepsis. Even more

preferably, the sepsis is septic shock.

The term "prognosing a systemic inflammatory response syndrome (SIRS)", as used herein, means determining whether an individual has a predisposition to develop SIRS, e.g.

after surgical intervention. In other words, the term

"prognosing a systemic inflammatory response syndrome

(SIRS)", as used herein, means determining whether an

individual will likely develop SIRS, e.g. after surgical intervention .

The term "individual", as used herein, refers to any subject for whom it is desired to know whether she or he suffers from SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.

Specifically, the term "individual", as used herein, refers to a subject suspected to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. The individual may be diagnosed to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. diseased, or may be diagnosed to be not affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. healthy with respect to SIRS.

The term "individual", as used herein, also refers to a subject that is affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. diseased. The individual may be retested for SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, and may be diagnosed to be still affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e.

diseased, or not affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, anymore, i.e. healthy with respect to SIRS, for example after therapeutic intervention. The individual may further be retested for SIRS and may be diagnosed as having developed an advanced form of SIRS, in particular sepsis, or as suffering from a serious form of SIRS, in particular sepsis, such as severe sepsis or septic shock.

The term "individual", as used herein, further refers to any subject for whom it is desired to know whether she or he may develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. In particular, the term

"individual", as used herein, refers to a subject who has a predisposition to develop SIRS or will likely develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. The individual may be prognosed to develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, as the present inventors surprisingly found that miRNAs representative for SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, are already present/deregulated in a biological sample before SIRS occurs or during the early stage of SIRS.

The term "individual", as used herein, preferably refers to a subject who has experienced, experiences, or will experience an event involving a risk of developing SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. This event may have caused, causes, or will likely cause SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. The term "event involving a risk of developing SIRS", as used herein, encompasses any incident that may be responsible for the development of SIRS or a trigger of SIRS. In particular, the event involving a risk of developing SIRS encompasses a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical

intervention/surgery, a trauma, burns, pancreatitis,

ischemia, tissue injury, hemorrhagic shock, and/or immune- mediated organ injury. The infection may be a bacterial, viral, fungal, and/or parasitic infection. SIRS may be SIRS with infection (sepsis) or SIRS without infection.

The individual may suffer from a disease that requires a surgery, suffer from an infection, experience a trauma, have burns, suffer from pancreatitis, suffer from ischemia, have a tissue injury, have a hemorrhagic shock, and/or have an immune-mediated organ injury. The individual may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human subjects as individuals are

particularly preferred. The term "(control) subject", as used herein, refers to a subject known to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection (positive control), i.e. diseased. The term "(control) subject", as used herein, also refers to a subject known to be not

affected by SIRS, in particular SIRS with infection (sepsis) and/or SIRS without infection (negative control), i.e.

healthy with respect to SIRS.

The term "(control) subject", as used herein, preferably refers to a subject not suffering from SIRS who has

experienced an event involving a risk of developing SIRS without developing SIRS. The event involving a risk of developing SIRS is preferably a surgical

intervention/surgery. As mentioned above, the inventors of the present invention have taken into account in their measurements and data analysis that also in (control) subj ects , i.e. in subj ects that did not develop SIRS, the miR A level slightly changed pre and post surgical

intervention . This might be caused by the fact that the immune system is specifically challenged and stressed during surgical intervention, which is also apparent on the miRNA level . Thus , the consideration of such treated control subj ects as references in the methods of the present invention lead to reliable results as well as to results with great significance .

The (control) subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human (control) subjects as

individuals are particularly preferred.

The term "treatment", in particular "therapeutic

treatment", as used herein, refers to any therapy which improves the health status and/or prolongs (increases) the lifespan of an individual. Said therapy may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or

severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who

previously has had a disease. The disease may be SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. The therapeutic treatment of SIRS includes, but is not limited to, antimicrobial therapy, fluid therapy, vasopressor therapy, inotropic therapy, and corticosteroid therapy . The term "biological sample", as used herein, refers to any biological sample from an individual or a (control) subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32. The biological sample may be a body fluid sample or a tissue sample. For example, biological samples encompassed by the present invention are tissue samples, blood (e.g. whole blood or blood fraction such as blood cell/cellular fraction, serum or plasma) samples, urine samples, or samples from other peripheral sources. Said biological samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and a urine sample. Said biological samples may be provided by removing a biological sample from an individual or (control) subject, but may also be provided by using a previously isolated sample. For example, a blood sample may be taken from an individual or (control) subject by conventional blood collection techniques, or a tissue sample may be taken from an individual or (control) subject by biopsy. The biological sample, e.g. urine sample, blood sample or tissue sample, may be obtained from an individual or (control) subject prior to the initiation of a therapeutic treatment, during the

therapeutic treatment, and/or after the therapeutic

treatment. If the biological sample is obtained from at least one (control) subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject(s), it is designated as "reference biological sample". Preferably, the reference biological sample is from the same source than the biological sample of the individual to be tested, e.g. both are blood samples, urine samples or tissue samples. It is further preferred that both are from the same species, e.g. from a human. It is also (alternatively or additionally) preferred that the measurements of the reference biological sample of the (control) subject and the biological sample of the individual to be tested are identical, e.g. both have an identical volume. It is particularly preferred that the reference biological sample and the biological sample are from (control) subjects/individuals of the same sex and similar age.

The term "body fluid sample", as used herein, refers to any liquid sample derived from the body of an individual or (control) subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32.

Particularly, the term "body fluid sample", as used herein, refers to any body fluid sample from an individual or

(control) subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32.

Said body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof. The term "body fluid sample" also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions. The body fluid samples may be mixed or pooled. Thus, a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample. Said body fluid sample may be provided by removing a body liquid from an individual or (control) subject, but may also be provided by using previously isolated body fluid sample material. The body fluid sample allows for a non ^¬ invasive analysis of an individual. It is further preferred that the body fluid sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml, and most preferably of between 1 and 5 ml . If the body fluid sample is obtained from at least one (control subject), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1.000 control subject(s), it is designated as "reference body fluid sample".

