A method for identifying a subset of polynucleotides from an initial set of polynucleotides correspo

专利摘要:
The present invention relates to a method for identifying a subset of polynucleotides from an initial set of polynucleotides corresponding to the human genome for in vitro determination of a severity of host response of a patient in an acute infectious and / or acute inflammatory condition A sample using a measuring device comprising a plurality of different gene probes representing the substantially entire human genome, the subjects being classified according to their infection and / or inflammation status into at least two of the clinically determined phenotype groups, the changes in gene expression signals be statistically compared between the phenotype groups and those gene probes are selected whose gene expression signals are statistically significantly changed between at least two phenotype groups. It determines a Master Score as a measure of Host Response severity and identifies a significantly reduced number of polynucleotides compared to baseline by establishing a score that does not exceed a given deviation from the Master Score. This score may be used to diagnose, predict the development or monitoring of an acute infectious and / or inflammatory condition of a patient and / or the course of the therapy and / or focus control.
公开号:CH706461B1
申请号:CH01508/13
申请日:2012-03-07
公开日:2016-12-15
发明作者:Möller Eva；Ruryk Andriy；Wlotzka Britta；Guillen Cristina；Felsmann Karen
申请人:Analytik Jena Ag；
IPC主号:

专利说明:

A subject not according to the invention relates to a method for identifying a subset of polynucleotides from an initial set of polynucleotides corresponding to the human genome for in vitro determination of the severity of a patient who is in an acutely infectious and / or acutely inflammatory state. The invention relates to the use of k-tuples of polynucleotides which are selected from the group consisting of m polynucleotides with SEQ ID No. 1 to SEQ ID 7704, where k is at least 7 and less than or equal to the number of polynucleotides m in the group is to record a score as a measure. for the severity of the host response of a test person who is in an acutely infectious and / or acutely inflammatory state, according to claim. The invention also relates to the uses of k-tuples on protein gene products from the polynucleotides according to claim 1.
Sepsis ("blood poisoning") is a life-threatening infection that affects the entire organism. It is associated with high mortality, occurs more and more frequently and affects people of all ages. Sepsis endangers medical progress in many areas of high-performance medicine and consumes a large part of the resources in health care. The mortality rate from severe sepsis has not improved significantly over the past few decades. The last two leaps in innovation after the introduction of blood culture (around 1880) were the introduction of antibiotics over 60 years ago and the beginning of intensive care medicine around 50 years ago. In order to achieve similarly decisive progress in treatment today, new types of diagnostics must be made available.
In the international literature, the criteria of the consensus conference of the American College of Chest Physicians / Society of Critical Care Medicine Consensus Conference (ACCP / SCCM) from 1992 have become the most widely accepted for the definition of the term sepsis [Bone et al., 1992]. The 2001 international sepsis conference also proposed a new concept (called PIRO) for describing sepsis, which is composed of the criteria of predisposition, infection, immune response and organ dysfunction [Levy et al., 2003]. Despite a new definition of SIRS / Sepsis with the acronym PIRO [Opal et al., 2005], the ACCP / SCCM consensus conference from 1992 is still used in most studies [Bone et al., 1992] to treat their patients classify.
Life-threatening bacterial infections and their consequences Sepsis and consecutive organ failure are common complications in hospital patients and are increasing worldwide by 2% to 7% per year. In Germany, 60,000 of the approximately 154,000 sufferers die each year from severe sepsis, which is one of the most common causes of death in intensive care units. Targeted antibiotic therapy, beginning in the first few hours after infection, is considered to be crucial for successful treatment [Ibrahim et al., 2000; Fine et al., 2002; Garracho-Montero et al., 2003; Valles et al., 2003]. The mortality rate is currently unacceptably high at 40% to 60%, although the risk is considerably increased in the case of delayed treatment based on resistance. The specific detection of pathogens in sepsis is currently unsatisfactory in clinical use. The “gold standard” blood culture suffers from a lack of sensitivity and remains negative in 80% to 90% of all sepsis cases. In addition, the blood culture results are only available after 24 to 72 hours and then only form the basis for further microbial diagnostics (species differentiation, creation of an antibiogram). Therapy with broad spectrum antibiotics is often initiated at the time of the first suspicion of sepsis without confirmed microbiological findings. On the one hand, this promotes the development of multi-resistant germs, and on the other hand, the antibiotic pretreatment reduces the success rate of a later blood culture. Epidemiological data show that in the case of inadequate therapy, mortality doubles [Valles et al., 2003] and, if the effective start of therapy is delayed, an increase in mortality of more than 5% per hour can be expected [Iregui et al., 2002; Ibrahim et al., 2000; Fine et al., 2002; Garnacho-Montero et al., 2003; Valles et al., 2003; Kumar et al., 2006].
The exact diagnosis of systemic-inflammatory and infectious disease states and their causes and associated risks for subsequent complications plays an important role in a number of other indications in addition to sepsis when making clinical decisions about the treatment of patients and subsequent monitoring of the course. The treatment of acutely and chronically ill patients as well as the peri-operative control can be seen in this context. It is known that in the case of acute pancreatitis the prognosis of a fatal outcome due to an infection worsens significantly from 16% to 40%. When a complex superinfection develops, there is a high risk of sepsis with a mortality of up to 90%. Furthermore, the follow-up of intra-abdominal inflammation and / or infection in chronically ill, post-operative and trauma patients is important. Even today there are difficulties in an unequivocal clinical diagnosis of intra-abdominal infections. Monitoring the course of the chronically ill, such as patients with cirrhosis of the liver or renal insufficiency, is of clinical relevance, since this group of patients can be predestined to take inflammatory and / or infectious disease courses depending on the level of organ compensation. In particular, patients with peritoneal dialysis with renal insufficiency are prone to chronic inflammation and infections [Blake, 2008]. The observation of patients with liver cirrhosis is of particular interest, as they can develop spontaneous bacterial peritonitis, which has a high mortality rate [Koulaouzidis et al., 2009]. The diagnosis of secondary peritonitis in the context of postoperative follow-up treatment is of great clinical value and can greatly influence the success of the operation. Postoperative infections are still a major problem in surgical treatment today. One percent of the laparotomies performed lead to complications after surgery. The complication rate can vary greatly between surgical procedures. Interventions in the gastrointestinal tract in particular can lead to a fulminant spread of bacteria into the sterile abdominal cavity due to suture insufficiency. Infectious courses also play a role in follow-up treatment after transplants, thoracotomies, extremity and joint corrections and neurosurgical interventions.
The person skilled in the art is aware that these statements are only an exemplary selection and that there are numerous other fields of application for which the detection and monitoring of the course of an infectious-inflammatory process and the assessment of its extent and the resulting risk of occurrence of corresponding complications is of great importance. The present invention provides a solution to this diagnostic problem.
The morbidity and lethality contribution of SIRS and sepsis is of interdisciplinary clinical-medical importance, because it is increasingly the treatment success of the most advanced therapy methods in numerous medical fields (e.g. traumatology, neurosurgery, heart / lung surgery, visceral surgery, transplant medicine, hematology) / Oncology etc.), for whom an increase in the risk of disease is inherent without exception due to unbalanced and uncontrolled infectious-inflammatory processes. This is also expressed in the continuous increase in the incidence of sepsis: between 1979 and 1987 an increase of 139%, namely from 73.6 to 176 cases of illness per 100,000 hospital patients was recorded [MMWR Morb Mortal Wkly Rep 1990]. The reduction in the morbidity and mortality of a large number of seriously ill patients is therefore linked to simultaneous progress in the prevention, treatment and, in particular, the detection and monitoring of the course of sepsis and severe sepsis.
In particular in patients who have survived the initial fulminant systemic inflammation response to highly virulent pathogens, there is a long-lasting phase with an increased risk of sepsis-induced multiple organ failure (up to several weeks). Induced immunosuppression is currently being discussed as the cause of this risk. In addition to the inability of the immune system to neutralize the primary infection, secondary infections with multiple antibiotic-resistant and less virulent germs often develop in intensive care patients or latent virus infections break out [Hotchkiss et al. 2009, 2010]. This results in a high clinical relevance, especially against the background of the increasing development of resistance to antibiotics and the lack of new effective antimicrobial agents. An important goal of clinical research is therefore the prevention of this sepsis-induced immunosuppression. Molecular targets for an intervention in the form of molecular immunotherapy are known (such as IL-15 and IL-7 as anti-apoptotic and immunostimulatory cytokines), but initial clinical studies show that the therapy decision should be based on the individual immune status. The close monitoring of innate and adaptive immune functions in further studies requires new and more complex measuring tools for immunosuppressed patients in order to shift the balance between pro- and anti-inflammatory signals in favor of the patient.
Mechanisms of immunosuppression are currently considered to be: production of anti-inflammatory cytokines, e.g. IL-10, this can induce the development of the so-called T-cell anergy (non-responsive behavior) - Dying of immune cells • Apoptotic depletion of immune effector cells (e.g. lymphocytes and dendritic cells) • The restoration of the normal population of specific cells is apparently associated with an improved prognosis - suppression of MHC class II molecules (suppression of induction of the adaptive immune response) - expression of negative costimulatory molecules (PD-1, CTLA-4)
A study [Meisel et al. 2009] provides the first example of a therapeutic intervention in immunocompromised patients. Molecular surface markers of monocytes (HLA-DR) were used to obtain information about the immune status. A greatly reduced monocyte population is a characteristic indicator of sepsis-associated immunosuppression (also shown in Venet et al. 2010). If HLA-DR expression in the blood of patients was low, they were treated with the growth factor GM-CSF or a placebo. GM-CSF has strong immunostimulatory properties, in particular phagocytosis, proliferation and the pathogen defense of neutrophils and monocytes / macrophages are stimulated. Previous studies have shown that immunostimulants can reverse long-term monocyte deactivation. As a result of the study, a numerical increase could be demonstrated for many immune cell populations: Both the monocytes and the neutrophils and lymphocyte populations benefited from the treatment. The patients in the treatment group also showed improvements in their clinical condition: shorter ventilation times and hospital stay became apparent. For the first time, the positive influence of a biomarker-guided immunostimulatory therapy could be demonstrated on the immunological and clinical level.
In a further study, the immunosuppressive phase of sepsis was characterized in more detail [Muenzer et al. 2010]. The initial hyperinflammatory phase, which can be associated with a so-called cytokine storm, resulting early organ damage and death of the patient, is followed by a phase with persistent immunosuppression. The balance of the immune system is therefore disturbed in both phases, the importance of an intervention in the second phase is underlined by the occurrence of secondary infections and extremely high mortality. In the mouse model it could be shown how the IL-10 synthesis could be blocked with an immune modulator and the production of proinflammatory cytokines could be stimulated during the course of sepsis for 7 days. In particular, it was found that the time of a so-called “second hit”, ie a second infection, is of decisive importance for the survival of the organism. The immune paralysis status lasted for 4 days in the mouse model, and on day 7 after the septic stimulus the immune response was partially restored. This was expressed in the survival rate of the animals after a septic stimulus, which was higher after 7 days than after 4 days. In the hypoinflammatory time window (4 days) the survival rate could also be increased by the immunomodulator AS101 or by blocking IL-10. Until the immune status normalizes, the innate immune cells return and the pro- and anti-inflammatory signal cascades are balanced, a critical gap arises with a high risk of further infections and the patient's survival.
Another study describes drastic changes in lymphocyte populations in patients with septic shock [Venet et al. 2010]. The fact that these extensive changes can mostly be detected at diagnosis suggests that they are a very early event in the chain of processes leading to immune paralysis and predisposition to further infection. In studies with patients, the exact start of the septic episode cannot be precisely defined, which means that the study populations cannot be reliably synchronized with regard to the course of the disease. However, it was found that after the onset of the septic shock, the immunosuppressed state lasted for about 48 hours despite intensive medical measures. There is an enormous need for a monitoring tool to qualify patients for immunomodulatory interventions.
The immune status of patients diagnosed with sepsis is therefore severely impaired. The impairment affects both the innate and adaptive immune systems. Features of this impairment are the loss of immune effector cells from the peripheral bloodstream due to apoptosis, the decrease in the expression of MHCII molecules and the decrease in the ability of monocytes to be stimulated by cytokines. This impairment of the immune status can be reversible. The consequence of the impairment is, on the one hand, the inability to eliminate infections and to control an infection focus so that it remains active. In addition, there is a high probability of developing secondary, nosocomial infections. Such infections are often caused by less pathogenic bacteria that do not pose a threat if the immune system is intact.
The macroscopic post-mortem examination of 235 critically ill patients whose cause of death was sepsis or septic shock showed that an active focus of infection was found in 80% of these cases [Torgersen et al., 2009]. The organs most commonly affected were the lungs, abdomen, and genitourinary tract. A large number of these patients were transferred to the intensive care unit because of a sepsis diagnosis and treated there for more than 7 days before their death. This period can be considered long enough to bring a focus of infection under control. Although immediate focus control in combination with antibiosis are the central measures of sepsis therapy, the focus control measures were unsuccessful in the majority of the study patients and seem to have been the cause of death. The recommendation of the authors of the publication is to develop better diagnostic and therapeutic methods to meet the medical needs in this area.
In a recently published study it is concluded that about 20% of the patients who are admitted to the clinic with a suspicion of sepsis, after careful examination, actually have non-infectious causes of the disease, but the appearance of sepsis same. The authors interpret their results to the effect that sepsis rather encompasses the continuum of a syndrome and is not a delimited specific disease [Heffner et al., 2010].
In a cohort of 857 patients, the endotoxin level was examined on the day of their admission to the intensive care unit. It was found that endotoxemia, a significantly increased level of endotoxin in the patient's blood, is widespread in critically ill patients. In more than half of all examined patients, an endotoxin level higher than 2 standard deviations of the value determined in healthy volunteers was measured. At the same time, a large discrepancy between a high endotoxin value and the number of confirmed infections with gram-negative pathogens was observed. It is concluded that the origin of the endotoxin must be of an endogenous nature and must lie in the intestinal flora, whereby both endotoxin and viable bacteria can enter the bloodstream due to translocation processes. High endotoxin values were correlated with higher APACHE II scores and a higher prevalence of severe sepsis, so that it is assumed that endotoxemia indicates a high-risk subpopulation in critically ill patients [Marshall et al., 2004].
Endotoxemia can also be viewed as a cause of excessive stimulation of the immune system.
A review gives an overview of clinical and immunological parameters that determine the risk of developing a septic complication and lethal consequences after severe operations and trauma [Kimura et al., 2010]. The current state of the art suggests that surgical interventions and traumatic injuries affect the so-called innate and adaptive immune response so severely that a suppression of the body's cellular immunity as a result of an excessive inflammatory reaction is responsible for the high susceptibility of a subsequent septic episode. The reaction cascades of the innate and adaptive immune response are initiated and modulated by so-called pathogen-associated molecular structures (PAMPS) and tissue damage-associated molecular structures (DAMPS) by the corresponding recognition receptors.
The spectrum of the disease process, which is thus covered by the invention, is the progression of an infectious-inflammatory reaction of the body, also called the host response, from the ability to effectively fight pathogens to the suppression of the immune defense, in which the pathogens at the Infection site persists and secondary and / or nosocomial infections occur.
