SYSTEMS AND METHODS FOR EARLY DETECTION,

MONITORING, AND TREATMENT OF SEPSIS

CROSS-REFERENCE TO RELATED APPLICATION

The benefits of United States Provisional Application No. 61/612,184 filed March 16, 2012 and entitled "Systems and Methods for Early Detection, Monitoring, and Treatment of Sepsis" are claimed under 35 U.S.C. § 119(e), and the entire contents of this provisional application are expressly incorporated herein by reference thereto.

TECHNICAL FIELD

The inventions relate generally to the field of diagnosis and treatment of sepsis and inflammation as well as the use of dialysis-like therapies.

BACKGROUND

Sepsis is a disease process or syndrome caused by an excessive inflammatory response of the body's immune defenses to an infection or toxin. Sepsis has a high incidence and high mortality in the general population. It occurs in 750,000 patients per year in the United States and in 35% of intensive care unit patients where it is the primary cause of death. There is a 27% mortality rate in hospitalized patients due to sepsis, a 54% mortality rate in patients with severe sepsis, and an 82% mortality rate in patients with septic shock, with a cost of care of about nineteen billion dollars per year. It is estimated that over 18 million people suffer from sepsis per year worldwide and several million of them die. The cause of the high mortality is thought to be multifactorial and includes factors such as late diagnosis of the disease and lack of testing the blood level of endotoxins. Because of the dismal outcome with the current therapy, there is a need for a new approach to the diagnosis and treatment of sepsis. There is a need for a system for the early diagnosis, monitoring and treatment of sepsis to decrease mortality. Sepsis is ultimately an inflammatory disease; even though it is initiated by a pathogen or toxin, the excessive and abnormal immune response is the cause of the disease process. There is an excessive and protracted stimulation of inflammation genes resulting in excessive release of pro-inflammatory or anti-inflammatory cytokines or an insufficient stimulation of anti- inflammation genes. At present, there is no effective treatment to prevent sepsis or modulate the immune response. Patients with sepsis typically receive a standard treatment that includes: hemodynamic support such as fluid administration and vasopressors; eradication of the infectious agent with antibiotics and surgery; measures to maintain adequate organ perfusion and function such as support of blood volume, cardiac output, blood pressure, and urine output; and treatment of damaged organs such as with a ventilator, oxygen, and dialysis. The severity and evolution of sepsis is monitored by several scoring systems such as APACHE (Acute Physiology And Chronic Health Evaluation) and SOFA (Sepsis-related Organ Failure Assessment), which evaluate clinical parameters related to multiple organ dysfunction syndrome. There is a need for an effective treatment to modulate the immune response or tools to monitor the immune response.

The clinical stages of sepsis are: sepsis, severe sepsis, and septic shock. Sepsis is represented by a fever, high/ low white blood cell count, and infection of tissue or blood, and an inflammatory response including hypothermia or fever, tachypnea, hypocapnia, tachycardia, leucopenia or leukocytosis. Severe sepsis is represented by sepsis plus organ dysfunction such as damage to the lungs, liver or kidney, and hypoperfusion or hypotension. Septic shock is represented by sepsis, organ dysfunction, and a mean arterial pressure (MAP) less than 60 mmHg. Lipopoly saccharide (LPS) is the most common endotoxin. Although normally LPS is scarcely detectable in blood, it is elevated in up to 80% of patients with sepsis. The blood level of LPS is very high in about 30% of patients with severe sepsis, and it can increase 1,000-fold. Blood levels are elevated even in the absence of an identified gram-negative infection and the level of LPS is associated with worsening of clinical outcome. LPS is carried in the blood by a specific carrier protein, lipopolysaccharide-binding protein, and interacts with cells of the innate immune system through a distinct receptor, D14.

The endotoxin-CD14 complex then engages a specific receptor, TLR4, to initiate intracellular signaling and the transcription of hundreds of genes. Endotoxin can be formed directly by gram-negative infections but its blood level can also be elevated in a setting of gram- positive infection, fungal infection, or in other cases of septic shock where no microbiologic source is identified. In these cases, the source of endotoxin is thought to be related to the movement of endotoxin across the gut mucosal barrier in the setting of shock, hypoxemia, and gut hypoperfusion. See Rachoin JS, Fostger D, Dellinger RP, Endotoxin removal: how far from the evidence? EUPHAS to EUPHRATES, Ronco C, Piccinni P, Rosner MH (Eds): Endotoxemia and Endotoxin Shock: disease, diagnosis and therapy. Contrib Nephrolo. Basel, Karger, 2010, vol 167, pp 111-118.