The term "blood sample", as used herein, encompasses a whole blood sample or a blood fraction sample such as a blood cell/cellular fraction, blood serum, or blood plasma sample. It is preferred that the blood serum or plasma sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml and most preferably of between 1 and 5 ml.

The term "blood cell/cellular fraction", as used herein, refers to a blood cell/cellular portion which has been produced from whole blood by removing the extracellular fraction (serum and/or plasma) . In other words, the blood cell/cellular fraction is depleted of the extracellular blood components, such as serum and/or plasma.

In one embodiment, the blood sample is a blood

cell/cellular fraction. Preferably, the blood cell/cellular fraction comprises/consists of erythrocytes, leukocytes, and thrombocytes .

In one alternative embodiment, the blood sample is a blood cell sample. Preferably, the blood cell sample is a sample of leukocytes. More preferably, the blood cell sample is a sample of lymphocytes.

The term "level", as used herein, refers to an amount (measured for example in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration) of the miRNA(s) described herein, in particular of the miRNA(s) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO : 32. The term "level", as used herein, also comprises scaled, normalized, or scaled and normalized amounts or values . Preferably, the level determined herein is the expression level .

The term "sensitivity", as used herein, refers to the number of true positive patients (%) with regard to the number of all patients (100%) . The individuals may be

subjects having SIRS, in particular SIRS with infection

(sepsis) or SIRS without infection. The sensitivity is calculated by the following formula: Sensitivity= TP/ (TP+FN) (TP= true positives; FN=false negatives).

The term "specificity", as used herein, relates to the number of true negative individuals (%) with regard to the number of all healthy subjects (100%) . The specificity is calculated by the following formula: Specificity= TN/ (TN+FP) (TN= true negatives; FP=false positives) .

The term "accuracy", as used herein, means a statistical measure for the correctness of classification or identification of sample types. The accuracy is the

proportion of true results (both true positives and true negatives) . The result of each analysis group is usually calculated from a plurality of isolated samples, i.e. from at least 2 isolated samples, preferably from between 2 and 20, more preferably from between 10 and 60, and even more preferably from between 50 and 100 isolated samples, e.g. selected from the group consisting of subjects not suffering from SIRS, in particular SIRS with infection and/or SIRS without infection, and subjects having SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. The methods of the present invention can be carried out in combination with other methods for diagnosing an individual as

having/suffering from SIRS or not, for prognosing SIRS in an individual or for determination the severity of SIRS in an individual to increase the overall sensitivity and/or

specificity. The determination of the level of the miRNA(s) of the present invention allows the diagnosis of SIRS in an individual, the prognosis of SIRS in an individual, or the determination of the severity of SIRS in an individual.

The term "AUC", as used herein, relates to an

abbreviation for the area under a curve. In particular, it refers to the area under a Receiver Operating Characteristic (ROC) curve. The term "Receiver Operating Characteristic (ROC) curve", as used herein, refers to a plot of the true positive rate against the false positive rate for the

different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity) . The area under an ROC curve is a measure for the accuracy of a diagnostic test (the larger the area the better, optimum is 1, a random test would have a ROC curve lying on the diagonal with an area of 0.5 (see, for reference, for example, JP . Egan.

Signal Detection Theory and ROC Analysis) . In the context of the present invention, the term "kit of parts (in short: kit)" is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components. Said kit may allow point-of-care testing (POCT) .

The term "point-of-care testing (POCT)", as used herein, refers to a medical diagnostic testing at or near the point of care that is the time and place of individual care. This contrasts with the historical pattern in which testing was wholly or mostly confined to the medical laboratory, which entailed sending off specimens away from the point of care and then waiting hours or days to learn the results, during which time care must continue without the desired

information. Point-of-care tests are simple medical tests that can be performed at the bedside. The driving notion behind POCT is to bring the test conveniently and immediately to the individual to be tested. This increases the likelihood that the individual, physician, and care team will receive the results quicker, which allows for immediate clinical management decisions to be made. POCT is often accomplished through the use of transportable, portable, and handheld instruments and test kits. Small bench analyzers or fixed equipment can also be used when a handheld device is not available - the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the individual so that the treatment plan can be adjusted as necessary before the individual leaves the hospital .

Embodiments of the invention

The present inventors studied the role of miRNAs as key modulators of the immune system. They found that miRNAs measured in biological samples, preferably in body fluids, more preferably in blood cells, reflect early and very specific immune responses against human pathologies. They considered this observation as an indication to further study the role of miRNAs in SIRS, e.g. sepsis. In particular, they analysed miRNA expression profiles of patients prior to surgical interventions and followed up the respective

patients over time. Afterwards, they classified said patients in patients not developing SIRS, in particular SIRS without infection as well as SIRS with infection (sepsis) (=

controls) , patients developing SIRS without infection, and patients developing SIRS with infection (sepsis) .

Subsequently, they performed a pairwise analysis of the groups: (i) controls pre-surgical intervention/post-surgical intervention, (ii) patients having SIRS without infection pre-surgical intervention/post-surgical intervention, and (iii) patients having SIRS with infection (sepsis) pre- surgical intervention/post-surgical intervention and

identified miRNAs that allow to diagnose SIRS, in particular sepsis, and to distinguish between SIRS without infection and SIRS with infection (sepsis) which high diagnostic power. The diagnosis can be performed following a severe intervention (e.g. surgery) or if first symptoms are present (e.g. in an acute care setting) . In contrast to former studies , the present inventors have taken into account in their

measurements and data analysis that also in controls , i.e. in patients that did not develop SIRS, the miRNA level slightly changed pre and post surgical intervention . This might be caused by the fact that the immune system is specifically challenged and stressed during surgical intervention, which is also apparent on the miRNA level . Thus , having regard to these circumstances , the inventors of the present invention determined miRNAs as biomarkers that allow to determine SIRS, in particular sepsis , with high reliability . Thus, in a first aspect, the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the step of: determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, or 25 miRNA(s)) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual.