When using molecular diagnostic DNA-based pathogen identification, clinically irrelevant results such as non-disease-associated bacteremia, the presence of freely circulating bacterial and fungal nucleic acids from colonization and the detection of non-vital pathogen cells are problematic for the evaluation of the result. The presence of circulating microbial DNA from translocation processes or the transient presence of non-disease-associated bacteria in blood have been demonstrated in vivo [Dagan et al., 1998; Isaacman et al., 1998]. The origin and the clinical significance of such false-positive results are mostly unclear and could result from previously unknown host-pathogen interactions [Schrenzel, 2007]. In addition, cases are known in which bacteria have been isolated from the blood of symptom-free blood donors, and even transient fungemia without visible clinical significance has been reported [Davenport et al., 2007, Rodero et al., 2002].
In the “unclear” cases just described, the measurement of the defined immune state can be used to more reliably assess the significance and clinical relevance of the findings from DNA-based pathogen detection.
The subject matter of the invention can be summarized as follows. Excessive stimulation of the immune system by PAMPS and DAMPS, e.g. due to an uncontrolled focus of infection or excessive inflammation after a serious surgical procedure, affects the innate and adaptive immune system. The resulting reaction of the body, also known as the host response, or the resulting "immune stress" depends on the extent, amount, duration and / or frequency of the infectious and / or inflammatory stimulation. This stimulation cannot be measured directly, but rather as a response of the body to the stimulation, as the severity of the host's response. This reaction shows a continuous change in the form of an increase from the healthy state to a maximum, as e.g. in the extreme case of bloodstream infection is present. A large number of studies have shown that in this state the body no longer has the protective mechanisms of the immune system. Existing infections can no longer be fought effectively. There is a high risk of developing a secondary infection at this stage. This is especially true in cases where sterile processes were the cause of the induction of immunosuppression. Clinical measures such as Identification of the cause of the excessive stimulation, control of the source of infection, operative focus adjustment, targeted antibiosis or preventive drug therapies to end / block them must be initiated promptly in these cases. The invention provides a diagnostic test that can be used to determine and monitor the course of the disease process described and to monitor the success of the therapeutic measures taken.
The excessive stimulation can have the following causes: - uncontrolled focus of infection - insult due to sterile inflammatory events • tissue damage from surgery • tissue damage from trauma • necrotic processes - endotoxemia • translocation from the intestine as a result of a serious pathological occurrence
The processes mentioned lead to transient, induced immunosuppression of the innate and adaptive immune system and are correlated with:High mortality due to an uncontrolled focus of infectionHigh risk of nosocomial or secondary infection with life-threatening complications
This pathophysiological occurrence must be countered by clinical measures suitable for the respective case:Identification of an infection focus by escalating the diagnostic measuresFocus restructuring, also through operative interventionsTargeted antibiosis for known pathogensCalculated antibiosis to prevent secondary infectionImmunostimulatory therapeutic measuresAnti-inflammatory therapeutic measures for sterile infectious events
Several approaches to diagnosing SIRS and sepsis have been developed.
One group contains score systems such as APACHE, SAPS and SIRS, which can stratify the patient on the basis of a large number of physiological indices. While some studies have demonstrated diagnostic potential for the APACHE II score, other studies have shown that APACHE II and SAPS II cannot differentiate between sepsis and SIRS [Carrigan et al., 2004].
In a review, Pierrakos and Vincent [Pierrakos et al., 2010] summarize the status of the biomarker search in the indication of sepsis. 3370 publications on 178 different biomarkers were viewed. The conclusion is that most biomarkers have been studied primarily in clinical trials and primarily as prognostic markers. Few have been tested as diagnostic markers. None of these candidates have demonstrated sufficient sensitivity or specificity for routine use in the clinic. No marker was tested for one question about a patient's immune status. Procalcitonin (PCT) and C-reactive protein (CRP) are used, but also show only limited properties for differentiating between sepsis and other inflammatory conditions, or for predicting certain outcomes.
Procalcitonin is a 116 amino acid long protein that plays a role in inflammatory reactions. Despite the widespread acceptance of the PCT biomarker, international studies have shown that the sensitivities and specificities achieved by the PCT sepsis marker are still inadequate, especially when differentiating between systemic bacterial SIRS, i.e. sepsis, and non-bacterial SIRS [Ruokonen et al ., 1999; Suprin et al., 2000; Ruokonen et al., 2002; Tang et al., 2007a]. The meta-analysis by Tang and colleagues [Tang et al., 2007a], in which 18 studies were taken into account, shows that PCT is only poorly suited to discriminating SIRS from sepsis. In addition, the authors emphasize that PCT has a very poor diagnostic accuracy with an Odd Ratio (OR) of 7.79.
C-reactive protein (CRP) is a 224 amino acid long protein that plays a role in inflammatory responses. The measurement of CRP should serve to track the course of the disease and the effectiveness of the selected therapy.
Several reports have described that PCT is a more suitable diagnostic marker than CRP in the intensive care sector [Sponholz et al., 2006; Kofoed et al., 2007]. In addition, PCT is considered to be more suitable than CRP to differentiate between non-infectious versus infectious SIRS and bacterial versus viral infection [Simon et al., 2004].
It will be apparent to those skilled in the art that the solution provided by this invention can with the aforementioned biomarkers such as e.g. but not exclusively PCT or CRP can be combined in order to expand the diagnostic information.
Another group contains biomarkers or profiles that have been identified at the transcriptome level. Gene expression profiles or classifiers are used to determine the severity of sepsis [WO 2004/087 949], to distinguish between a local or systemic infection [unpublished DE 10 2007 036 678.9], to identify the source of infection [WO 2007/124 820] or from Gene expression signatures suitable for differentiating between several etiologies and pathogen-associated signatures [Ramilo et al., 2007]. However, due to the insufficient specificity and sensitivity of the consensus criteria according to [Bone et al., 1992], the currently available protein markers and the time required to detect the cause of infection by blood culture, there is an urgent need for new methods that take the complexity of the disease into account. Many gene expression studies that use either individual genes and / or combinations of genes that are named as classifiers, as well as numerous descriptions of statistical methods for deriving a score and / or index [WO 2003/084 388; US 6,960,439] belong to the prior art.
When using gene expression markers for the determination of a pathophysiological state, the amounts of the corresponding mRNA present in a sample, the gene expression level, are always determined quantitatively. The information determined by these gene expression levels is the respective over- or under-expression of these mRNAs, which is determined experimentally in relation to a control state or in relation to control genes. The determination of over- or under-expression can be seen analogously to the determination of the concentration of a protein biomarker.
Several uses of gene expression profiles are known in the art.
Pachot and colleagues [Pachot et al., 2006] examined whether differential gene expression from whole blood could be used to predict the outcome variable survival vs. non-survival in septic shock patients. To answer this question, they identified a signature of 28 differentially expressed genes by screening on an Affimetrix array. In a very small test data set, they showed that this signature could be used to differentiate between survivors and non-survivors with high sensitivity and specificity. For the plausibility of the result, they argue that the late phase of septic shock is characterized by the development of an immunosuppressed state and that restoration of immune function is necessary for the patient's survival. They point out that a number of the overexpressed genes in survivors are to be assigned to the innate immune system and justify the observed overexpression with the recovery of the immune system.
US 2008/0 020 379 A1 relates to the diagnosis and prognosis of infectious diseases, clinical phenotypes and other physiological conditions using host gene expression biomarkers in the blood.
According to the abstract, US 2008/0 020 379 A1 is concerned with the fact that specific sets of gene expression markers from peripheral blood (leukocytes) can provide an indication of a host response to exposure, response and recovery from infections with infectious pathogens.
In US 2008/0 020 379 A1 reference is only made in a general way to the fact that it is possible with the unique technology of US 2008/0 020 379 A1 to diagnose a large number of different diseases. Furthermore, in [0293] on page 22, right column of US 2008/0 020 379 A1, reference is only made to the usual statistical methods.
In paragraph [0324] on page 24, US 2008/0 020 379 A1 lists possibilities in the field of diagnostics and here refers to inflammatory diseases, 48 inflammatory genes for rheumatoid arthritis being used which come from a commercial source, namely « Source Precision Medicine ». Paragraph [0608] on page 45, right column to page 46, left column, first paragraph, contains a list of genes which was carried out as a “batch search” in the “Genetic Association database”.
Paragraph [0533] on page 44, left column, discloses that the biomolecular pathways which are differentially expressed at the cellular level can distinguish between adenovirus infections and non-adenovirus infections. In order to find these paths, reference was then made to the analysis following in Paragraph [0533] using the KEGG path and the Genetic Association databases using EASE (70) in order to examine the functions of these genes with regard to molecular issues.
The above-mentioned list of genes in paragraph [0608] also belongs to this and thus US 2008/0 020 379 A1 relates to a distinction between adenovirus infections and non-adenovirus infections.
Nowhere in the description of US 2008/0 020 379 A1 is the test subjects divided into local and systemic depending on their infection and / or information status.
Document US 2009/0 307 181 A1 relates to genetic analyzes and the determination of genetic health scores for specific phenotypes, such as diseases, disorders, treatments and conditions, both for organ systems and for certain medical specialties and the overall state of health.
In paragraph [0195] of US 2009/0 307 181 A1, with reference to FIGS. 15 to 24, 26 to 33 and 39, it is said that so-called panels of phenotype groups can be examined within the scope of this document. The panels mentioned are of a general nature and relate - without individual evidence - to more or less the entire clinical diagnosis and include, for example, inflammatory diseases as diverse as gastrointestinal diseases of unclear aetiology, viral hepatitis, rheumatoid arthritis, systemic lupus erythematosus, malaria, chronic obstructive pulmonary diseases, autoimmune diseases and an infection panel (page 42, right column). In FIG. 15R of US 2009/0 307 181 A1, for example, rheumatoid arthritis is treated with a list of genes in column 2 and so-called “reflex testing phenotypes”. However, no subdivision into systemic or non-systemic was made, but the risk of developing rheumatoid arthritis in connection with cigarette smoke exposure is investigated.
15V of US 2009/0 307 181 A1 includes Crohn's disease as an inflammatory bowel disease and / or ulcerative colitis. Furthermore, age and onset of Crohn's disease and the location and / or severity of the colitis are listed as phenotypes. In paragraph [0232] reference is made to a set of phenotypes which can be identified according to US 2009/0 307 181 A1 for the connection between infectious diseases and pulmonology. Such phenotypes can include two or more phenotypes. However, reference is only made to general panels, for example the World Infectious Disease Panel, HIV Panel, Malaria Panel, Viral Hepatitis Panel, Infection Panel, etc.
In paragraph [0408] on page 94, left column of US 2009/0 307 181 A1, among many other possibilities, for example, in addition to atrial fibrillation, acute and chronic infections, sepsis and SIRS are mentioned.
The abundance of examples given in US 2009/0 307 181 A1, most of which are available without biostatistical data, mean that the person skilled in the art lacks a reproducible teaching. This view is supported, for example, by feature b) in claim 1 of US 2009/0 307 181 A1, where it says "using a computer to determine the predisposition or carrier status of said individual for at least two phenotypes ..." using a computer to determine the predisposition or carrier status of the individual for two phenotypes ... »]. Since no reference is made to any algorithms as to how this determination is to be carried out, the teaching of US 2009/0 307 181 A1 - if it can be recognized at all - is not comprehensible and cannot be carried out.
Document US 2010/0 293 130 A1 relates to genetic analysis systems and methods for this. According to the abstract, this document is essentially concerned with determining methods for determining a genetic composition index score for estimating the connection between the genotype of an individual and at least one disease or condition.
[0050] In particular, US 2010/0 293 130 A1 compares the gene profile of an individual with a database of medically relevant genetic variations which was created in order to be associated with a specific disease or a specific pathophysiological condition.
In paragraph [0115] on page 12, right column, document US 2010/0 293 130 A1 discloses that a specific phenotype can be associated with corresponding genotypes correlated therewith. According to US 2010/0 293 130 A1, this can include Crohn's disease, lupus, psoriasis and rheumatoid arthritis as inflammatory diseases.
Claim 1 of US 2010/0 293 130 A1 relates to a general method for generating at least one genetic composition index score based on a phenotype gene correlation without explicitly specifying which genes are to be used. For lupus and rheumatoid arthritis, in addition to a number of other diseases, claim 133 on page 33 indicates that a special gene expression profile is used to indicate between an SNP and a phenotype and a list of specific SNPs that associate with a certain phenotype.
Boldrick et al. (2002): Stereotyped and specific gene expression programs in human innate immune responses to bacteria, PNAS 99, 972-977 describes in particular on page 973, left column, last paragraph the host response to immunological provocation with gram-negative bacteria, examined using a group of 206 Genes, however, are not found anywhere in Boldrick et al. (2002) a phenotype classification in local and systemic.
Tang et al. (2007b): The Use of Gene Expression Profiling to Identify Candidate Genes in Human Sepsis, Am J Respir Crit Care Med 176, 676-684 relates to the use of gene expression profiles to identify gene candidates in human sepsis.
According to the abstract and the box on page 676, right column relates to Tang et al. (2007b) diagnosed sepsis using gene expression profiles and also speaks of a mechanistic biological insight into the host response in sepsis.
Tang et al. (2007b) thus concerns the “classic” approach of looking for certain “lead genes” for sepsis and correlating their gene expression with a prediction of the course of sepsis.
This follows from the fact that a set of 50 signature genes according to Tang et al. (2007b) correctly identified sepsis, with a prediction probability of 91% and 88% for the training and validation sets. Tang et al. (2007b) also claim that certain genes that play a role in immune modulation and inflammatory response show decreased expression in sepsis patients.
In particular, Tang et al. (2007b) found that the activation of the core factor Kappa B metabolic pathway was reduced, whereas the corresponding inhibitor gene NFKBIA was significantly upregulated.
Tang et al. (2007b) have concluded that the signature genes found suppress a suppression of the immune and inflammatory function of the neutrophils in sepsis. According to the authors of Tang et al. (2007b) thus offer gene expression profiles a new approach to understand the host response in sepsis.
According to page 678 of Tang et al. (2007b), using the keyword “Statistical Analysis”, it is shown that the authors developed a prediction model for sepsis using the data from the training set. According to page 679, left column, paragraph below the table and FIG. 3A, Tang et al. (2007b) identified three clusters of coordinated genes. According to the heat map in FIG. 3A, these clusters relate to a mitochondrial function cluster, an immune regulation cluster and an inflammatory response cluster.
Nowhere within Tang et al. (2007b), however, it can be seen that phenotype groups are to be formed according to claim 1 of the present application.
Warren et al. (2009): A Genomic Score Prognostic of Outcome in Trauma Patients, Mol Med 15, 220-227 relates to a genomic score that is supposed to be prognostic for the outcome in trauma patients.
Finally, Xu et al. (2010): Human transcriptome array for high-throughput clinical studies, PN AS 108, 3707-3712 a transcriptome array for high-throughput in clinical studies and describes in particular oligonucleotide arrays with 6.9 million oligonucleotides.
The present invention can be distinguished from the prior art discussed at the beginning. The object of the invention is to determine and monitor the course of the body's reaction to infectious and / or inflammatory stimulation, also referred to as the host response, or the "immune stress" resulting therefrom. It is independent of the presence of septic shock and is not restricted to this patient group. It is used to determine a certain condition and not to distinguish between survival vs. non-survival after septic shock. The present invention is also independent of the presence of an infection in accordance with the current definition of sepsis. It is shown in the accompanying document that a critical condition, a maximum "immune load" even without infection, e.g. excessive stimulation of the innate system by other causes may be present. The use of the invention lies in deriving suitable therapeutic measures and monitoring the progress, but not in predicting which of the patients will survive.
In a review [Monneret et al., 2008], the importance of the effectively functioning immune system is presented on the basis of a series of scientific results and summarized by which investigations this assumption is made plausible. At the same time, it is stated that suitable procedures for the routine determination of the immune status still have to be determined.
The prior art contains numerous studies on the identification of gene expression markers [Tang et al., 2007b] or gene expression profiles for the determination of a systemic infection [Johnson et al., 2007].
Tang and colleagues [Tang et al., 2007b] searched a certain blood cell population, the neutrophils, for a signature that enables a distinction between patients with SIRS and sepsis. 50 markers from this cell population are sufficient to reproduce the immune response to a systemic infection and to enable new insights into the pathophysiology and the signaling pathways involved.
The classification of patients with and without sepsis succeeds with a high degree of certainty (PPV 88% and 91% in the training and test data set). The applicability for clinical diagnosis is limited by the fact that this signature can be superimposed in the blood by signals from other blood cell types. In terms of applicability, the preparation of this blood cell population is associated with considerably increased effort. However, the informative value of the results published in this study is limited for practical applications because the patient selection was very heterogeneous. The study included patients with widely differing comorbidities such as 11% to 16% had tumor diseases or were subject to very different therapeutic measures (e.g. 27% to 64% vasopressor therapy), which strongly influenced the gene expression profiles.
Johnson and colleagues [Johnson et al., 2007] describe in a collective of trauma patients that the severity of sepsis can be measured up to 48 hours before the clinical diagnosis on the basis of molecular changes. The trauma patients were examined over several days. Some of the patients developed sepsis. Non-infectious SIRS patients were compared with preseptic patients. The identified signature from 459 transcripts is composed of markers of the immune response and inflammation markers. Sample material was whole blood, the analyzes were carried out on a microarray. It is unclear whether this signature can also be extended to other collectives of septic or preseptic patients. A classification and the diagnostic benefit of this signature has not been shown.
This work is all aimed at the identification of an infectious event by differential gene expression. Thus, these publications can be well delimited from the subject matter of the present invention, the identification and progress monitoring of the body's response to infectious and / or inflammatory stimulation, also referred to as the host response, or the resulting “immune stress”
The aim of Feezor and colleagues [Feezor et al., 2003] was to identify differences between infections with gram-negative and gram-positive pathogens. Blood samples from three different donors were stimulated ex vivo with E. coli LPS and heat-inactivated S. aureus. Gene expression studies were carried out using microarray technology. The working group found both genes that were upregulated after S.aureus stimulation and downregulated after LPS stimulation, as well as genes that were more strongly expressed after LPS treatment than after the addition of heat-inactivated S.aureus germs. At the same time, many genes were upregulated to the same extent by gram-positive and gram-negative stimulation. This applies, for example, to the cytokines TNF-α, IL-1β and IL-6. The differentially expressed genes were unfortunately not published by name, so that only an indirect comparison with other results is possible. In addition to gene expression, Feezor and colleagues also examined the plasma concentrations of some cytokines. The gene expression data did not necessarily correlate with the plasma concentrations. The amount of mRNA is measured during gene expression. However, this is subject to post-transcriptional regulation before protein synthesis, from which the observed differences can result.
The most interesting publication on this topic was published by a Texas research group led by Ramilo [Ramilo et al., 2007]. Here, too, gene expression studies were carried out on human blood cells, which revealed differences in the host's molecular response to various pathogens. Pediatric patients with acute infections such as acute respiratory diseases, urinary tract infections, bacteremia, local abscesses, bone and joint infections and meningitis are examined. Microarray experiments were carried out with RNA samples isolated from peripheral mononuclear blood cells from ten patients each with E. coli and S. aureus infection. The pathogen was identified by means of blood culture. On the basis of the training data set, 30 genes were identified, through the use of which the causative pathogenic germs could be diagnosed with high accuracy.
This work can be clearly distinguished from the present invention, since here the causative pathogens are to be determined on the basis of gene expression signatures of the host response, but the invention is intended to determine and monitor the course of the body's reaction to infectious and / or inflammatory stimulation or the the resulting «immune stress» should be used.
None of these publications offer the reliability, accuracy, and robustness of the invention disclosed herein. These studies concentrate on identifying the “best” multigene biomarker (classifier) from a scientific point of view, but not, as in the present invention, the optimum multigene biomarker for a specific clinical problem [Simon et al., 2005].
It is therefore the object of the present invention to provide a test system with which a quick and reliable statement about a pathophysiological condition, in the present case it is the determination and monitoring of the reaction of the body to infectious and / or inflammatory stimulation, also referred to as the host response, or the resulting «immune stress» can be met, but without having to rely on condition-specific biomarkers.
This object is achieved by the features of claim 1.
In particular, an object not according to the invention relates to a method for identifying a subset of polynucleotides from a starting set of polynucleotides corresponding to the human genome for in vitro determination of the severity of the host response of a patient who is in an acutely infectious and / or acutely inflammatory state is located in a sample, using a measuring device having a plurality of different gene probes which represent essentially the entire human genome; in whichNucleic acid samples from a plurality of test subjects who have a known phenotypic physiological state are brought into contact with the probes of the measuring device in order to obtain expression signals of a respective expression of a gene;From the total number of gene probes used, those are selected which deliver an expression signal with a detectable intensity for at least one nucleic acid sample of a subject;the subjects are divided into at least two of the following clinically determined phenotype groups depending on their infection and / or inflammation status:
[0078]
where «a» represents an AND link between the properties S, L and N;the changes in the gene expression signals between the phenotype groups are statistically compared and it is assessed whether there is a significant difference between at least two of the phenotype groups;those gene probes are selected whose gene expression signals are statistically significantly changed between at least two phenotype groups and an estimated number of such gene probes is excluded which give a false positive result in relation to a predetermined threshold value;a master score as a measure of the severity of the host response of a subject who is in an acutely infectious and / or acutely inflammatory state is determined by quantifying the increase and decrease in the gene expression intensity of the selected gene probes; anda number of polynucleotides that is significantly reduced compared to the initial amount is identified by determining a score which has at most a predetermined deviation from the master score and which is also used as a measure of the severity of the host response of a subject who is in an acutely infectious and / or acute state inflammatory condition is used.
The present invention relates to the use of k tuples on polynucleotides which are selected from the group consisting of m polynucleotides with SEQ ID No. 1 to SEQ ID 7704, where k is at least 7 and less than or equal to Number of polynucleotides is m in the group; to record a score as a measure of the severity of the host response of a subject who is in an acutely infectious and / or acutely inflammatory state.
Use of k-tuples of polynucleotides which are selected from the group consisting of m polynucleotides with SEQ ID No. 1 to SEQ ID 7704, where k is at least 7 and less than or equal to the number of polynucleotides m in the group is; for carrying out the method according to the invention.
The subclaims relate to preferred embodiments of the present invention.
In the applicant's practice, it has been found that such a use is particularly suitable, which is characterized in that the gene activities by means of enzymatic methods, in particular amplification methods, preferably polymerease chain reaction (PCR), preferably real-time PCR, especially probe-based methods such as Taq-Man, Scorpions, Molecular Beacons; and / or by means of hybridization processes, in particular those on microarrays; and / or direct mRNA detection, in particular sequencing or mass spectrometry; and / or isothermal amplification, can be detected [Valasek et al., 2005; Klein, 2002]. This classic method can be used to detect highly sensitive DNA and, via reverse transcription (RT), also RNA [Wong et al., 2005; Bustin, 2002].
Real-time PCR, also known as quantitative PCR (qPCR), is a method for the detection and quantification of nucleic acids in real time [Nolan et al., 2006]. It has been an established standard technique in molecular biology for several years.
The quantitative determination of a template can take place by means of absolute or relative quantification. In the case of absolute quantification, the measurement is based on external standards, e.g. Plasmid DNA in different dilutions instead. On the other hand, relative quantification uses so-called housekeeping or reference genes as reference [Huggett et al., 2005].
For the method according to the invention (array technique and / or amplification method) the sample is selected from: tissue, body fluids, in particular blood, serum, plasma, urine, saliva or cells or cell components; or a mixture of these.
It is preferred that samples, in particular cell samples, are subjected to a lytic treatment in order to release their cell contents.
It is clear to the person skilled in the art that the individual features of the invention set out in the claims can be combined with one another as desired without restriction.
Further advantages and features of the present invention emerge from the description of exemplary embodiments and from the drawing.
Another subject matter not according to the invention is a use which is characterized in that an index is formed from the individual specific gene activities which, after appropriate calibration, is a measure of the severity and / or the course of the pathophysiological condition, preferably the index is displayed on an easily interpretable scale.
It is also preferred that the gene activity data obtained are used to produce software for the description of at least one pathophysiological condition and / or an investigation question and / or as an aid for diagnostic purposes and / or for patient data management systems, in particular for use in patient stratification and as an inclusion criterion for clinical studies.
In addition, a use is preferred in which, to create the gene activity data, such specific gene loci, sense and / or antisense strands of pre-mRNA and / or mRNA, small RNA, in particular scRNA, snoRNA, micro RNA, siRNA, dsRNA, ncRNA or transposable elements, genes and / or gene fragments with a length of at least 5 nucleotides are used, which have a sequence homology of at least about 10%, in particular about 20%, preferably about 50%, particularly preferably about 80% to the Polynucleotide sequences according to SEQ ID No. 1 to 7704 have.
The sample nucleic acid is preferably RNA, in particular total RNA or mRNA, or DNA, in particular cDNA.
It must be emphasized, however, that the primers mentioned are only exemplary.
The amplicons mentioned can be used, for example, as probes for hybridization methods.
As part of an optimized computerized hospital management as well as for further research in the field of sepsis, it has been found to be advantageous that the gene activity data obtained for the production of software for the description of at least one pathophysiological condition and / or an investigation question and / or as an aid for diagnostic purposes and / or for patient data management systems.
The multigene biomarker is preferably a combination of several polynucleotide, in particular gene sequences, on the basis of whose gene activities a classification is carried out by means of an interpretation function and / or an index or score is formed.
For the purposes of the present invention it has also been found to be advantageous that the gene activities can be carried out by means of enzymatic processes, in particular amplification processes, preferably polymerease chain reaction (PCR), preferably real-time PCR; and / or by means of hybridization methods, in particular those on microarrays.
Differential expression signals of the polynucleotide sequences contained in the multigene biomarker that occur during the detection of the gene activities can advantageously and unambiguously be assigned to a pathophysiological condition, a course and / or therapy monitoring.
This score can provide the attending physician with a quick diagnostic aid.
[0100] The applicant has developed several methods that use different sequence pools to determine and / or differentiate states or to answer defined examination questions. Examples can be found in the following patent specifications: Differentiation between SIRS, sepsis and sepsis-like conditions [WO 2004/087 949; WO 2005/083115], creation of criteria for predicting the course of the disease in sepsis [WO 05/106 020], differentiation between non-infectious and infectious causes of multi-organ failure [WO 2006/042 581], in vitro classification of gene expression profiles of patients with infectious disease / non-infectious multi-organ failure [WO 2006/100 203], determination of the local causes of a fever of unclear genesis [WO 2007/144 105], polynucleotides for recording gene activities to differentiate between local and systemic infection [DE 10 2007 036 678.9].
With regard to the nucleotide sequences used in the present application, the following should be noted:RefSeq is a public database that contains nucleotide and protein sequences with their properties as well as bibliographic information.The RefSeq database was created by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine belonging to the US National Institute of Health, and is continuously maintained and updated [Pruitt et al., 2007].NCBI creates RefSeq from the sequence data of the archive database «GenBank» [Benson et al., 2009], an extensive public database of sequences that are set up by GenBank in the USA, the EMBL data library in Great Britain and the DNS database of Japan and also be exchanged between these databases.The RefSeq collection is unique when it comes to providing error-corrected, non-redundant, explicitly linked nucleotide and protein databases. The entries are non-redundant with the aim of representing the various biological molecules that are characteristic of the organism, strain or haplotype.
If certain entries appear multiple times in the collection, there can be several reasons for this:alternative spliced transcripts code for the same protein product (so-called transcript variants),there are several genomic regions within a species or between species which code for the same protein product,when RefSeqs are created that represent alternative haplotypes, and some mRNA and protein sequences are identical in all haplotypes.
RefSeq database provides the critical foundation for sequence integration, genetic and functional information and is internationally recognized as the standard for genome annotation. When searching for sequences by BLAST, RefSeq information is available in several NCBI resources including Entrez Nucleotide, Entrez Protein, Entrez Gene, Map Viewer, by FTP download; or by networking with PubMed [Pruitt et al., 2007; The NCBI handbook 2002]. RefSeq information can be identified by the unique accession format, which contains the underscore (_).Working groups use different methods and protocols and compile the RefSeq collection for different organisms. RefSeq records are produced by several different methods [The NCBI handbook 2002]:1. Scientific cooperation2. Computer-aided genome annotation methods3. Error correction by the NCBI staff4. Extracts from GenBank
Each item of information has a comment which shows the status of the respective error correction and the assignment of the cooperating working group. As a result, the RefSeq information contains either the essentially unchanged, initially valid copy of the original GenBank entries or corrected and additional information that was added by cooperation partners or experts [The NCBI handbook 2002].If a molecule is represented by several sequences in GenBank, the NCBI staff will make a decision on the “best” sequence, which is then presented as RefSeq.
The decision to use the marker population named in the present application on the basis of its RefSeq identity for the purposes of the present invention was made on the basis of the properties of the RefSeq database described above. The characteristics of this database, the creation, quality, maintenance and updates of the biological sequences, as well as the availability of functional information at the nucleic acid level, also for alternative splice variants, were decisive.
As already explained, the biological mechanism of the alternative splicing offers scope for extensions of the scope of protection which are well known to those skilled in the art. It is conceivable that completely new primary structures will be identified with new transcript variants or that sequence changes of the known transcript variants will result. On the other hand, the genomic regions are claimed which encompass all these known and unknown variants of the coding transcripts, together with their cis-regulatory sequences, as complete genomic functional units and thus fall within the scope of the present invention or at least equivalents to those in make available to the claims, description and the sequences mentioned in the sequence listing.
Definitions:
For the purposes of the present invention, the following definitions are used:
SIRS:
Systemic Inflammatory Response Syndrome, according to Bone [Bone et al., 1992] and Levy [Levy et al., 2003], a generalized, inflammatory, non-infectious condition of a patient.
sepsis
According to Bone [Bone et al., 1992] and Levy [Levy et al., 2003], a generalized, inflammatory infectious condition of a patient.
Inflammation / Inflammation:
It is a reaction of the body, caused by injury or tissue destruction, that serves to clear, thin, or isolate the injuring agent or tissue.The inflammatory process can be caused by physical, chemical, or biological agents, including mechanical trauma, exposure to sun, x-ray and radioactive radiation, corrosive chemicals, extreme heat or cold, and infectious agents such as bacteria, viruses, fungi and other pathogenic organisms. However, inflammation and infection cannot be used as synonyms.The classic signs of inflammation are warmth, redness, swelling, pain, and loss of function in the affected tissue. These are manifestations of the physiological changes that occur during the inflammatory process. The three main components of this process are. 1) Changes in the diameter of blood vessels and the speed of blood flow through these vessels (hemodynamic changes) 2) Increased permeability of the capillaries, and 3) Leukocyte migration
Infection:
The penetration of pathogenic microorganisms into the body and their reproduction there, which cause disease through damage to cells or cell aggregates, the secretion of toxins or the antigen-antibody reaction of the host.
A systemic infection is an infection in which the pathogens have spread throughout the organism via the bloodstream.
Biological fluid:
Biological fluids in the context of the invention are understood to mean all body fluids of mammals, including humans.