The method may be performed after an event involving a risk of an individual of developing SIRS, or before and after an event involving a risk of an individual of developing SIRS. Preferably, the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,

hemorrhagic shock, immune-mediated organ injury, or any combination thereof.

SIRS may be SIRS with infection (sepsis) or SIRS without infection. Sepsis is preferably severe sepsis, more

preferably septic shock. The infection may be selected from the group consisting of bacteremia, viremia, fungemia, and parasitemia .

Preferably, the level of the at least one miRNA (e.g. 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto. More preferably, the level of the at least one miRNA (e.g. 1, 2,

3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and a sequence having at least 80% sequence identity thereto . In particular, the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 and a sequence having at least 80% sequence identity thereto. Said at least one miRNA allows to determine whether the individual suffers from SIRS. Said at least one miRNA, however, does not allow to determine whether SIRS is SIRS with infection

(sepsis) or SIRS without infection.

In contrast thereto, the at least one miRNA (e.g. 1, 2, 3, 4,

5, or 6 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS with infection (sepsis) .

Moreover, the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS without infection. Preferably, the at least one miRNA (e.g. 1, 2, 3, 4, 5,

6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. More preferably, the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. In particular, said at least one miRNA allows to determine whether the individual does not have SIRS (SIRS with infection (sepsis) and/or SIRS without infection).

In one embodiment, the level of the at least one miRNA is compared to a reference level of said at least one miRNA. Thus, in one particular embodiment, the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the steps of:

(i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, or 25 miRNA ( s ) ) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more

preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual, and

(ii) comparing the level of the at least one miRNA to a

reference level of said at least one miRNA.

The above comparison allows to determine whether an

individual has/suffers from SIRS or not.

The reference level may be any level which allows to determine whether an individual suffers from SIRS or not. It may be obtained from a (control) subject (i.e. a subject different from the individual to be tested/diagnosed) or from the same individual.

In one preferred embodiment, the reference level is the level determined by measuring at least one reference

biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject not suffering from SIRS, in particular not suffering from SIRS with

infection (sepsis) and/or SIRS without infection, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject (s) not suffering from SIRS, in particular not suffering from SIRS with infection (sepsis) and/or SIRS without infection. The at least one subject not suffering from SIRS can be considered as being healthy with respect to SIRS. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS.

Preferably, the at least one subject not suffering from SIRS is a subject who has experienced an event involving a risk of developing SIRS without developing SIRS. It is preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,

hemorrhagic shock, immune-mediated organ injury, or any combination thereof. The infection may be a bacterial, viral, fungal, and/or parasitic infection. It is more preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery. It is particularly preferred that the reference level is the level taken from the subject after the experience of an event involving a risk of developing SIRS.

It is practicable to take one reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested. Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof. It is preferred that the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed.

As mentioned above, the level of the at least one miRNA is compared to a reference level of said at least one miRNA. Said reference level is the level determined by measuring a reference biological sample. For example, if the level of the miRNA according to SEQ ID NO: 1 is determined in a biological sample from an individual , it is compared to a reference level of the miRNA according to SEQ ID NO : 1 determined in a reference biological sample . Alternatively, if the level of the miRNA according to SEQ ID NO : 1 and the level of the miRNA according to SEQ ID NO : 2 is determined in a biological samp1e from an individual , both levels are compared to the respective reference levels , i.e. the level of the miRNA according to SEQ ID NO : 1 is compared to the reference level of the miRNA according to SEQ ID NO : 1 and the level of the miRNA according to SEQ ID NO : 2 is compared to the reference level of the miRNA according to SEQ ID NO : 2 determined in a reference biological sample .

Preferably,

the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s)) selected from the group

consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the

individual has SIRS, and/or the level of the at least one miRNA (e.g. 1, 2, 3, 4, or 5 miRNA(s)) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS, and/or

the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80%

sequence identity thereto is comparable with the reference level, which indicates that the individual does not have SIRS . In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold

below/above the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

In this respect, the term "comparable" means that the level may vary between 0 and 10%, e.g. 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

Preferably, both levels are identical with each other (i.e. 0% variation) . The term "comparable" in this respect also means that the detected level variation may be within the accuracy of a measurement. The accuracy of a measurement depends on the measurement method used. In case it is determined whether the individual does not have SIRS, the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .

In one (additional or alternative) preferred embodiment, the reference level is the level determined by measuring a reference biological sample isolated from the individual before an event involving a risk of developing SIRS, and wherein the biological sample is isolated from the individual after the event involving a risk of developing SIRS.

The reference biological reference sample may be isolated from the individual 1, 2, 3, 4, 5, or 6 day(s) before an event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) before an event involving a risk of developing SIRS, or immediately before an event involving a risk of developing SIRS. Preferably, the reference biological sample is isolated from the individual between 6 days and 1 hour, more preferably between 2 days and 1 hour, and even more preferably between 1 day and 1 hour before an event involving a risk of developing SIRS.

The biological sample may be isolated from the individual immediately after the event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) after the event

involving a risk of developing SIRS, or 1, 2, 3, 4, 5, 6, day(s) after the event involving a risk of developing SIRS. Preferably, the biological sample is isolated from the individual between 1 hour and 6 days, more preferably between 1 hour and 4 days, and even more preferably between 1 hour and 2 days after the event involving a risk of developing SIRS .

It is preferred that reference biological sample isolated from the individual before an event involving a risk of developing SIRS and the biological sample isolated from the individual after the event involving a risk of developing SIRS are from the same source (e.g. blood samples) .