Gene:
A gene is a section on deoxyribonucleic acid (DNA) which contains the basic information for the production of a biologically active ribonucleic acid (RNA) as well as regulatory elements which activate or inactivate this production. Genes in the context of the invention are also understood to mean all derived DNA sequences, partial sequences and synthetic analogs (for example peptide nucleic acids (PNA)). The description of the invention relating to the determination of gene expression at the RNA level therefore expressly does not represent a restriction, but only an exemplary application.
Genetic Locus:
Gene locus is the position of a gene in the genome. If the genome consists of several chromosomes, the position within the chromosome is meant on which the gene is located. Different expressions or variants of this gene are called alleles, which are all located in the same place on the chromosome, namely the gene locus. Thus, the term "gene locus" contains, on the one hand, the pure genetic information for a specific gene product and, on the other hand, all regulatory DNA segments and all additional DNA sequences that are functionally related to the gene at the gene locus. The latter connect to sequence regions that are in the immediate vicinity (1Kb), but outside the 5- ’and / or 3 - end of a gene locus. The gene locus is specified by the accession number and / or RefSeq ID of the main RNA product which is derived from this locus.
Gene activity:
Gene activity is understood to mean the extent to which a gene is able to be transcribed and / or to form translation products.
Gene expression:
The process of generating a gene product and / or expressing a genotype into a phenotype.
Multigene biomarkers:
Combination of several gene sequences, the gene activities of which form a combined overall result (e.g. a classification and / or an index) by means of an interpretation function. This result is specific to a condition and / or an investigation question.
Hybridization conditions:
Physical and chemical parameters well known to the person skilled in the art, which can influence the establishment of a thermodynamic equilibrium of free and bound molecules. In the interests of optimal hybridization conditions, the duration of contact between the probe and sample molecules, the cation concentration in the hybridization buffer, temperature, volume and concentrations and ratios of the hybridizing molecules must be coordinated with one another.
Amplification conditions:
Constant or cyclically changing reaction conditions which enable the starting material to be replicated in the form of nucleic acids. The reaction mixture contains the individual building blocks (deoxyribonucleotides) for the resulting nucleic acids, as well as short oligonucleotides that can attach to complementary areas in the starting material, as well as a nucleic acid synthesis enzyme, called polymerase. Cation concentrations, pH value, volume and the duration and temperature of the individual reaction steps well known to the person skilled in the art are important for the course of the amplification.
Primer:
In the present invention, an oligonucleotide is referred to as a primer which acts as a starting point for nucleic acid replicating enzymes such as e.g. the DNA polymerase serves. Primers can consist of both DNA and RNA (Primer3, see e.g. http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi from MIT)
Probe:
In the present application, a probe is a nucleic acid fragment (DNA or RNA) which is labeled with a molecular label (for example fluorescent label, in particular Scorpion ®, molecular beacons, minor groove binding probes, TaqMan ® probes, isotope labeling etc.) can be provided and is used for the sequence-specific detection of target DNA and / or target RNA molecules.
PCR:
[0122] is the abbreviation for the English term “Polymerase Chain Reaction” (polymerase chain reaction). The polymerase chain reaction is a method to replicate DNA in vitro outside a living organism with the help of a DNA-dependent DNA polymerase. According to the present invention, PCR is used in particular to amplify short parts - up to about 3000 base pairs - of a DNA strand of interest. This can be a gene or just part of a gene or non-coding DNA sequences. It is well known to the person skilled in the art that a number of PCR methods are known in the prior art, all of which are encompassed by the term “PCR”. This applies in particular to “real-time PCR” (see also the explanations below).
Transcript:
For the purposes of the present application, a transcript is understood to mean any RNA product which is produced using a DNA template.
Small RNA:
Small RNAs in general. Representatives of this group are in particular, but not exclusively: a) scRNA (small cytoplasmatic RNA), which is one of several small RNA molecules in the cytoplasm of a eukaryote. b) snRNA (small nuclear RNA), one of the many small forms of RNA that only occur in the nucleus. Some of the snRNAs play a role in splicing or other RNA-processing reactions. c) small non-protein codinq RNAs, which are the so-called small nucleolar RNAs (snoRNAs), microRNAs(miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs (dsRNAs), which include gene expression at many levels, including chromatin architecture, RNA editing, RNA stability, translation and possibly also transcription and splicing. In general, these RNAs are processed in multiple ways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Although only 1.2% of the human genome codes for proteins, a large part is still transcribed. In fact, about 98% of the transcripts found in mammals and humans consist of non-protein-coding RNAs (ncRNA) from introns of protein-coding genes and the exons and introns of non-protein-coding genes, including many which are or are anti-sense to protein-coding genes overlap with these. Small nucleolar RNAs (snoRNAs) regulate the sequence-specific modification of nucleotides in target RNAs. Two types of modifications occur here, namely 2'-0-ribose methylation and pseudouridylation, which are regulated by two large snoRNA families, called box C / D snoRNAs on the one hand and box H / ACA snoRNAs on the other. Such snoRNAs are about 60 to 300 nucleotides in length. miRNAs (microRNAs) and siRNAs (short interfering RNAs) are even smaller RNAs with generally 21 to 25 nucleotides. miRNAs originate from endogenous short hairpin precursors and usually use other loci with similar - but not identical - sequences as targets for translational repression. siRNAs arise from longer double-stranded RNAs or long hairpins, often of exogenous origin. They usually target homologous sequences at the same locus or elsewhere in the genome, where they are involved in gene silencing, a phenomenon also known as RNAi. However, the boundaries between miRNAs and siRNAs are fluid. d) In addition, the term “small RNA” can also include so-called transposable elements (TEs) and in particular retro elements, which are also understood by the term “small RNA” for the purposes of the present invention.
RefSeq ID:
This designation refers to entries in the NCBI database (www.ncbi.nlm.nih.gov). This database provides non-redundant reference standards for genomic information. This genomic information includes chromosomes, mRNAs, RNAs and proteins, among others. Each RefSeq ID represents a single, naturally occurring molecule of an organism. The biological sequences that represent a RefSeq are derived from GenBank entries (also NCBI), but are a compilation of information elements. These information elements come from primary research at the DNA, RNA and protein levels.
Accession number:
An accession number represents the entry number of a polynucleotide in that known to the person skilled in the artNCBI-GenBank. In this database, both RefSeq ID’s and less well-characterized and redundant sequences are managed as entries and made available to the public (www.ncbi.nlm.nih.gov/genbank/index.html).
Local infection:
The infection is limited to the entry portal of the pathogen (e.g. wound infection).
Generalized infection:
Pathogens penetrate into the vascular system and affect the entire organism. Generalized infections can lead to sepsis.
Colonization:
The presence of microorganisms does not trigger any symptoms of illness in the organism.
Bacteremia:
A condition in which bacteria are temporarily and briefly present in the blood without this having to be associated with the occurrence of bacterial-related clinical symptoms.
Alternative splicing:
A process in which the exons of the primary gene transcript (pre-mRNA) are reconnected in different combinations after the introns have been excised.
BLAST:
Basic Local Alignment Search Tool [according to Altschul et al., J Mol Biol 215: 403-410: 1990]. Sequence comparison algorithm, speed-optimized, is used for searching in sequence databases for optimal local adaptation to the query sequence.
cDNA:
Complementary DNA. DNA sequence, product of reverse transcription of mRNA.
Coding sequence:
Protein-coding section of a gene or an mRNA, differentiated from introns (non-coding sequences) and 5- 'or 3'-untranslated sections. Coding sequences of the cDNA or mature mRNA encompass the region between the start (AUG or ATG) and stop codons.
EST:
Expressed Sequence Tag. Short ssDNA sections of the cDNA (usually ~ 300–500 bp), usually produced in large quantities. Represent the genes that are expressed in certain tissues and / or during certain developmental phases. Partly coding or non-coding identifiers of the expression for cDNA libraries. Valuable for determining the size of complete genes and in the context of mapping.
Exon:
Coding sequence region corresponding to the mRNA of typical eukaryotic genes. Exons can include the coding sequences, the 5 'untranslated region or the 3' untranslated region. Exons encode specific sections of the complete protein and are usually separated by long sections (introns), which are sometimes referred to as "junk DNA" and whose function is not exactly known, but probably encode short, untranslated RNAs (snRNA) or regulatory information.
GenBank:
Nucleotide sequence database with sequences from more than 100,000 organisms. Entries which are annotated with properties of the coding regions also include the translation products. GenBank is part of the international cooperation of the sequence databases, which also includes EMBL and DDBJ.
Intron:
The non-coding sequence region of a typical eukaryotic gene is cut out of the primary transcript during the RNA splicing and is therefore no longer in the mature, functional mRNA, rRNA or tRNA.
mRNA:
Messenger RNA or sometimes just “message”. RNA that contains the sequences necessary for protein coding. In contrast to the (unspliced) primary transcript, the term mRNA is only used for the mature transcript with a polyA tail (excluding the introns removed by splicing). Has 5'-untranslated, amino acid-coding, 3 - untranslated areas and (almost always) a poly (A) tail. Typically represents about 2% of the total cellular RNA.
Poly (A) tail:
SsAdenosine extension (~ 50-200 monomers) attached to the 3 'end of the mRNA during splicing. The polyA tail probably increases the stability of the mRNA (possibly protection against nucleases). Not all mRNA have the construct, e.g. the histone mRNA.
RefSeq:
NCBI database of reference sequences. Error-corrected, non-redundant sequence collection of genomic DNA contigs, mRNA and protein sequences or of known genes and complete chromosomes.
SNPs:
Single Nucleotide Polymorphisms. Genetic differences between alleles of the same gene based on individual nucleotide deviations. Arise at specific individual positions within a gene.
Transcript variants:
Alternative splicing products. The exons of the primary gene transcript (pre-mRNA) were reconnected in different ways and are subsequently translated.
3 'non-translated area:
Transcribed 3'-terminal mRNA region without protein-coding information (region between stop codon and polyA tail). Can affect the translation efficiency or the stability of the mRNA.
5 'non-translated area:
Transcribed 5'-terminal mRNA region without protein-coding information (region between the initial 7-methylguanosine and the base immediately before the ATG start codon). Can affect the translation efficiency or the stability of the mRNA.
Polynucleotide Isoforms:
Polynucleotides with the same function, but different sequence.
Abbreviations
CRP C-reactive protein OR Odd Ratio PCT Procalcitonin Sensitivity Proportion of correct tests in the group with a given disease (infectious SIRS or sepsis) Specificity Proportion of correct tests in the group without a given disease (non-infectious SIRS)
In addition, DE 10 2009 044 085, which was not previously published, discloses a system that comprises the following elements:A set of gene activity markersReference genes as internal controls for gene activity marker signals in whole bloodDetection mainly via real-time PCR or other amplification methods or hybridization methodsUse of an algorithm to convert the individual results of the gene activity markers to a common numerical value, index or scoreRepresentation of this numerical value on a correspondingly graded scaleCalibration, i.e. Classification of the scale according to the intended application through previous validation experiments.
The system provides a solution to the problem of diagnosing disease states such as the distinction between infectious and non-infectious multi-organ failure, but also for other applications and issues relevant in this context.However, all approaches for the diagnostic / prognostic detection of inflammatory and / or infectious conditions, which were previously published in the prior art and contained in the above-mentioned document DE 10 2009 044 085 not yet published at the time of registration, have so far only - as explained at the beginning - been included in the clinical routine found.
Using a broad spectrum of inflammatory-infectious clinical phenotypes from healthy volunteers to patients with local inflammation and local infection to intensive care patients with systemic inflammation (SIRS) and systemic infection (sepsis) and a measurement platform, which in the form of 25,000 If different probes represent the entire human genome, a study on the differential gene expression from peripheral whole blood samples was carried out. As a result, a transcriptomic signature was surprisingly identified which, instead of sudden changes depending on the various phenotypes, represents an inflammatory-infectious continuum of differential gene expression. Another surprising result that followed from this finding was the lack of infection-specific gene groups. In the group with systemic inflammation, a differential gene expression comparable to that of patients with bloodstream infections could be determined.
These results can provide an explanation for the determination made below that in less than half of the suspected sepsis cases, pathogen detection from blood samples is successful. A condition that is represented by extensive, simultaneous over- and under-expression of certain gene transcripts can be viewed as critical and include the characteristics of sepsis listed in the following sections. It characterizes the body's reaction to infectious and / or inflammatory stimulation or the resulting “immune stress”, also known as the host response. The information about this condition, which is represented by all significantly differentially expressed gene transcripts, can be computationally summarized to a non-dimensional numerical value, a score, and represented as the distance from the condition of the healthy person. The larger the numerical value of the score, the greater the extent of the body's reaction to infectious and / or inflammatory stimulation or the resulting “immune stress”. Comparable information about the body's reaction to infectious and / or inflammatory stimulation or the resulting “immune stress” can also be obtained using scores formed from sub-selections from all differentially expressed genes.
The reaction of the body to infectious and / or inflammatory stimulation or the resulting "immune stress" can not only increase or worsen, but also work in the opposite direction, i.e. move back or improve to the state of the healthy. This return can be seen as a recovery process. If this recovery immediately follows a therapeutic measure, it indicates the success of the therapy.
A progression of the condition into the critical area indicates a high mortality risk for the patient. In this state, the probability is high for the patient to suffer a life-threatening complication in the form of an uncontrolled primary infection and / or a secondary infection and / or to die as a result of the uncontrolled inflammatory-infectious host response.
A progression in the direction of a critical condition can be used as early detection and on the basis of this the necessary therapeutic measures and interventions can be initiated.
The reaction of the body to infectious and / or inflammatory stimulation or the “immune stress” resulting therefrom can be used as an individual determination for determining a condition.
The immune status can be used on the basis of successive multiple determinations for monitoring the course and monitoring therapy in patients.
Medical measures can be drug treatment and its escalation or de-escalation, invasive measures such as surgical interventions to reorganize the focus and / or further diagnostic measures.
The determination of the immune status can be used for differential diagnosis in that it is determined whether the immune system is making a contribution to the acute disease process or is out of the question as the cause of a life-threatening condition of a patient.
The aim of most gene expression studies for the diagnosis of sepsis so far has been to find markers which distinguish whether a systemic inflammatory response syndrome (SIRS, ACCP / SCCM, 1992) was caused by pathogens or not. In these studies, samples from intensive care patients were examined in particular. The differentiation in groups “sterile SIRS” vs. "Sepsis" (SIRS + positive pathogen detection) occurred particularly after the microbiological detection. These studies yielded very heterogeneous results, as was stated in a recent comparative publication [Tang et al. (2010)]. The subject not according to the invention illuminates the causes for this with an experimental approach. The inventors' experimental plan was based on the following consideration:
The previous approach to sepsis diagnosis was taken from the procedure for local inflammation. Here, too, one wants to distinguish whether the inflammation in an organ was caused by pathogens or not (one then speaks, for example, of bacterial or sterile myocarditis or pancreatitis). Infection-specific gene markers have to show increased or reduced expression even in the case of an infection that does not necessarily manifest itself as a systemic inflammation, but only causes inflammation in the affected organ / site. However, this could not be shown in the prior art for the gene markers found so far.
Another explanation for heterogeneous results would be that the gene expression studies use RNA samples from blood. Thus, the best chances of finding infection-specific gene markers had to be present when only samples with a positive blood culture, which is a “gold standard” for bloodstream infection, are measured. In previous studies, sepsis patients were considered as one study group regardless of the spread of the infection
The applicant bases its studies on a test plan in which the characteristics of infection and inflammation were classified independently of one another in terms of their spatial distribution. A distinction was made as to whether the inflammation was systemic, local or not at all. Systemic inflammation was determined by the definition of the systemic inflammatory response syndrome (SIRS). In addition, it was checked whether pathogens were found in the blood (systemic), on an organ (local) or not at all. The 9 possible phenotypes were defined from the combination of the spread of infection and inflammation. They are summarized in Table 1.