The event involving a risk of developing SIRS is preferably a surgical intervention/surgery.

Preferably,

the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s)) selected from the group

consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the

individual has SIRS, and/or

the level of the at least one miRNA (e.g. 1, 2, 3, 4, or 5 miRNA(s)) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS, and/or

the level of the at least one miRNA (e.g. 1, 2, 3, or 4 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the

individual does not have SIRS, and/or

the level of the at least one miRNA (e.g. 1 or 2 miRNA(s)) selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the individual does not have SIRS.

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold

below/above the reference level. For example, the level of the at. least one miRNA is: at. least 0 6-fold, at. least 0.7- fold, at least 0 .8 -fold, at least 0. 9 -fold, at least 1 .0- fold, at least 1 .1 -fold, at least 1. 2 -fold, at least 1 .3- fold, at least 1 .4 -fold, at least 1. 5 -fold, at least 1 .6- fold, at least 1 .7 -fold, at least 1. 8 -fold, at least 1 .9- fold, at least 2 .0 -fold, at least 2. 1 -fold, at least 2 .2- fold, at least 2 .3 -fold, at least 2. 4 -fold, at least 2 .5- fold, at least 2 .6' -fold, at least 2. 7 -fold, at least 2 .8- fold, at least 2 .9' -fold, or at least 3.0-fold below/above the reference : level

In case it is determined whether the individual does not have SIRS, the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .

In one more preferred embodiment, SIRS is selected from the group consisting of SIRS with infection (sepsis) and SIRS without infection.

Preferably,

the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, or 9 miRNA(s)) is selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto. Said at least one miRNA allows to determine whether the individual suffers from SIRS with infection (sepsis) or SIRS without infection .

It is further preferred that SIRS is SIRS with infection (sepsis) and the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%

sequence identity thereto.

It is particularly preferred that the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the

individual has SIRS with infection (sepsis) .

It is also preferred that SIRS is SIRS without infection and the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto.

It is particularly preferred that the level of the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS without infection.

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold

below/above the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

Alternatively, the method of the first aspect of the present invention is formulated as follows: a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the steps of:

(i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 miRNA ( s ) ) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more

preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or

99%, sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, (ii) determining the level of the at least one miRNA in a

biological sample isolated from the (same) individual at least one time (e.g. one, two, or three time(s)) after the event involving a risk of developing SIRS, and (iii) comparing the levels of the at least one miRNA

determined in steps (i) and (ii) with each other.

The above comparison allows to determine whether an

individual has/suffers from SIRS or not.

The reference biological reference sample may be

isolated from the individual 1, 2, 3, 4, 5, or 6 day(s) before an event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) before an event involving a risk of developing SIRS, or immediately before an event involving a risk of developing SIRS. Preferably, the reference biological sample is isolated from the individual between 6 days and 1 hour, more preferably between 2 days and 1 hour, and even more preferably between 1 day and 1 hour before an event involving a risk of developing SIRS. The biological sample may be isolated from the

individual immediately after the event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) after the event involving a risk of developing SIRS, or 1, 2, 3, 4, 5, 6, day(s) after the event involving a risk of developing SIRS. Preferably, the biological sample is isolated from the individual between 1 hour and 6 days, more preferably between 1 hour and 4 days, and even more preferably between 1 hour and 2 days after the event involving a risk of developing SIRS .

It is preferred that the reference biological sample isolated from the individual before an event involving a risk of developing SIRS and the biological sample isolated from the individual after the event involving a risk of developing SIRS are from the same source (e.g. blood samples) .

The event involving a risk of developing SIRS is

preferably a surgical intervention/surgery.

SIRS may be SIRS with infection (sepsis) or SIRS without infection. Sepsis is preferably severe sepsis, more

preferably septic shock. The infection may be selected from the group consisting of bacteremia, viremia, fungemia, and parasitemia .

Preferably,

the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 miRNA ( s ) ) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto. More preferably, the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and a sequence having at least 80% sequence identity thereto. In particular, the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, and a sequence having at least 80% sequence identity thereto. Said at least one miRNA allows to determine whether the individual suffers from SIRS. Said at least one miRNA, however, does not allow to determine whether SIRS is SIRS with infection

(sepsis) or SIRS without infection.

In contrast thereto, the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS with infection (sepsis) .

Moreover, the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS without infection.

Preferably,

the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. More preferably, the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. In particular, said at least one miRNA allows to determine whether the individual does not have SIRS (SIRS with

infection (sepsis) and/or SIRS without infection).

Even more preferably, the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s)) selected from the group

consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual has SIRS, and/or

the level of the at least one miRNA (e.g. 1, 2, 3, 4, or 5 miRNA(s)) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the

individual has SIRS, and/or

the level of the at least one miRNA (e.g. 1, 2, 3, or 4 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the individual does not have SIRS, and/or

the level of the at least one miRNA (e.g. 1 or 2 miRNA(s)) selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual does not have SIRS. In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold below/above the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

In case it is determined whether the individual does not have SIRS, the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .

Most preferably, the method is a method of diagnosing an individual as having/suffering from SIRS with infection

(sepsis) . In particular, the method of diagnosing an

individual (suspected of having SIRS with infection (sepsis) ) as having/suffering from SIRS with infection (sepsis)

comprises the steps of: (i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group

consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, in particular SIRS with infection (sepsis) ,

(ii) determining the level of the at least one miRNA in a

biological sample isolated from the individual at least one time after the event involving a risk of developing SIRS, in particular SIRS with infection (sepsis) , and (iii) comparing the levels of the at least one miRNA determined in steps (i) and (ii) with each other.

The above comparison allows to determine whether an

individual has/suffers from SIRS with infection (sepsis).

Specifically, the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual has SIRS with

infection (sepsis) .