Table 1: Representation of the phenotypes which result from the spatial expression of inflammation and / or infection. Each phenotype is identified by a three-letter abbreviation. The first capital letter indicates the spread of the infection, the second the spread of the inflammation. Both were connected with an "a" (for and).
From the applicant's point of view, this division is clear, complete and independent of other factors. I.e. any test subject can be assigned to one of the groups at the time the sample is taken.
While inflammation is not necessarily caused by pathogens, an infection without an inflammatory reaction is not diagnostically relevant. A systemic infection that causes only a localized inflammation (so-called bacteremia, e.g. in endocarditis) is a rare phenomenon and is a special case. Therefore the combinations LaN, SaN and SaL do not form any clinically relevant phenotypes and were not considered for the purposes of the present invention.
For the study, the inventors divided diagnostically relevant patient samples according to the spatial spread of an inflammation and / or infection. 6 study groups were created (in Tab. 1 written in bold). These groups represent the most important and most common infection-inflammatory phenotypes. In particular, the 4 phenotypes with a local infection with local and systemic inflammation (LaL and LaS) as well as the corresponding control groups without an infection (NaL and NaS) enable the detection of infection-specific gene markers in a statistical comparison. The comparison of the SaS and LaS groups provides information on whether, in the case of systemic inflammation, the infection of the circulating cells (blood flow infection) indicates a different gene expression pattern than the locally restricted infection.
The present application is accompanied by a sequence listing with the SEQ ID numbers 1-7718, the content of which belongs entirely to the disclosure content of the present application.
Further advantages and features emerge on the basis of the description of exemplary embodiments and on the basis of the drawing. 1 shows a heat map in which the expression patterns are sorted according to study groups; 2 shows a sorted heat map in which the expression patterns are sorted according to the score (master score); 3 shows distance triangles for the study samples; 4 shows distance triangles for two patient courses; 5 shows the course of the score for all study samples; and FIG. 6 shows the course of the score which was calculated from Delta-Ct values of the real-time PCR gene expression measurement in comparison to the master score.
Examples
Example 1: Determination of the severity of the host's response to stress on the immune system by acute inflammation
Patient groups
Samples from the following test subjects were included in the selection: healthy donors, patients from the otolaryngology clinics (ENT) and anesthesia and intensive care (KAI) clinics at the Jena University Clinic. The groups were assigned as follows:SaS: 6 intensive care patients diagnosed with severe sepsis / septic shock. The examined sample was taken on the day on which the same pathogen was confirmed in the blood in two independent tests (blood culture and DNA detection).LaS: 13 intensive care patients with the diagnosis of severe sepsis / septic shock, in whom at least one blood sample was checked for pathogens with two independent tests (blood culture and DNA detection) during the illness, but all findings remained negative. The examined sample was taken on the first day on which a pathogen was locally confirmed.NaS: 13 intensive care patients diagnosed with SIRS without any signs of infection. The examined sample was taken within the first 3 days with the SIRS diagnosis.LaL: 7 patients at the ENT clinic with an acute peritonsillar abscess (PTA). The examined sample was taken immediately before the surgical removal of the infectious abscess. The associated microbiological blood test (as with LaS) was negative.NaL: 8 patients at the ENT clinic with chronic tonsillitis without an acute focus of infection. The examined sample was taken within the first 3 days after the tonsillectomy (surgical removal of the tonsils). The patients showed no SIRS symptoms; there was a sterile wound infection at the surgical site. Furthermore, 4 patients with chronic non-infectious pancreatitis without SIRS were included in the group.NaN: 7 healthy donors, 3 patients with chronic tonsillitis without an acute focus of infection. The examined sample was taken before the tonsillectomy, the postoperative samples of these patients were not included in the NaL group.Follow-up samples: In the study, samples from 2 patients were examined over a period of 6 consecutive days. In both cases, the first sample was taken before a planned surgical procedure, the subsequent samples were taken in the intensive care unit. Case 1: patient does not recover after surgery. The inflammation-relevant parameters increase from the 2nd postoperative day, on the 3rd day an infection was diagnosed, the patient died after 10 days. Case 2: After the operation, the patient recovers very slowly. On the third day, some inflammation-related parameters increase. After the medical measures, the condition improves and the patient is transferred after a total of 6 days.The most important clinical parameters for the samples examined are summarized in Table 2.
Table 2: Summary of the clinical parameters of the subjects included in the study. If a feature was not queried or a parameter was not determined, it is indicated with the abbreviation n.a. Mistake.
Experimental implementation
73 RNA samples from the whole blood of 63 people were measured. Commercial Microarray BeadChips HumanHT-12 v3 from the Illumina company were used for this. The measurement platform used contained 48,803 different gene probes that represent the entire human genome, regardless of the tissue.The samples were processed and measured in the following steps: 1. Isolation and stabilization of the totaIRNA from whole blood samples: The starting material for the analysis of the transcriptome of blood samples is 2.5 ml of whole blood. This was removed in a PAXgene tube (PAXgene Blood RNA Tube PreAnalytiX # 762165 (Becton Dickinson)) and stored at −80 ° C. until processing. 2. Standard automatic RNA isolation from PAXgene blood samples: The QIAcube (Qiagen, Hilden) and the PAXgene Blood RNA Kit (PreAnalytiX # 762174) were used to isolate the total RNA using the “PAXgene Blood RNA (CE)” program. After the end of the protocol, the elution tubes with the RNA isolates were closed. Remaining DNase enzyme activities were inactivated by heating the samples for 5 min at 65 ° C, the samples then immediately cooled on ice and stored at -80 ° C. 3. Quality control of the total RNA: Checking the isolated RNA is an important measure for quality assurance of the hybridization results. Only an intact RNA can give excellent hybridization results. The integrity of the isolated total RNA was checked by capillary electrophoresis with the Bioanalyzer 2100 from Agilent Technologies using the RNA 6000 Nano LabChip Kit (Agilent Technologies, catalog number 5067-1511) according to the manufacturer's specifications. A RIN value of 7.5 on a scale from 1–10 is a guideline. All samples used achieved a RIN> 5, which is sufficient for the purposes of gene expression analysis (cf. Fleige and Paffl, 2006). 4. Reduction of the globin mRNA: To improve the sensitivity of the gene expression measurements in whole blood, the highly abundant globin mRNA was recommended. The kit “GLOBINclear TM-Human”, from Ambion / Applied Biosystems # AM 1980) was used. According to estimates by the manufacturer, globin's transcripts, with their 70% share of all mRNAs in blood, overlay significantly less present transcripts. 1 µg of total RNA was used for processing from each sample. 5. Amplification of total RNA to cRNA: The globin-reduced RNA was prepared for hybridization with the Ambion / Applied Biosystems Illumina TotalPrep RNA amplification kit (AMIL 1791) according to the manufacturer's instructions and with an amount of 500 ng. The cRNA eluates were cooled on ice and the concentration of the cRNA was measured spectrophotometrically on the Nanodrop ND-2000. 6. Hybridization on Illumina BeadChips: The pangenomic Illumina BeadChips, version human HT-12v3 were used. 750 ng of the cRNA samples were applied in a volume of 5 μl per array and hybridized at 58 ° C. overnight. The signal detection of successfully hybridized probes takes place via CY3-streptavidin-staining (GE-Amersham) according to the manufacturer's specifications, Illumina protocol: Whole-Genome Gene Expression with IntelliHybTM Seal; Experienced User Card; Part # 11 226 030 Rev. B, Illumina Inc. The fluorescence signals were read out with the Illumina BeadArray Reader 500 and the corresponding Illumina software “BeadScan” (version 3.6.17). 7. Image analysis of the microarrays: The fluorescent dye colored BeadChips are scanned with the Illumina <®> BeadArray Reader. The resulting images are analyzed using the software from lllumina <®> «Genome-Studio» (version Genome Studio 2009.2). For the first assessment of the hybridization, technical control signals are queried. A first qualitative overview is obtained from the number of genes detected and the mean signal strength per array. The raw data is subjected to quality control and statistical analysis.
Data analysis and statistical evaluation
The data analysis was carried out using the free software R Project Version R 2.8.0 (R.app GUI 1.26 (5256), S. Urbanek & SMIacus, © R Foundation for Statistical Computing, 2008), which is available at www.r -project.org is available [R Development Gore Team (2006)]. This software is preferred when analyzing gene expression data because it provides numerous algorithms for manipulating this type of data. In particular, the following software packages could be used in our study: lumi for reading in the lllumina measurement values and the associated gene annotation, vsn for data normalization (both in Du et al., 2008), fdrtool for determining the false positive rate (Strimmer, 2008) and stats for performing statistical significance tests, for clustering and for visualizing the results.The analysis was carried out in the following steps. The raw data as well as additional available annotation data were imported. Due to technical variations in sample processing as well as deviations from reagent batches, the fluorescence intensities of different samples cannot usually be compared directly with one another. In order for this to be possible, such variations are compensated for by suitable normalization. A variance-stabilizing transformation was used [Huber et al., 2002].Normalized and base 2 logarithmic measurement data were included in the data analysis. In the statistical analysis 61 samples from the 6 study groups were examined. The gene expression between these groups was compared by means of the one-way analysis of variance [Mardia et al. a., 1979]. It was checked whether there was a significant difference between at least 2 of the groups examined. The associated significance test was calculated, gene by gene, for 18,517 gene probes which delivered a signal with a detectable intensity for at least one RNA sample. The number of false positive tests was determined using the false discovery rate (FDR) [Storey et al. (2003)]. The so-called q-value was calculated for each gene probe, which is defined as the minimum FDR below which the probe appears to be significantly changed. The method used for FDR control estimated 87.5% of the gene probes to be significantly changed. This corresponds to 16,204 probes from the pool of 18,517 examined.The gene expression patterns of the 6 patient groups were further compared in pairs using the t-test. The test was applied gene by gene for a total of 15 group combinations. The same gene probe selection was taken into account and the FDR control carried out, which was described in the one-way analysis of variance. The results are summarized in Table 3.
Table 3: Summary of the false positive rates (FDR) when comparing all study groups in pairs. The table shows how many gene probes achieved an FDR value less than the specified threshold in a comparison. The interpretation is explained using the example: When comparing NaS vs. NaL, a group of 265 genes with FDR of 1% and less was determined, i.e. 2–3 of the 265 gene probes are false positive.
From the table (last column) it follows that the clearest differences exist between the patients with sepsis (LaS and SaS) and the groups NaN (no inflammation) and NaL (non-infectious local inflammation). In contrast, there were few clear differences between the group pairs NaS and LaL, SaS and LaS, and LaS and NaS. When comparing the amounts of gene probes that were compared with LaS vs. NaS and NaS vs. If LaL reached an FDR <0.1, no gene probes could be found which had the same change in gene expression upon infection. Thus, no gene probes could be discovered that show an infection-specific expression pattern. It should be mentioned that further statistical comparisons, including the 2-way analysis of variance [Mardia et al., 1979 between the groups NaL, NaS, LaL and LaS, as well as the comparison of the groups LaL and LaS vs. NaS, gave the same negative result.
Surprisingly, the pairwise comparisons made it possible to arrange the study groups as follows: (1) NaN, (2) NaL, (3) LaL, (4) NaS, (5) LaS, (6) SaS. This arrangement is characterized by the fact that the neighboring groups differ only little statistically in their expression. More precisely, in a statistical comparison, a sufficient number of altered gene probes were found; gene probe groups with a sufficiently low false positive rate (approx. 5%) could not be found between the neighboring groups. This phenomenon occurs when the group mean values differ, but the scatter of the groups is so high that it leads to an overlap between the two groups compared. The further the groups are from one another, the greater the differences. It should be mentioned that without determining the gene expression, a directed arrangement of the study groups was possible.In order to show the revealed change in gene expression within the study groups, 8537 gene probes were included in the further analysis from the first statistical comparison, in which group differences were generally examined using a one-way analysis of variance. This selection was based on an estimate of the false positive rate of 0.3%. This is statistically interpreted in such a way that approx. 26 probes in the selection list of 8537 probes (0.3%) are false positive. Any increase in choice would result in a higher false positive rate. The selected 8357 gene probes address a total of 7694 different RNA transcripts.The gene expression of these gene probes within the study was grouped according to their similarity into 9 gene clusters using the k-means algorithm (Hartigan and Wong, 1979). The R-function kmeans was used for this, the expression signals were standardized in advance, gene by gene, with regard to the mean value and the spread. The estimation of the number of clusters took place after the 2nd derivation of the error function (cost function), which results from the repetition of the clustering procedure for 1 to 20 clusters [Goutte u. a., 1999].The patient groups were arranged in the order (1) NaN, (2) NaL, (3) LaL, (4) NaS, (5) LaS, (6) SaS. At the end, the 12 gene expression patterns from the 8537 gene probes of the progression samples from both patients were lined up. The sorted expression matrix was visualized in a so-called heat map. In FIG. 1, each row represents a gene probe and each column represents an RNA sample. It shows the relative change in gene expression in gray levels. For example, dark gray to black codes a lower expression and light gray to white a higher expression than was determined on average per gene probe. The clusters were numbered from 1 to 9, the number in brackets under the cluster number indicates the number of gene probes in the cluster. Within a cluster, the gene probes were sorted in descending order, so that probes with the greatest differences within the study groups are arranged at the upper end of the cluster.
It can be seen from Figure 1 that there is a trend in expression from left to right. From the NaN group to the SaS group, expression increases in gene clusters 1 to 4 and decreases in gene clusters 5 to 9. However, there is also a high degree of variability within the individual groups, especially the NaS group. The transitions between individual groups are fluid, the groups overlap.
The mean difference between the NaN (no acute inflammation) and SaS (SIRS patients with confirmed bloodstream infection) groups is most evident. In the next step, this difference was quantified using a distance measure (see next section). All other samples were arranged according to their distance from these two groups. This arrangement is visualized in the sorted heat map, which is shown in FIG. 2. Samples that were more similar to the pattern of healthy subjects were sorted on the left, and samples that were more similar to the pattern of ICU patients with a blood infection were sorted on the right.
The sorted heat map shows that the patient samples were predominantly sorted according to their group membership in the order set out above or are mixed into neighboring groups. However, individual samples are sorted into significantly higher or lower ranks than most group representatives. The gene expression pattern of the heat map shows a simultaneous increase and decrease in gene activity from NaN to SaS. The individual gene clusters differ only in the progression of the deviation.
This result indicates that the gene expression particularly reflects the strength of the host response to the burden on the organism caused by inflammation. In fact, a bloodstream infection with systemic inflammation is the greatest burden on the immune system. A similar burden will arise from a local infection if the immune system is not able to fight the pathogen at the source of the infection. But also a traumatic event, e.g. A serious operation can briefly push the immune system to its limits. In addition, the gene expression maps the severity of the host's response to a lower exposure caused by local inflammation.As already mentioned, the selected genes are only divided into 2 groups. In one group of 3041 gene probes (36%), gene expression increases with the load on the immune system (clusters 1 to 4), in the other group of 5496 gene probes (64%) it decreases (clusters 5 to 9). Based on known references [(cf. Calvano et al. (2005) and Foteinou et al., 2008] one would speak of an (pro- and anti) inflammatory response in the case of an increase in expression and an energetic response in the case of a decrease in expression to an irrepressible immune response.
Quantifying the severity of the host response
The heat map was sorted according to the following score.For an RNA sample, let X be the associated gene expression vector that summarizes the expression signals of the selected gene probes. Let d (X1, X2) denote the Euclidean distance between two vectors X1 and X2. Further, let cor (X1, X2) denote the correlation coefficient between X1 and X2 according to Pearson, which corresponds to the cosine value of the angle between X1 and X2 [Mardia et al., 1979].The score is calculated according to the following formula
calculated, where mH and mS are the two gene expression vectors that summarize the means of the groups NaN (mH) and SaS (mS) gene for gene. The score can be illustrated as follows. A triangle is formed from the distances d (mS, mH), d (mS, X) and d (X, mH), the corners of which are designated X, mH and mS for the sake of simplicity (see FIG. 3). The value
defines the position of the base point of the perpendicular from corner X to the straight line that is determined by d (mS, mH). The value of LX corresponds to the distance between mH and this plumb line. It also indicates how far the corner X of the triangle is from the corner mH, the height of the triangle is not evaluated.Finally, the score, which is defined by formula 1, gives the relative proportion of the distance LX the distance d (mS, mH). In fact, one obtains from formula 1 for X = mH: score (mH) = 0% and for X = mS: score (mS) = 100%.In FIG. 3, sample 3 is further away from mS than mH and receives a score of -9.3%. Sample 59 lies further from mH than mS and receives a score of 127.8%. Finally, sample 37 lies between mH and mS and receives a score of 27.7%. A sample that has the same distance to the mH and mS would get the value of 50%.The distance d (mS, mH) is made up of the distances between the individual gene probes. This total distance is broken down into two components, one component d <+> (mS, mH) being calculated from the gene probes of the clusters, 1 to 4 and the other dn (mS, mH) from the gene probes of the clusters, 5 to 9 , one obtains information about the contribution of the increase and decrease in expression to the total deviation. In the data set we examined, the increase, which is represented by 36% of the gene probes, was 51% of the total distance. The decrease, represented by 64% of all probes, was 49% of the total distance. Thus, on average, the expression of a gene probe decreased more from clusters 1 to 4 than in clusters 5 to 9. The overall ratio of the increase and decrease was about the same. If the ratio of the increase and decrease is calculated for each sample, information is obtained about the progression of the deviation in the marked direction.4 shows how the distance triangle moves for the two patient cases in the course of the disease, the index above the tip of the triangle indicating the day of the decrease and 0 denoting the pre-operative sample.The score for all study samples is shown in FIG. 5. The study groups and the two courses were arranged as in FIG. 1. The black dots mark the score, the bars the deviation of 7.5 percentage points up and down.It should be noted that the proposed score is not the only one that can be used to quantify the differences in gene expression between the study groups. The advantage of this score is that it defines a relative measure, namely a percentage of the difference that was determined between the gene expression and the group without acute inflammation and SIRS patients with a blood infection. The score is independent of the measurement platform and the number of gene markers used.Although the score only quantifies a simultaneous increase and decrease in gene activity, it is calculated from the expression of several thousand genes. The reason for this lies in the phenomenon examined. The genes used are generally responsible for different processes. Therefore, the expression deviation from normal for a single gene can have various causes. However, the swarm behavior of many genes in the marked direction reflects the quantitative extent of the immune load.The examples in which the postoperative condition was observed for 2 patients shows that the score records the current extent of the host response. Therefore it can be used for surveillance / monitoring. In fact, the score for a patient changes from about 20% to about 90 percentage points within 6 days. In addition, it is suitable for general assessment of immune stress. In fact, the pre-operative samples from both patients show an increased score of over 20%. This can be one of the causes of the complication-rich postoperative course.
Example 2: Determination of the severity of the host response in patients with acute inflammation with a reduced number of markers
Gene marker selection based on simulations
A high number of gene markers was used to determine the strength of the host response. The genes examined are generally responsible for different processes and the deviation from normal expression for a single gene has various causes. The more genes that are observed, the more easily another cause other than the burden on the immune system for the expression deviation can be excluded. The observation of all relevant genes completely rules out other causes. However, there is a well-founded assumption that a reduced number of gene probes would also sufficiently reflect the stress level of the immune system. The gene number reduction is only credible if the resulting score is sufficiently close to the original score (master score). In our study, the master score could be determined with great accuracy from the 4372 gene probes, the mean expression of which between 2 study groups was at least 0.8. In fact, the difference was no more than 1 percentage point. If the score was calculated from the remaining 4165 gene probes, the deviation was less than 8 percentage points.Since there is no algorithm for marker selection, it makes sense to use computer simulations to check whether there is a preferred number and amount of genes that represent the master score. In order to estimate the number of gene probes required, a maximum of 1,500 were randomly selected from the 8357 gene probes of the master score in the first simulation runs. Of these, 36% were from clusters 1 to 4 and 64% from clusters 5 to 9. For each selection, the score was calculated according to formula 1 for all 73 RNA samples. The gene probe sets whose scores did not differ by more than ± 7.5 percentage points from the master score were retained. 241 such gene marker sets were found in 500 repetitions. All 8375 gene probes were represented in these sets. The shortest set contained 138 gene probes. In the next 5000 repetitions, a maximum of 150 gene probes per run were randomly selected; their distribution over the clusters was as in the first run. As a result, we received 24 sets with a length of 86 to 148 gene probes, with the associated score values not deviating more than ± 7.5 percentage points from the master score. The results of the preliminary investigation show that the number of gene markers can be reduced considerably if a reasonable deviation from the master score is accepted. In the simulations described below, the maximum number of gene probes was further reduced.
The first simulation was carried out as follows. The expression matrix of 3357 gene markers and 61 expression vectors from the 6 study groups was subjected to a principal component analysis (Mardia et al., 1979). The R function prcomp was used for this. 512 gene probes were selected that correlated most strongly with the first main component. There were 183 (36%) from gene clusters 1 to 4 and 329 (64%) from gene clusters 5 to 9. This restricted the selection to gene probes that most clearly represent the trend in gene expression changes investigated Decrease in the same proportion as in the primary selection.From this preselection (512 gene probes) 40 to 50 gene probes were randomly selected in 5000 simulation steps and the score was calculated from them according to formula 1 for all 73 gene samples. The selection was discarded if the absolute difference between the master score and the new score was greater than 7.5 percentage points for at least one sample. Otherwise the selection was saved. This simulation yielded 2 sets of gene probes that fulfilled the condition. These quantities were described as Set 1 and Set 2 via the associated sequence number. They contained 49 and 47 gene sequences.In the next 5000 simulation steps, the gene probe tuples were retained, in which the reduced score for 70 samples (95%) did not differ from the master score by more than 7.5 percentage points. 14 different combinations were found in this simulation step. These amounts, described as set 3 to set 16 in the corresponding sequence number, contained 46 to 49 gene sequences.In a third simulation, the number of randomly selected probes was reduced to a maximum of 20 and the selection procedure was repeated 50,000. The gene probe tuples were also retained, in which the reduced score for 70 samples (95%) did not differ from the master score by more than 7.5 percentage points. In this simulation, 20 different combinations were found that met the selection condition.These amounts, described as set 17 to set 36 in the associated sequence number, contained 18 to 20 gene sequences.The results of these simulations show that the score can be determined with sufficient accuracy from a significantly lower number of probes. In fact, an error bar of 15 percentage points (which corresponds to an error of ± 7.5 percentage points) appears acceptable if it leads to a considerable reduction in the number of gene markers. It must be taken into account that the original score is also subject to random fluctuations and depends on the underlying sample.In addition, the simulations show that there are no preferred gene probes that determine the score. In fact, different gene probe tuples lead to a similar estimate of the master score.
Table 4: Summary of the gene probe tuples which met the selection criteria described in simulations. The sets were numbered from 1 to 36, the number n in brackets indicates the number of sequences in the set. The subsequent sequence of numbers indicates the associated sequence numbers from the sequence listing.
[0182] Set 1 (n = 49) 508, 553, 611, 679, 734, 769, 851, 860, 871, 896, 1117, 1263, 1646, 1647, 1648, 1675, 1688, 1975, 2011, 2077, 2415, 2516, 2560, 2581, 3381, 3491, 3820, 3947, 4156, 4230, 4506, 4576, 5012, 5235, 5614, 5730, 5803, 5873, 6114, 6262, 6265, 6301, 6689, 6738, 6820, 6847, 6879, 7069, 7230
[0183] Set 2 (n = 47) 160, 309, 374, 428, 462, 911, 937, 1039, 1092, 1105, 1458, 1533, 1604, 1895, 1917, 1997, 2002, 2055, 2242, 2332, 2369, 2386, 2427, 2516, 2541, 2560, 2785, 3359, 3407, 3624, 4230, 4587, 4636, 5164, 5235, 5247, 5371, 5776, 6278, 6328, 6497, 6636, 7156, 7201, 7230, 7314, 7450
[0184] Set 3 (n = 48) 10, 366, 411, 462, 493, 495, 567, 1204, 1226, 1409, 1414, 1449, 1487, 1583, 1724, 1744, 2013, 2055, 2064, 2208, 2248, 2692, 2891, 3051, 3624, 4156, 4205, 4510, 4587, 4923, 5176, 5373, 5400, 5435, 5873, 5912, 5954, 6041, 6073, 6247, 6301, 6478, 6525, 6923, 7207, 7450, 7670, 7681
[0185] Set 4 (n = 47) 160, 359, 441, 493, 522, 541, 652, 691, 1128, 1408, 1583, 1651, 1652, 1664, 1688, 2002, 2077, 2248, 2273, 2415, 2675, 2690, 2755, 2876, 3053, 3623, 4216, 4327, 4525, 4587, 4765, 4870, 5013, 5164, 5431, 5614, 5950, 6098, 6265, 6432, 6497, 6981, 7062, 7202, 7314, 7450, 7607
[0186] Set 5 (n = 49) 97, 428, 441, 543, 611, 851, 1136, 1384, 1533, 1868, 1997, 2077, 2183, 2208, 2226, 2260, 2329, 2386, 2475, 2686, 2690, 2876, 3054, 3821, 4000, 4357, 4479, 4530, 4636, 4765, 4923, 5013, 5137, 5204, 5760, 5778, 5819, 5873, 5908, 6005, 6099, 6242, 6417, 6499, 6585, 6847, 7450, 7670, 7681
[0187] Set 6 (n = 48) 10, 97, 359, 475, 495, 627, 928, 1039, 1117, 1248, 1384, 1408, 1472, 1652, 1675, 1744, 1868, 1918, 2370, 2423, 2537, 2742, 2865, 3051, 3086, 3408, 3916, 4030, 4078, 4274, 4294, 4362, 4751, 5129, 5235, 5247, 5431, 5734, 5803, 5811, 5908, 5950, 6005, 6417, 6497, 6525, 6923, 7456
[0188] Set 7 (n = 49) 32, 160, 383, 414, 493, 611, 652, 679, 734, 885, 896, 946, 1177, 1640, 1650, 1704, 1382, 2077, 2248, 2250, 2260, 2415, 2551, 3086, 3488, 3623, 3624, 4135, 4156, 4160, 4510, 4525, 4530, 4742, 5137, 5204, 5247, 5730, 5950, 6114, 6210, 6225, 6430, 6478, 6497, 6545, 6668, 7314, 7607
[0189] Set 8 (n = 48) 359, 383, 515, 538, 544, 691, 769, 813, 1024, 1039, 1092, 1409, 1519, 1640, 1649, 1665, 1696, 1731, 1744, 2167, 2183, 2226, 2260, 2273, 2425, 2516, 2618, 2634, 2672, 3051, 3168, 3202, 4160, 4754, 4966, 5373, 5465, 5493, 5541, 5574, 5912, 6005, 6216, 6432, 6636, 6748, 6847, 7423
Set 9 (n = 46) 160, 352, 544, 691, 802, 885, 1126, 1147, 1163, 1336, 1416, 1639, 1969, 2002, 2058, 2077, 2183, 2331, 2332, 2426, 2526, 2742, 2855, 2860, 2891, 3054, 3138, 3488, 3947, 4560, 4576, 4707, 4776, 5235, 5371, 5400, 5431, 5760, 5873, 6247, 6301, 6417, 6673, 6820, 7447, 7604
Set 10 (n = 49) 8, 164, 462, 494, 495, 510, 545, 567, 611, 679, 941, 1039, 1105, 1128, 1147, 1318, 1533, 1649, 1918, 1973, 1975, 2011, 2077, 2080, 2370, 2537, 3051, 3202, 3676, 4274, 4587, 4928, 5204, 5373, 5431, 5465, 5541, 5734, 5908, 5912, 5950, 6278, 6417, 6497, 6668, 6673, 7156, 7230, 7670
Set 11 (n = 46) 89, 97, 160, 355, 359, 366, 374, 411, 462, 475, 515, 538, 543, 691, 1384, 1647, 1649, 1651, 1724, 2011, 2058, 2064, 2242, 2369, 2859, 3414, 4000, 4742, 4765, 4870, 4966, 5040, 5232, 5247, 5276, 5373, 5431, 5760, 5873, 5954, 6417, 6419, 6497, 6545, 6636, 7484
[0193] Set 12 (n = 49) 8, 89, 515, 543, 585, 769, 969, 1126, 1163, 1526, 1583, 1639, 1744, 2019, 2393, 2415, 2453, 2618, 2690, 2692, 2810, 2855, 2863, 3153, 3158, 3190, 3408, 4000, 4083, 4104, 4248, 4479, 4491, 4550, 4661, 4877, 4995, 5176, 5276, 5599, 5695, 6073, 6114, 6265, 6417, 6499, 6585, 6632, 6673
[0194] Set 13 (n = 48) 414, 538, 946, 1263, 1384, 1512, 1895, 2077, 2248, 2260, 2516, 2676, 2975, 3168, 3414, 4083, 4274, 4776, 4800, 4919, 4923, 5179, 5204, 5431, 5493, 5541, 5619, 5695, 5819, 6005, 6073, 6099, 6210, 6247, 6265, 6350, 6417, 6432, 6499, 6536, 6545, 6636, 6668, 6689, 7040, 7062, 7472, 7604
[0195] Set 14 (n = 47) 383, 428, 538, 553, 691, 814, 871, 896, 911, 937, 1426, 1639, 1685, 1688, 1983, 2093, 2253, 2260, 2454, 2516, 2587, 2672, 2761, 2865, 2975, 3086, 3781, 4000, 4030, 4308, 4510, 4636, 4923, 5137, 5235, 5574, 5776, 5819, 5908, 6226, 6278, 6417, 6632, 7202, 7230, 7315, 7456
[0196] Set 15 (n = 46) 97, 366, 383, 802, 1426, 1514, 1558, 1685, 1744, 1975, 2011, 2013, 2369, 2415, 2454, 2510, 2516, 2577, 2587, 2759, 2968, 3168, 3364, 3641, 3780, 4083, 4230, 4294, 4587, 4638, 4817, 5040, 5164, 5276, 5371, 5465, 5541, 6073, 6098, 6114, 6184, 6216, 6497, 6515, 7062, 7202
Set 16 (n = 49) 10, 504, 541, 553, 567, 652, 802, 1024, 1092, 1136, 1197, 1519, 1646, 1647, 1648, 1652, 2055, 2058, 2260, 2273, 2330, 2331, 2415, 2491, 2581, 2618, 2676, 2742, 3053, 3408, 3652, 3915, 4216, 4870, 5235, 5641, 5695, 5954, 6114, 6278, 6419, 6461, 6791, 6820, 6847, 6923, 7428, 7604, 7670
Set 17 (n = 20) 10, 160, 428, 871, 941, 1136, 1197, 1416, 1558, 1786, 1951, 2386, 2510, 2560, 3488, 3652, 3781, 5176, 5400, 6515
Set 18 (n = 20) 871, 1163, 1414, 1416, 1426, 1487, 2001, 2055, 2369, 2386, 2552, 2577, 2865, 3051, 4550, 4577, 5614, 6098, 7369, 7423
Set 19 (n = 20) 567, 958, 2226, 2250, 2260, 2427, 2516, 3364, 4030, 4135, 5235, 5574, 5950, 6114, 6226, 6267, 6278, 6418, 7069, 7518
Set 20 (n = 20) 409, 1647, 1648, 1770, 1883, 1951, 2013, 2386, 2423, 3152, 3491, 4205, 4577, 4661, 4765, 4919, 7428, 7604