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold above the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8- fold, at least 0.9-fold, at least 1.0-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4- fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3- fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9- fold, or at least 3.0-fold above the reference level.

Most preferably, the method is also a method of

diagnosing an individual (suspected of having SIRS without infection) has having /suffering from SIRS without infection. In particular, the method of diagnosing an individual

(suspected of having SIRS without infection) as

having/suffering from SIRS without infection comprises the steps of: (i) determining the level of at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, in particular SIRS without infection, (ii) determining the level of the at least one miRNA in a

biological sample isolated from the individual at least one time after the event involving a risk of developing SIRS, in particular SIRS without infection, and (iii) comparing the levels of the at least one miRNA

determined in steps (i) and (ii) with each other.

The above comparison allows to determine whether an

individual has/suffers from SIRS without infection.

Specifically, the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the individual has SIRS without infection .

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold below the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0. fold, at least 0. 9 -fold, at least 1. 0 -fold, at least 1. 1- fold, at least 1. 2 -fold, at least 1. 3 -fold, at least 1. 4- fold, at least 1. 5 -fold, at least 1. 6 -fold, at least 1. 7- fold, at least 1. 8 -fold, at least 1. 9 -fold, at least 2. 0- fold, at least 2. 1 -fold, at least 2. 2 -fold, at least 2. 3- fold, at least 2. 4 -fold, at least 2. 5 -fold, at least 2. 6- fold, at least 2. 7 -fold, at least 2. 8 -fold, at least 2. 9- fold, or at least 3.0-fold below the reference level .

The transition from a systemic inflammatory response syndrome (SIRS) to a severe form of SIRS is a gradual and fast moving process. Thus, biomarkers which allow a quick and reliable determination of the degree of severity of SIRS are highly desirable. The present inventors identified miRNAs which can be used to determine the severity of SIRS in an individual suffering from SIRS in a fast and reliable way.

Thus, in a second aspect, the present invention relates to a method of determining the severity of a systemic

inflammatory response syndrome (SIRS) in an individual

(having SIRS) comprising the step of: determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample

isolated from an individual.

In one embodiment, the level of the at least one miRNA is compared to a reference level of said at least one miRNA. Thus, in one particular embodiment, the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual

(having SIRS) comprising the steps of:

(i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group

consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO:

13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual, and (ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA.

The above comparison allows to determine the severity of SIRS in the individual .

The reference level may be any level which allows to determine the severity of SIRS in the individual. In one preferred embodiment, the reference level is the level determined by measuring at least one reference

biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200,

250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject not suffering from SIRS, in particular not suffering from SIRS with

infection (sepsis) and/or SIRS without infection, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject (s) not suffering from SIRS, in particular not suffering from SIRS with infection (sepsis) and/or SIRS without infection. The at least one subject not suffering from SIRS can be considered as being healthy with respect to SIRS. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS. Preferably, the at least one subject not suffering from SIRS is a subject who has experienced an event involving a risk of developing SIRS without developing SIRS. It is preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,

hemorrhagic shock, immune-mediated organ injury, or any combination thereof. The infection may be a bacterial, viral, fungal, and/or parasitic infection. It is more preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery. It is particularly preferred that the reference level is the level taken from the subject after the experience of an event involving a risk of developing SIRS.

In another preferred embodiment, the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject suffering from SIRS without infection, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject(s) suffering from SIRS without infection. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects suffering from SIRS without infection. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects suffering from SIRS without infection. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects suffering from SIRS without infection .

It is practicable to take one reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested. Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof. It is preferred that the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed. Preferably, the level of the at least one miRNA above the reference level indicates that the individual has a severe form of SIRS/has SIRS with infection (sepsis) .

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold above the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8- fold, at least 0.9-fold, at least 1.0-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4- fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3- fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9- fold, or at least 3.0-fold above the reference level. Sepsis is preferably severe sepsis, more preferably septic shock.

Biomarkers, in particular miRNA biomarkers, which allow to determine whether the individual will likely develop SIRS (predictive biomarkers, in particular predictive miRNA biomarkers) are not known yet. The present inventors

identified miRNAs that are already dysregulated prior to surgical intervention/surgery. These miRNAs allow to

determine whether the individual will likely develop SIRS or has a predisposition to develop SIRS, e.g. after surgical intervention/surgery. This might be important in cases where a surgical intervention/surgery is planned on the individual. If the surgical intervention/surgery is not in an acute setting, it may be shifted for several days. If the surgical intervention/surgery is in an acute setting, the monitoring of the respective individual has to be done very specifically as well as carefully. Thus, in a third aspect, the present invention relates to a method of prognosing/predicting a systemic inflammatory response syndrome (SIRS) in an individual comprising the step of:

determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, or 7 miRNA(s)) selected from the group consisting of

SEQ ID NO: 26 to SEQ ID NO: 32 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual.

In one embodiment, the level of the at least one miRNA is compared to a reference level of said at least one miRNA. Thus, in one particular embodiment, the present invention relates to a method of prognosing a systemic inflammatory response syndrome (SIRS) in an individual comprising the steps of: (i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, or 7 miRNA(s)) selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual, and

(ii) comparing the level of the at least one miRNA to a

reference level of said at least one miRNA. The above comparison allows to determine whether the

individual will likely develop SIRS or has a predisposition to develop SIRS.

The reference level may be any level which allows to determine the severity of SIRS in the individual.

In one preferred embodiment, the reference level is the level determined by measuring at least one reference

biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject not suffering from SIRS, in particular not suffering from SIRS with infection (sepsis) and/or SIRS without infection, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject (s) not suffering from SIRS, in particular not suffering from SIRS with infection (sepsis) and/or SIRS without infection. The at least one subject not suffering from SIRS can be considered as being healthy with respect to SIRS. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS.

It is practicable to take one reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested. Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof. It is preferred that the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed.

Preferably, the level of the at least one miRNA below the reference level indicates that the individual has a predisposition to develop SIRS or that the individual will likely develop SIRS.

In particular, the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, and more preferably at least 1.2-fold or 1.3-fold below the reference level. For example, the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1-fold, at least 1.2-fold, or at least 1.3-fold below the reference level. In the methods of the first to third aspect of the present invention, it is preferred that the individual is a mammal, preferably a human.

In the method of the first to third aspect of the present invention, it is further preferred that the

biological sample is a body fluid or tissue sample.

Preferably, the body fluid sample is selected from the group consisting of a blood sample, a urine sample, and a

combination thereof. More preferably, the blood sample is a whole blood sample or a blood fraction sample. Even more preferably, the blood fraction sample is a blood

cell/cellular fraction sample, a blood serum sample, or a blood plasma sample.

In one embodiment, the blood cell/cellular fraction

comprises/consists of erythrocytes, leukocytes, and

thrombocytes . In one alternative embodiment, the blood sample is a blood cell sample of leukocytes, in particular a sample of

lymphocytes .

If in the context of the first to third aspect of the present invention, the level of more than one miRNA is determined, e.g. of two or more miRNAs, it is referred herein to a set comprising at least two miRNAs.

The determination of the level of the at least one miRNA may be carried out by any convenient means for determining the level of a nucleotide sequence such as miRNA. For this purpose, qualitative, semi-quantitative and quantitative detection methods can be used. Quantitative detection methods are preferred. A variety of techniques are well known to the person skilled in the art. For example, the level of the at least one miRNA can be determined in the methods of the first to third aspect of the present invention by nucleic acid hybridization, nucleic acid amplification, polymerase

extension, sequencing, mass spectroscopy, an immunochemical method, or any combination thereof.

Preferably,

(i) the nucleic acid hybridization is performed using a

microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real- time PCR (RT-PCR) or quantitative real-time PCR (qPCR) ,

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked

immunosorbent assay (ELISA) .

Nucleic acid amplification, for example, may be

performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative PCR (RT qPCR) . The real time polymerase chain reaction (RT-PCR) may include the following steps: (i) extracting total RNA from the biological sample isolated from the individual, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using miRNA-specific primers, (iii) designing miRNA-specific cDNA forward primers and providing universal reverse primers to amplify the cDNA via polymerase chain reaction (PCR) , (iv) adding a

fluorescent probe to conduct PCR, and (v) detecting and comparing the variation in levels of miRNAs in the biological sample isolated from the individual relative to those of miRNAs in a reference biological sample isolated from a

(control) subject. A variety of kits and protocols to determine the miRNA level by real time polymerase chain reaction (RT-PCR) such as real time quantitative PCR (RT qPCR) are available. For example, reverse transcription of miRNAs may be performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied

Biosystems) according to manufacturer's recommendations.

Nucleic acid hybridization, for example, may be

performed using a microarray/biochip or in situ

hybridization. For nucleic acid hybridization, for example, the polynucleotides (probes) described herein with

complementarity to the corresponding miRNAs to be detected are attached to a solid phase to generate a

microarray/biochip. Said microarray/biochip is then incubated with miRNAs, isolated (e.g. extracted) from the biological sample, which may be labelled or unlabelled. Upon

hybridization of the labelled miRNAs to the complementary polynucleotide sequences on the microarray/biochip, the success of hybridisation may be controlled and the intensity of hybridization may be determined via the hybridisation signal of the label in order to determine the level of each tested miRNA in said biological sample.

Alternatively, the miRNA level may be determined using an immunochemical method, e.g. using an ELISA. Said method may include the following steps: (i) isolating miRNAs from a biological sample, (ii) hybridizing polynucleotide probes (complementary) to the miRNAs to obtain hybrids of said polynucleotides probes and said miRNAs, and (iii) binding said hybrids to antibodies capable of specifically binding hybrids of said polynucleotide probes and said miRNAs, and (iv) detecting the antibody-bound hybrids.

In the methods of the first to third aspect of the present invention, it is further preferred that the level of the at least one miRNA is the expression level of said at least one miRNA. In a fourth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual (suspected of having SIRS) for diagnosing the individual as

having/suffering from a systemic inflammatory response syndrome (SIRS) or not,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25.

It is preferred that at least one polynucleotide

(probe/primer, in particular primer pair) is used for

detecting at least one miRNA in a biological sample isolated from an individual for diagnosing the individual as

having/suffering from SIRS with infection (sepsis),

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, and SEQ ID NO: 25. It is alternatively preferred that at least one

polynucleotide (probe/primer, in particular primer pair) is used for detecting at least one miRNA in a biological sample isolated from an individual for diagnosing the individual as having/suffering from SIRS without infection,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 15.

As to further preferred miRNA combinations, it is referred to the first aspect of the present invention.

Preferably

(i) the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or

(ii) the at least one polynucleotide has at least 80

preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the

polynucleotide according to (i) .

It is particularly preferred that the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for

conducting the method according to the first aspect of the present invention.

In a fifth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual (having SIRS) for determining the severity of a systemic inflammatory response syndrome (SIRS) in the individual,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25.

Preferably

(i) the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or

(ii) the at least one polynucleotide has at least 80 ~6 , more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, or 99%, sequence identity to the

polynucleotide according to (i) .

It is particularly preferred that the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for

conducting the method according to the second aspect of the present invention.

In a sixth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in

particular primer pair) for detecting at least one miRNA in a biological sample isolated from an individual for

prognosing/predicting a systemic inflammatory response syndrome (SIRS) in the individual,

wherein the at least one miRNA is selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32.

Preferably

(i) the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or

(ii) the at least one polynucleotide has at least 80 ~6 , more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the

polynucleotide according to (i) . It is particularly preferred that the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for

conducting the method according to the third aspect of the present invention.