Set 21 (n = 20) 355, 480, 494, 667, 1492, 2475, 2855, 2948, 3155, 3158, 3408, 3780, 4661, 5113, 5232, 5368, 5574, 6114, 6419, 6499
[0203] Set 22 (n = 19) 769, 1163, 1472, 2077, 2370, 2759, 3488, 3567, 3737, 3780, 4230, 4245, 4274, 4550, 5950, 6497, 7069, 7109, 7681
Set 23 (n = 20) 160, 164, 355, 411, 1106, 1408, 1675, 1679, 2386, 2453, 2516, 2810, 3168, 3202, 3652, 4230, 5574, 5986, 7428, 7484
Set 24 (n = 19) 10, 164, 885, 1263, 1318, 1416, 1492, 1508, 1647, 1951, 2250, 2560, 2785, 2827, 3086, 4506, 5137, 5575, 5954
Set 25 (n = 19) 32, 522, 679, 1519, 2001, 2491, 2516, 2876, 3412, 3737, 4205, 4294, 4560, 5235, 5954, 6005, 6114, 6499, 6525
Set 26 (n = 19) 958, 1449, 1472, 1582, 2332, 2516, 2552, 2891, 2975, 3168, 3190, 3683, 3820, 3947, 4245, 4530, 7040, 7069, 7145
Set 27 (n = 20) 428, 515, 544, 562, 567, 1263, 2002, 2332, 2526, 3438, 4577, 4754, 5574, 5614, 5912, 6328, 6515, 7156, 7423, 7456
Set 28 (n = 20) 355, 508, 937, 1263, 1973, 2002, 2510, 4078, 4156, 4550, 4673, 4817, 5247, 5368, 5730, 6005, 6247, 6515, 7201, 7207
Set 29 (n = 20) 10, 544, 871, 1408, 1487, 1649, 2002, 2415, 2690, 2859, 2975, 3126, 4577, 4636, 5541, 6073, 6417, 6432, 6866, 6879
Set 30 (n = 20) 896, 1248, 1318, 1472, 1786, 1830, 1983, 2386, 2865, 2975, 3641, 3916, 4030, 4530, 4995, 5472, 5619, 6099, 6247, 6265
Set 31 (n = 20) 355, 462, 1416, 1983, 2011, 2183, 2248, 2618, 3190, 3412, 4490, 4576, 4776, 4923, 5164, 6101, 6114, 6278, 7314, 7369
Set 32 (n = 20) 310, 1226, 1895, 2248, 2427, 2516, 2552, 2690, 3086, 3438, 3915, 4216, 4587, 5235, 5276, 5954, 6265, 6478, 6515, 7207
Set 33 (n = 20) 493, 584, 633, 937, 2330, 2377, 2491, 2587, 3153, 3683, 4216, 4248, 4530, 6114, 6419, 6478, 6525, 6689, 7202, 7456
Set 34 (n = 20) 10, 359, 383, 478, 626, 1472, 1487, 1647, 2475, 3683, 3780, 4490, 4636, 5179, 5247, 5371, 5950, 6748, 6923, 7670
Set 35 (n = 20) 97, 626, 1039, 1163, 1426, 1617, 1704, 2002, 2248, 2690, 3168, 4216, 4638, 5247, 5614, 5950, 6265, 6461, 6632, 7428
Set 36 (n = 20) 366, 414, 544, 734, 1263, 1416, 2167, 2208, 2250, 2370, 2491, 2526, 2855, 3190, 3488, 4083, 4248, 6673, 6845, 6847
It should be noted that the individual sets 1 to 36 mentioned in Table 4 and the two following sets: Set 37 (n = 10): SEQ ID numbers: 1983, 507, 5431, 3043, 1665, 5776 , 2902, 6585, 3167 and 745; andSet 38 (n = 7): SEQ ID numbers: 1983, 507, 5431, 3043, 1665, 5776 and 7695 each represent preferred embodiments of the present invention taken individually, with which a score value can be obtained that only is within the limits of the present invention specified deviation from a master score, so that the individual sets 1 to 38 each provide exact statements regarding the in vitro determination of the severity of the host response of a patient who is in an acutely infectious and / or acute inflammatory condition in a sample of a subject or patient.
Preferred gene marker tuples
In an earlier unpublished patent application DE 10 2009 044 085 of the applicant, gene expression markers were found which can indicate an infection in patients with a systemic inflammatory response syndrome (SIRS) (DE 10 2009 044 085). Of the 13 gene markers examined there, 6-7 markers can be found in the list of 8537 gene probes examined here. The explanation for this is that SIRS patients with an infection are more frequently exposed to high immune stress than SIRS patients without an infection. In the following steps it is shown that the score calculated from the expression of these markers and according to formula 1 depicts the host response similar to the master score introduced in the 1st application example. In addition, it is shown that the deviation from the master score can be reduced very significantly by expanding the marker set.The gene selection of the 1st application example contains 6 gene sequences from an application (DE 10 2009 044 085) not previously published by the applicant, which are indicated here by their symbols: TLR5 and CD59 in heatmap cluster 2, CPVL and FGL2 in the heatmap Cluster 5, HLA-DPA1 and IL7R in heat map cluster 6. The gene probe used on the microarray bead chip HumanHT-12 v3 did not deliver any signals for another gene marker (CLU). For these 7 gene markers, expression values were available for 67 patient samples from Table 2, which were measured on an alternative platform, the so-called real-time PCR. The substitute representative TFPI for CLU was selected from the list of 8537 gene probes, whose expression values on the microarray reached a correlation of 0.8 (correlation coefficient according to Pearson) with the expression values associated with the marker CLU on the real-time PCR.From the gene expression of the 73 investigated RNA samples, which was determined on the microarray for the 7-tuple of gene probes, we calculated the score according to formula 1. Its deviation from the master score was a maximum of ± 6 percentage points for half of the samples, a maximum of ± 11 percentage points for three quarters of the samples and ± 18.3 percentage points for 90% of the samples.In the next step, 1 gene probe from each of the remaining 8530 gene probes was added to this set of 7 markers, and the associated score was calculated using Formula 1. The set was expanded to include the probe that produced the smallest error. The error was defined as the 95% quantile of all absolute deviations from the master score. The procedure was repeated until this error was less than ± 10 percentage points. This case occurred after the gene marker tuple was expanded to 10 gene probes. The values of the scores for 7 and 10 gene probes as well as the master score were summarized in Table 5. This table also shows the expression signals of the associated gene sequences, logarithmized to base 2.
Table 5: Score values for 7 and 10 selected gene probes in comparison to the master score (columns 2 to 4). Base 2 logarithmized expression values for 10 selected gene probes, which are described by the associated sequence number (2nd line), accession (3rd line) and symbol (4th line). The 5th line shows the heat map cluster into which the respective probe was classified.
Example 3: Determination of the severity of the host response on alternative measurement platforms
As already mentioned in the 2nd example, gene expression signals were measured for 7 relevant gene markers from Table 5 and for 67 RNA samples by means of a real-time PCR. The following measurement and analysis steps were carried out for this purpose.
Real-time PCR
The Platinum SYBR Green qPCR SuperMix-UDG kit from Invitrogen (Invitrogen Germany, Karlsruhe, FRG) was used. The patient cDNA was diluted 1:25 with water, 1 µl of which was used in the PCR. The samples were each pipetted in 3 replicates. PCR mixture per well (10 µl) 2 µl template cDNA 1: 1001 µl forward primer, 10 mM1 µl reverse primer, 10 mM1 µl fluorescein reference dye5 µl Platinum SYBR Green qPCR SuperMix-UDG
A master mix was prepared without a template, this was aliquoted in 9 μl aliquots into the PCR plate, and the patient cDNAs were pipetted in each case.
The subsequent PCR program consisted of the following steps:
The iQ <TM> 5 Multicolor Real-Time-PCR Detection System from BIORAD was used with the associated evaluation software. The so-called Ct values (estimated number of cycles when the threshold is exceeded) were automatically calculated as a measurement result by the program in the area of the linear rise of the curves. The measured values were saved in string format.
Data analysis:
The data analysis was carried out using the free software R Project Version R 2.8.0 (R.app GUI 1.26 (5256), S. Urbanek & SMIacus, © R Foundation for Statistical Computing, 2008), which is available at www.r -project.org is available (see R Development Core Team, 2006).The data matrices of the measured Ct values used in the analysis were processed as follows. Together with the marker genes, 3 so-called housekeeper genes were measured, which were used as references. For normalization, the mean value of the 3 selected housekeeper genes was calculated for each sample. The Ct value of each individual marker was subtracted from this value. Each Delta Ct value obtained in this way reflects the relative abundance of the target transcript in relation to the calibrator, whereby a positive Delta Ct value means an abundance higher than the mean value of the references and negative Delta Ct value means an abundance smaller than the mean value of the references. The data matrix of the normalized Delta Ct values is summarized in Table 6.
According to formula 1 and the description in the 1st example, the associated score for quantifying the severity of the host response was calculated from the expression signals, which were measured by means of real-time PCR. The comparison of this score with the master score is demonstrated in FIG. 6, the master score being shown by the associated error bar of 10 percentage points up and down and the score determined from the PCR measurement as a diamond symbol. As already described in the 1st example, the calculation rule for the score is independent of the number of gene markers used and the measurement platform; only the mean expression signals of the phenotype groups NaN and SaS, which were defined in Table 1, are required for its calculation.
As can be seen from FIG. 6, the score, which was determined from the real-time PCR measurement of 7 markers, shows a similar trend to the master score and thus provides information on the severity of the host's response in an acute inflammatory condition Load on the organism. It should be mentioned that real-time PCR is a simpler, faster and cheaper measurement platform for determining gene expression than a microarray. Their limitation lies in the lower number of simultaneously measurable gene markers.
Table 6: Summary of the Delta Ct values normalized with respect to 3 references for 7 selected gene sequences and 67 RNA samples. The sample ID refers to the patient samples shown in Table 2.
Example 4: Differential expression of proteins for the in vitro determination of a severity of the host response (host response) of patients
The differential expression of markers for the in vitro determination of a severity of the host response of patients can take place not only on the basis of transcriptomic markers, but also at the protein level. There are numerous examples of the use of proteins as biomarkers, which have already been briefly discussed (Pierrakos, 2010). It is also pointed out that individual protein markers have so far not provided satisfactory or only mediocre results and that combinations of protein markers should preferably be used.In the following, experiments are described by way of example, with the result of which it can be shown that protein markers are equally suitable for determining the in vitro determination of a severity of the host response of patients. Proteins for the gene transcripts of which it has already been shown in the previous examples that they are suitable for the stated purpose were preferably selected for the investigation.
Examined patient groups
Samples from 9 sepsis patients, 3 patients after cardiac surgery and 7 healthy volunteers were examined (EDTA blood samples). The patients after cardiac surgery (ICU patients) were classified according to clinical criteria at the time of sample collection with the diagnosis SIRS (Systemic Inflammatory Response Syndrome). The three patient groups represent the phenotypes LaS and SaS (together), NaS and NaN from Table 1.
Experimental implementation
From the blood samples, two cell populations of white blood cells were isolated with the aid of a density gradient (Lymphocyte Separation Medium LSM 1077, PAA Laboratories GmbH, Cölbe): peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (polymorphonuclear cells) PMNs). The cells used for the experiments represented two subpopulations of the PBMCs: the T lymphocytes and monocytes.The proteins were detected by means of flow cytometry and Western blotting, with flow cytometry being used for surface proteins and Western blotting for intracellular proteins. Monoclonal antibodies were preferred for both methods.Using flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson GmbH Heidelberg), the expression of the proteins of the following genes on T lymphocytes and monocytes was examined: CD59, HLA-DPA1, IL7R, TLR5 and HLA-DR complex. The analyzed blood samples were made up as follows: sepsis patients (n = 9), ICU patients (n = 3) and healthy controls (n = 7). The distinction between T lymphocytes and monocytes was made using two surface markers: CD3 coreceptor for T lymphocytes [Tsoukas et al., 1985] and CD14 receptor for monocytes [Goyert et al., 1988]. In addition, the cells were identified based on their cell size (FSC-H) and their granularity (SSC-H).The expression of proteins from the following genes in the lysate of the total population of PBMCs in sepsis patients and healthy volunteers was examined by means of Western blotting: FGL2, CLU and CPVL. The experiments were carried out using standard Western blot protocols, positive controls (lysates of transfected cells) were always included and the experiments were normalized using β-actin. The anti-fibrinogen-like protein 2- (product of FGL2-Gen), anti-clusterin- (product of CLU-Gen) and anti-vitellogenic carboxypeptidase-like protein (product of CPVL-Gen) antibodies were mouse monoclonal Antibody, the secondary antibody was an HRP-coupled rabbit anti-mouse antibody. The antibody for β-actin was a rabbit monoclonal (13E5) antibody (Cell Signaling Technology Inc., Danvers USA).
Data analysis
The measurement data were made available by the software of the respective measurement device. They are summarized in Table 7. The expression of individual proteins was tested for statistically significant differences for the 2 to 3 phenotype groups examined. As in the first embodiment, the one-way analysis of variance (Anova) and the paired t-test were used. The results of the comparisons are listed at the end of Table 7. They are summarized below.In T lymphocytes, the protein expression from the IL7R gene was significantly lower in sepsis patients than in healthy donors. This showed a change in the same direction as in gene expression. The expression of MHC class II HLA-DPA1 antigen (product of the HLA-DPA1 gene) was significantly higher in sepsis patients than in healthy donors. This showed a change in the opposite direction than in gene expression. The T lymphocytes showed no significant differences between the examined phenotype groups in the protein expression from CD59, TLR5 and HLA-DR genes.The monocytes showed no differences in the protein expression from the genes HLA-DPA1 and TLR5 in the 3 groups; however, the protein expression from the CD59 and HLA-DR genes was significantly higher in healthy volunteers than in SIRS and sepsis patients. An IL7R gene product was not detectable in monocytes.The Western blot analysis of PBMC lysates showed that the protein expression from the FGL2 gene in sepsis patients is significantly increased compared to healthy controls, but the protein expression from the CLU gene is reduced in sepsis patients. This showed a change in expression in the opposite direction than in gene expression for both proteins. A CPVL gene product was not detectable in the lysates.
Table 7: Protein expression values for selected markers. Unmeasured values were given with n.a. designated. Patient mean values that differed significantly from healthy subjects were marked accordingly (“**” for p <0.01, “*” for p <0.05, and “+” for p <0.1).