In a seventh aspect, the present invention relates to a kit comprising:

(i) means for determining the level of at least one miRNA selected from the group consisting of

(a) SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85~6 , more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto,

SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto, and/or (c) SEQ ID NO: 26 to SEQ ID NO: 32, and a sequence having at least 80%, preferably at least 85~6 , more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual, and (ii) optionally at least one reference.

As to the specific miRNA combinations of the miRNAs mentioned in (ia) , it is referred to the first aspect of the present invention.

It is preferred that the kit comprising the means referred to in

(ia) is for diagnosing an individual as having/suffering from a systemic inflammatory response syndrome (SIRS) or not,

(ib) is for determining the severity of a systemic

inflammatory response syndrome (SIRS) in an individual, and/or

(ic) is for prognosing/predicting a systemic inflammatory

response syndrome (SIRS) in an individual.

It is further preferred that the kit is useful for conducting the methods according to the first to third aspect of the present invention.

In particular, the kit comprising the means referred to in

(ia) is for conducting the method according to the first aspect of the present invention,

(ib) is for conducting the method according to the second aspect of the present invention, and/or (ic) is for conducting the method according to the third aspect of the present invention.

The at least one reference may be any reference which allows to diagnose whether an individual suffers from SIRS or not, to determine the severity of SIRS in an individual, and/or to prognose/predict SIRS in an individual.

It is more preferred that the means for determining the level of the at least one miRNA in a biological sample isolated from an individual of

(ia) comprise at least one polynucleotide (probe/primer, in particular primer pair) as defined in the fourth aspect of the present invention,

(ib) comprise at least one polynucleotide (probe/primer, in particular primer pair) as defined in the fifth aspect of the present invention, and/or

(ic) comprise at least one polynucleotide (probe/primer, in particular primer pair) as defined in the sixth aspect of the present invention.

It is even more preferred that the means in (ia) , (ib) , and/or (ic) further comprise at least one polynucleotide (probe) and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.

In particular, the means in (ia) , (ib) , and/or (ic) comprise at least one polynucleotide (probe) (see third to sixth aspect of the present invention) ,

at least one primer pair (see third to sixth aspect of the present invention), and/or

at least one polynucleotide (probe) and at least one antibody capable of binding a hybrid of said at least one

polynucleotide (probe) and said at least one miRNA. Said means allow to determine the level of the at least one miRNA in a biological sample isolated from an individual and, thus, to diagnose whether the individual suffers from SIRS or not, to determine the severity of SIRS in the

individual, and/or to determine the prognosis of SIRS in the individual .

The at least one polynucleotide may be part of a

microarray/biochip or may be attached to beads of a beads- based multiplex system.

The kit may further comprise (iii) a container, and/or

(iv) a data carrier.

The data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD) , a digital versatile disk (DVD) , a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized

database. The access code may also allow access to an

application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

Said data carrier may further comprise the at least one reference, e.g. the reference level of the level of the at least one miRNA determined herein. In case that the data carrier comprises an access code which allows the access to a database, said at least one reference, e.g. said reference level may be deposited in this database. The data carrier may also comprise information or instructions on how to carry out the methods according to the first to third aspect of the present invention. Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer (s), a reagent (s) and/or a diluent (s) for determining the level mentioned above. Various modifications and variations of the invention will be apparent to those skilled in the art without

departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific

embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

BRIEF DESCRIPTION OF THE FIGURES

The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

Figure 1: It presents the effects of the surgery and a developing disease as consequence for the controls, SIRS and sepsis patients. miRNAs that are affected in controls are caused by the surgery but not by a developing SIRS or sepsis. The markers of class 1 (only controls) are markers to rule out diseases. The markers of classes 2 and 3 are positive examples where effects also in SIRS / sepsis are observed.

The markers of class 3 allow to distinguish between SIRS with infection (sepsis) and SIRS without infection. In particular, the miRNA markers shown in Figure 1 allow to determine whether a patient suffers from a systemic

inflammatory response syndrome (SIRS) or not. In addition, the miRNA markers shown in Figure 1 allow to distinguish between SIRS with infection (sepsis) and SIRS without

infection .

Figure 2: It shows significant markers in the comparison controls versus SIRS / sepsis with median concentrations, p- values and fold changes. These miRNA markers allow to

prognose/predict a systemic inflammatory response syndrome (SIRS) in a patient.

Figure 3: It shows an overview of the identified and claimed miRNA biomarkers with their nucleic acid sequences.

Figure 4: It is a Box-plot representation. It shows as an example one miRNA, namely hsa-miR-199a-5p (SEQ ID NO: 24), that is only up-regulated in sepsis patients following surgery and development of the sepsis.

Figure 5: It is a Box-plot representation. It shows as an example one miRNA, namely hsa-miR-338-3p (SEQ ID NO: 25), that is only up-regulated in sepsis patients following surgery and development of the sepsis.

EXAMPLES The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Example 1 Identification of miRNAs as biomarkers for the diagnosis of a systemic inflammatory response syndrome (SIRS) in a patient

1. Materials and methods Patient samples

In the present study, patients undergoing significant surgical interventions were included. Blood samples (2.5 mL per patient) were collected in PAXgene tubes prior to and following surgery in time intervals (3 days) . Sample preparation Prior to RNA extraction, PAXgene tubes were thawed overnight at room temperature to ensure complete lysis of blood cells. Total RNA, including miRNA, was

extracted and purified using the PAXgene Blood miRNA Kit in accordance with the manufacturer' s instructions

(Qiagen GmbH, Hilden, Germany) . Quantification of purified RNA was performed with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) . The quality and integrity of the RNA (RIN value) was

evaluated using Agilent Bioanalyzer and the Nano RNA Kit in accordance with the manufacturer's protocols (Agilent