Summary
From the example described it can be seen that the severity of the host response in the case of acute inflammation is also reflected in the expression of proteins from selected genes. The results of the gene expression analysis provide an extensive collection of marker candidates. It therefore makes sense to determine a suitable severity score from the protein expression which is sufficiently similar to the master score introduced in Example 1 from the gene expression.
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权利要求:
Claims (6)
[1]
Use of k-tuples on polynucleotides and / or fragments thereof selected from the group consisting of m polynucleotides of SEQ ID NO: 1 to SEQ ID NO 7704, wherein k is at least 7 and less than or equal to the number the polynucleotide m is in the group and at least one of the following subset sets of polynucleotides are used, where "n" indicates the number of polynucleotides of each set; for in vitro acquisition of a score as a measure of the severity of the host response of a subject who is in an acute infectious and / or acute inflammatory conditionSet 1 (n = 49)) SEQ ID NOS: 508, 553, 611, 679, 734, 769, 851, 860, 871, 896, 1117, 1263, 1646, 1647, 1648, 1675, 1688, 1975, 2011, 2077, 2415, 2516, 2560, 2581, 3381, 3491, 3820, 3947, 4156, 4230, 4506, 4576, 5012, 5235, 5614, 5730, 5803, 5873, 6114, 6262, 6265, 6301, 6689, 6738, 6820, 6847, 6879, 7069, 7230Set 2 (n = 47) SEQ ID NOS: 160, 309, 374, 428, 462, 911, 937, 1039, 1092, 1105, 1458, 1533, 1604, 1895, 1917, 1997, 2002, 2055, 2242 , 2332, 2369, 2386, 2427, 2516, 2541, 2560, 2785, 3359, 3407, 3624, 4230, 4587, 4636, 5164, 5235, 5247, 5371, 5776, 6278, 6328, 6497, 6636, 7156, 7201 , 7230, 7314, 7450Set 3 (n = 48) SEQ ID NOS: 10, 366, 411, 462, 493, 495, 567, 1204, 1226, 1409, 1414, 1449, 1487, 1583, 1724, 1744, 2013, 2055, 2064 , 2208, 2248, 2692, 2891, 3051, 3624, 4156, 4205, 4510, 4587, 4923, 5176, 5373, 5400, 5435, 5873, 5912, 5954, 6041, 6073, 6247, 6301, 6478, 6525, 6923 , 7207, 7450, 7670, 7681Set 4 (n = 47) SEQ ID NOS: 160, 359, 441, 493, 522, 541, 652, 691, 1128, 1408, 1583, 1651, 1652, 1664, 1688, 2002, 2077, 2248, 2273 , 2415, 2676, 2690, 2755, 2876, 3053, 3623, 4216, 4327, 4525, 4587, 4765, 4870, 5013, 5164, 5431, 5614, 5950, 6098, 6265, 6432, 6497, 6981, 7062, 7202 , 7314, 7450, 7607Set 5 (n = 49) SEQ ID NOS: 97, 428, 441, 543, 611, 851, 1136, 1384, 1533, 1868, 1997, 2077, 2183, 2208, 2226, 2260, 2329, 2386, 2475 , 2686, 2690, 2876, 3054, 3821, 4000, 4357, 4479, 4530, 4636, 4765, 4923, 5013, 5137, 5204, 5760, 5776, 5819, 5873, 5908, 6005, 6099, 6242, 6417, 6499 , 6585, 6847, 7450, 7670, 7681Set 6 (n = 48) SEQ ID NOS: 10, 97, 359, 475, 495, 627, 928, 1039, 1117, 1248, 1384, 1408, 1472, 1652, 1675, 1744, 1868, 1918, 2370 , 2423, 2537, 2742, 2865, 3051, 3086, 3408, 3916, 4030, 4078, 4274, 4294, 4362, 4751, 5129, 5235, 5247, 5431, 5734, 5803, 5811, 5908, 5950, 6005, 6417 , 6497, 6525, 6923, 7456Set 7 (n = 49) SEQ ID NOS: 32, 160, 383, 414, 493, 611, 652, 679, 734, 885, 896, 946, 1177, 1640, 1650, 1704, 1882, 2077, 2248 , 2250, 2260, 2415, 2561, 3086, 3488, 3623, 3624, 4135, 4156, 4160, 4510, 4525, 4530, 4742, 5137, 5204, 5247, 5730, 5950, 6114, 6210, 6225, 6430, 6478 , 6497, 6545, 6668, 7314, 7607Set 8 (n = 48) SEQ ID NOS: 359, 383, 515, 538, 544, 691, 769, 813, 1024, 1039, 1092, 1409, 1519, 1640, 1649, 1665, 1696, 1731, 1744 , 2167, 2183, 2226, 2260, 2273, 2425, 2516, 2618, 2634, 2672, 3051, 3168, 3202, 4160, 4754, 4966, 5373, 5465, 5493, 5541, 5574, 5912, 6005, 6216, 6432 , 6636, 6748, 6847, 7423Set 9 (n = 46) SEQ ID NOS: 160, 352, 544, 691, 802, 885, 1126, 1147, 1163, 1336, 1416, 1639, 1969, 2002, 2058, 2077, 2183, 2331, 2332 , 2426, 2526, 2742, 2855, 2860, 2891, 3054, 3138, 3488, 3947, 4560, 4576, 4707,4776, 5235, 5371, 5400, 5431, 5760, 5873, 6247, 6301, 6417, 6673, 6820, 7447, 7604Set 10 (n = 49) SEQ ID NOS: 8, 164, 462, 494, 495, 510, 545, 567, 611, 679, 941, 1039, 1105, 1128, 1147, 1318, 1533, 1649, 1918 , 1973, 1975, 2011, 2077, 2080, 2370, 2537, 3051, 3202, 3676, 4274, 4587, 4928, 5204, 5373, 5431, 5465, 5541, 5734, 5908, 5912, 5950, 6278, 6417, 6497 , 6668, 6673, 7156, 7230, 7670Set 11 (n = 46) SEQ ID NOS: 89, 97, 160, 355, 359, 366, 374, 411, 462, 475, 515, 538, 543, 691, 1384, 1647, 1649, 1651, 1724 , 2011, 2058, 2064, 2242, 2369, 2859, 3414, 4000, 4742, 4765, 4870, 4966, 5040, 5232, 5247, 5276, 5373, 5431, 5760, 5873, 5954, 6417, 6419, 6497, 6545 , 6636, 7484Set 12 (n = 49) SEQ ID NOS: 8, 89, 515, 543, 585, 769, 969, 1126, 1163, 1526, 1583, 1639, 1744, 2019, 2393, 2415, 2453, 2618, 2690 , 2692, 2810, 2855, 2863, 3153, 3158, 3190, 3408, 4000, 4083, 4104, 4248, 4479, 4491, 4550, 4661, 4877, 4995, 5176, 5276, 5599, 5695, 6073, 6114, 6265 , 6417, 6499, 6585, 6632, 6673Set 13 (n = 48) SEQ ID NOS: 414, 538, 946, 1263, 1384, 1512, 1895, 2077, 2248, 2260, 2516, 2676, 2975, 3168, 3414, 4083, 4274, 4776, 4800 , 4919, 4923, 5179, 5204, 5431, 5493, 5541, 5619, 5695, 5819, 6005, 6073, 6099, 6210, 6247, 6265, 6350, 6417, 6432, 6499, 6536, 6545, 6636, 6668, 6689 , 7040, 7062, 7472, 7604Set 14 (n = 47) SEQ ID NOS: 383, 428, 538, 553, 691, 814, 871, 896, 911, 937, 1426, 1639, 1685, 1688, 1983, 2093, 2253, 2260, 2454 , 2516, 2587, 2672, 2761, 2865, 2975, 3086, 3781, 4000, 4030, 4308, 4510, 4636, 4923, 5137, 5235, 5574, 5776, 5819, 5908, 6226, 6278, 6417, 6632, 7202 , 7230, 7315, 7456Set 15 (n = 46) SEQ ID NOS: 97, 366, 383, 802, 1426, 1514, 1558, 1685, 1744, 1975, 2011, 2013, 2369, 2415, 2454, 2510, 2516, 2577, 2587 , 2759, 2968, 3168, 3364, 3641, 3780, 4083, 4230, 4294, 4587, 4638, 4817, 5040, 5164, 5276, 5371, 5465, 5541, 6073, 6098, 6114, 6184, 6216, 6497, 6515 , 7062, 7202Set 16 (n = 49) SEQ ID NOS: 10, 504, 541, 553, 567, 652, 802, 1024, 1092, 1136, 1197, 1519, 1646, 1647, 1648, 1652, 2055, 2058, 2260 , 2273, 2330, 2331, 2415, 2491, 2581, 2618, 2676, 2742, 3053, 3408, 3652, 3915, 4216, 4870, 5235, 5641, 5695, 5954, 6114, 6278, 6419, 6461, 6791, 6820 , 6847, 6923, 7428, 7604, 7670Set 17 (n = 20) SEQ ID NOS: 10, 160, 428, 871, 941, 1136, 1197, 1416, 1558, 1786, 1951, 2386, 2510, 2560, 3488, 3652, 3781, 5176, 5400 , 6515Set 18 (n = 20) SEQ ID NOS: 871, 1163, 1414, 1416, 1426, 1487, 2001, 2055, 2369, 2386, 2552, 2577, 2865, 3051, 4550, 4577, 5614, 6098, 7369 , 7423Set 19 (n = 20) SEQ ID NOS: 567, 958, 2226, 2250, 2260, 2427, 2516, 3364, 4030, 4135, 5235, 5574, 5950, 6114, 6226, 6267, 6278, 6418, 7069 , 7518Set 20 (n = 20) SEQ ID NOS: 409, 1647, 1648, 1770, 1883, 1951, 2013, 2386, 2423, 3152, 3491, 4205, 4577, 4661, 4765, 4919, 7428, 7604Set 21 (n = 20) SEQ ID NOS: 355, 480, 494, 667, 1492, 2475, 2855, 2948, 3155, 3158, 3408, 3780, 4661, 5113, 5232, 5368, 5574, 6114, 6419 , 6499Set 22 (n = 19) SEQ ID NOS: 769, 1163, 1472, 2077, 2370, 2759, 3488, 3567, 3737, 3780, 4230, 4245, 4274, 4550, 5950, 6497, 7069, 7109, 7681Set 23 (n = 20) SEQ ID NOS: 160, 164, 355, 411, 1106, 1408, 1675, 1679, 2386, 2453, 2516, 2810, 3168, 3202, 3652, 4230, 5574, 5986, 7428 , 7484Set 24 (n = 19) SEQ ID NOS: 10, 164, 885, 1263, 1318, 1416, 1492, 1508, 1647, 1951, 2250, 2560, 2785, 2827, 3086, 4506, 5137, 5575, 5954Set 25 (n = 19) SEQ ID NOS: 32, 522, 679, 1519, 2001, 2491, 2516, 2676, 3412, 3737, 4205, 4294, 4560, 5235, 5954, 6005, 6114, 6499, 6525Set 26 (n = 19) SEQ ID NOS: 958, 1449, 1472, 1582, 2332, 2516, 2552, 2891, 2975, 3168, 3190, 3683, 3820, 3947, 4245, 4530, 7040, 7069, 7145Set 27 (n = 20) SEQ ID NOS: 428, 515, 544, 562, 567, 1263, 2002, 2332, 2526, 3438, 4577, 4754, 5574, 5614, 5912, 6328, 6515, 7156, 7423 , 7456Set 28 (n = 20) SEQ ID NOS: 355, 508, 937, 1263, 1973, 2002, 2510, 4078, 4156, 4550, 4673, 4817, 5247, 5368, 5730, 6005, 6247, 6515, 7201 , 7207Set 29 (n = 20) SEQ ID NOs: 10, 544, 871, 1408, 1487, 1649, 2002, 2415, 2690, 2859, 2975, 3126, 4577, 4636, 5541, 6073, 6417, 6432, 6866 , 6879Set 30 (n = 20) SEQ ID NOS: 896, 1248, 1318, 1472, 1786, 1830, 1983, 2386, 2865, 2975, 3641, 3916, 4030, 4530, 4995, 5472, 5619, 6099, 6247 , 6265Set 31 (n = 20) SEQ ID NOS: 355, 462, 1416, 1983, 2011, 2183, 2248, 2618, 3190, 3412, 4490, 4576, 4776, 4923, 5164, 6101, 6114, 6278, 7314 , 7369Set 32 (n = 20) SEQ ID NOS: 310, 1226, 1895, 2248, 2427, 2516, 2552, 2690, 3086, 3438, 3915, 4216, 4587, 5235, 5276, 5954, 6265, 6478, 6515 , 7207Set 33 (n = 20) SEQ ID NOS: 493, 584, 633, 937, 2330, 2377, 2491, 2587, 3153, 3683, 4216, 4248, 4530, 6114, 6419, 6478, 6525, 6689, 7202 , 7456Set 34 (n = 20) SEQ ID NOs: 10, 359, 383, 478, 626, 1472, 1487, 1647, 2475, 3683, 3780, 4490, 4636, 5179, 5247, 5371, 5950, 6748, 6923 , 7670Set 35 (n = 20) SEQ ID NOS: 97, 626, 1039, 1163, 1426, 1617, 1704, 2002, 2248, 2690, 3168, 4216, 4638, 5247, 5614, 5950, 6265, 6461, 6632 , 7428Set 36 (n = 20) SEQ ID NOS: 366, 414, 544, 734, 1263, 1416, 2167, 2208, 2250, 2370, 2491, 2526, 2855, 3190, 3488, 4083, 4248, 6673, 6845 , 6847Set 37 (n = 10): SEQ ID NOS: 1983, 507, 5431, 3043, 1665, 5776, 2902, 6585, 3167 and 745;Set 38 (n = 7): SEQ ID NOS: 1983, 507, 5431, 3043, 1665, 5776 and 7695.
[2]
2. Use according to claim 1, characterized in that fragments of the polynucleotides are used, in particular those having lengths of 20 to 1000, in particular 20 to 500 nucleotides, preferably 20 to 200 nucleotides.
[3]
3. Use according to claim 1 or 2, characterized in that the score is detected by expression signals, wherein the expression signals are obtained by hybridization and / or amplification, in particular PCR, preferably quantitative PCR, preferably real-time PCR and / or protein detection.
[4]
4. Use according to one of claims 1 to 3, characterized in that the score is used for diagnosis, prediction of the development or monitoring of the acute infectious and / or acute inflammatory condition of a patient and / or the course of the therapy and / or focus control.
[5]
5. Use according to one of claims 1 to 4, characterized in that the score is used to indicate an opportunity for recovery or non-recovery of a patient with acute infectious and / or acute inflammatory condition.
[6]
Use of a plurality of k-tuples of protein gene products selected from the group consisting of TLR5, CD59, CPVL, FGL2, IL7R, HLA-DPA1, HLA-DR, and CLU, where k is at least 7, for Recording a score as a measure of the severity of the host response of a subject who is in an acute infectious and / or acute inflammatory state.

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法律状态:
2017-10-31| PL| Patent ceased|

优先权:

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申请号 | 申请日 | 专利标题

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DE102011005235.6A|DE102011005235B4|2011-03-08|2011-03-08|A method for identifying a subset of polynucleotides from an initial set of polynucleotides corresponding to the human genome for in vitro determination of a severity of the host response of a patient|

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PCT/EP2012/053870|WO2012120026A1|2011-03-08|2012-03-07|Method for identifying a subset of polynucleotides from an initial set of polynucleotides corresponding to the human genome for the in vitro determination of the severity of the host response of a patient|

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