Technologies, Santa Clara, California, USA) . Sample measurement For miRNA expression, profiling samples were analysed on

Agilent Sureprint G3 Human miRNA (8><60k) microarray slides with the latest miRBase v21 content. Each array targets 2,549 microRNAs with 20 replicates per probe. Extracted miRNA was labeled and hybridized using the miRNA Complete Labeling and Hybridization Kit from

Agilent, in accordance with the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA) . After rotating hybridization for 20 hours at 55 C, the slides were washed twice and scanned on Agilent's

SureScan Microarray Scanner. Image files from the scanner were transformed into text raw data using

Feature Extraction Software (Agilent Technologies) for bioinformatics analysis. Data analysis, statistics

For data processing, the profiled samples were subjected to normalization. The 2,549 human miRNAs available on the Agilent miRBase v21 arrays were then used for the bioinformatics analysis. For subsequent data analysis different methods were applied (e.g. unsupervised clustering or analysis of variance) . For pairwise comparisons, the t-test was used for comparisons between the control group and the other classes. In addition, multiple comparison was also carried out using the analysis of variance (ANOVA) test. All P values

underwent Benj amini-Hochberg adjustment to control the false discovery rate. In addition to the significance values, the area under the receiver operating

characteristics curve (AUC) value was computed.

Results

As mentioned above, patient samples prior to surgical interventions were collected. The respective patients were followed up for some days and measured again. Then three groups were build, patients that have not

developed SIRS and sepsis (controls) , patients that have developed SIRS but no sepsis (SIRS) and patients that have developed sepsis. For all three groups, samples prior surgery and following surgery were taken such that a total of 6 groups could be measured. A paired analysis of the respective groups ((1) controls pre versus controls post, (2) SIRS pre versus SIRS post, (3) sepsis pre versus sepsis post) allowed the identification of miRNA biomarkers that changed in time because of the surgical intervention (dysregulated in all groups, especially the controls) and represent exclusion markers (denoted in the following as class 1 markers) , that are specific for SIRS or sepsis but not in controls (referred to as class 2 markers) and that distinguished between SIRS and sepsis (referred to as class 3

markers) . The most relevant markers (significant in at least one comparison and matching to at least one of the three groups or presenting substantial differences in the AUC) are summarized in Figure 1 with the

significance values and AUC values for each of the three comparisons. The numbers in bold and italic represent significant associations, italic numbers represent downregulation and bold numbers represent upregulation dependent on the condition. Both, the italic and bold numbers, have a grey background.

Please note that the matching of the markers to the three groups is not ambiguous, since there are three conditions and each marker can be upregulated,

downregulated or not be dysregulated in each condition so that several combinations are possible (e.g. up sepsis, down control, not dysregulated SIRS) .

Combination of these markers to signatures allows for highly accurate disease diagnosis.

Selected examples of miRNA biomarkers identified in this study are shown in Figures 1, 4, and 5.

Example 2

Identification of miRNAs as biomarkers for the prediction of a systemic inflammatory response syndrome (SIRS) in a patient 1. Materials and methods

1.1 Patient samples

In the present study, patients undergoing significant surgical interventions were included. Blood samples (2.5 mL per patient) were collected in PAXgene tubes prior to and following surgery in time intervals (3 days) . Sample preparation

Prior to RNA extraction, PAXgene tubes were thawed overnight at room temperature to ensure complete lysis of blood cells. Total RNA, including miRNA, was

extracted and purified using the PAXgene Blood miRNA Kit in accordance with the manufacturer' s instructions (Qiagen GmbH, Hilden, Germany) . Quantification of purified RNA was performed with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) . The quality and integrity of the RNA (RIN value) was evaluated using Agilent Bioanalyzer and the Nano RNA Kit in accordance with the manufacturer' s protocols (Agilent Technologies, Santa Clara, California, USA) . Sample measurement

For miRNA expression, profiling samples were analysed on Agilent Sureprint G3 Human miRNA (8><60k) microarray slides with the latest miRBase v21 content. Each array targets 2,549 microRNAs with 20 replicates per probe. Extracted miRNA was labeled and hybridized using the miRNA Complete Labeling and Hybridization Kit from

Agilent, in accordance with the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA) . After rotating hybridization for 20 hours at 55 C, the slides were washed twice and scanned on Agilent's

SureScan Microarray Scanner. Image files from the scanner were transformed into text raw data using

Feature Extraction Software (Agilent Technologies) for bioinformatics analysis. Data analysis, statistics

For data processing, the profiled samples were subjected to normalization. The 2,549 human miRNAs available on the Agilent miRBase v21 arrays were then used for the bioinformatics analysis. For subsequent data analysis different methods were applied (e.g. unsupervised clustering or analysis of variance) . For pairwise comparisons, the t-test was used for comparisons between the control group and the other classes. In addition, multiple comparison was also carried out using the analysis of variance (ANOVA) test. All P values

underwent Benj amini-Hochberg adjustment to control the false discovery rate. In addition to the significance values, the area under the receiver operating

characteristics curve (AUC) value was computed. Results

Physicians consider the condition of patients prior to a surgery as factor. Beyond this, it would be of benefit to have a molecular marker that predicts a likely systematic response such as SIRS, in particular sepsis, prior to surgery. There is no molecular marker that allows to predict, prior to surgery, whether SIRS, in particular sepsis, is likely for an individual patient.

A cohort of patients prior to surgical intervention was measured and followed up. Then two groups were build, a group with normal recovery and a group developing a systematic response, including SIRS/sepsis. For the two cohorts, all human miRNAs were profiled and miRNA biomarkers that are already dysregulated prior to the intervention, i.e. predictive miRNA biomarkers, were indeed observed. The results are summarized in Figure 2.

Such miRNA biomarkers can be used to decide whether an intervention is postponed (in a non-acute care setting) or at least whether patients get special attention in an acute care seeing on an ICU.