The inflammation response of sepsis has an early hyperimmune phase with a large production and release of pro-inflammatory mediators, i.e. cytokines IL-1, IL-6, TNF-a, often followed by a hypoimmune or immunosuppression phase in which there is hyper production and release of anti-inflammatory mediators, i.e. cytokines IL-10, IL-4, IL-lra, a suboptimal proinflammatory response, increased apoptosis, and anergy. Often death occurs in this phase due to opportunistic bacterial or viral infections or organ failure. The inflammation response is initiated by the activation of toll-like receptors (TLRs) which detect a wide range of microbial components including pathogen-associated molecular patterns (PAMPs), lipopolysaccharides (LPSs), or viruses, and cellular constituents released due to tissue injury. The activated TLRs in millions of immune cells trigger the activation, or upregulation, of multiple genes through complex receptor signaling including mediators like high morbidity group box-1 (HMGB1) and adapters like myeloid differentiation factor 88 (MyD88), which produce and release multiple factors including fNH-kB, and nuclear factor kB (NfkB) which controls the release of pro-inflammatory cytokines. These pro-inflammatory cytokines initiate a cascade of reactions in multiple interdependent pathways and the release of over 200 factors including additional pro -inflammatory cytokines, anti-inflammatory cytokines, coagulation cascade factors, oxidants, proteolytic factors, endothelial factors (i.e., endothelin-1, and nitric oxide), proteases that contribute to apoptosis, and factors influencing hemodynamics (i.e. catecholamines). These mediators all have local and systemic effects that can cause or contribute to major organ failure, which often leads to death. On the other hand, there is also activation of microRNA (miRNA) genes and formation and release of miRNA factors that down- regulate inflammation genes and the immune response.

Therefore, the inappropriate inflammatory response of sepsis appears to be caused by the malfunction of regulatory mechanisms that result in a loss of control of inflammation, eventually leading to excessive inflammatory response in the early phase, profound immunosuppression in the late phase, and host damage. Ultimately, the magnitude of inflammation depends on the number of activated white cells, the intensity of upregulation of each inflammation gene in each activated white cell, and the intensity of upregulation of the miRNA' s genes which down- regulate inflammation genes. The illness is usually diagnosed 1-3 days after it starts which is sufficient time for the body to mount a hyperimmune response, and in many cases, early organ damage occurs during this three-day time period. The early diagnosis and early initiation of treatment would decrease the magnitude and the duration of the pro-inflammatory phase.

The blood level of endotoxin is not tested. This prevents the implementation of a specific therapy to remove endotoxins and improve the outcome.

At present, there is no effective treatment methodology to predict sepsis or modulate the immune response during sepsis. There is a need for methodologies and therapies to address this and to modulate the immune response.

SUMMARY

An exemplary method for diagnosis and treatment of sepsis includes: measuring the severity of sepsis; measuring the severity of inflammation; choosing a modality of treatment based on the severity of sepsis and inflammation; evaluating use of the modality of treatment; monitoring response to the modality of treatment; and monitoring the performance of dialysis-like therapies (DLT) to remove at least one component of blood selected from the group consisting of an endotoxin and a mediator of inflammation. The modality of treatment may include leukapheresis of white blood cells or infusion of mesenchymal stem cells. Evaluating use of the modality of treatment may include determination of when to use the modality of treatment, the length of time to use the modality of treatment, or the frequency of use of the modality of treatment.

Because of the dismal mortality rate associated with the current approach to sepsis, disclosed is a new approach to the diagnosis and treatment of sepsis. In one embodiment, a system for the early diagnosis, monitoring and treatment of sepsis uses a series of technologies. This system is expected to decrease the incidence of mortality due to sepsis. The system may include a portable diagnostic apparatus for: the rapid detection of bacteria, endotoxemia, and rapid measurement of multiple analytes including pro-inflammatory and anti-inflammatory plasma and intracellular pro-inflammatory and anti-inflammatory cytokines, oxidants and antioxidants, CD4 and CD8 cells count, markers of apoptosis (caspases and mitochondrial membrane potential) and of proteolysis (gelsolin, actin, ubiquitin, proteasome), gene expression profiling, and plasma levels of several miRNAs. Some analytes could be removed and other analytes could be added. This system may integrate several apparatuses.

The system may be further used in a method that determines: (1) the phase and severity of inflammation on the basis of data from a portable diagnostic apparatus such as described above; (2) the clinical phase of sepsis on the basis of data from the patient's charts, such as body temperature, white cells count, cultures, endotoxemia, liver tests, mean arterial pressure, P0 ₂ , P0 ₂ /FI0 ₂ , and kidney function; and (3) the severity of sepsis on the basis of data from the diagnostic device and data from the patient's chart. An exemplary method processes the data and provides quantitative scores of the severity of inflammation (and prognosis), severity of oxidative stress, severity of sepsis, severity of sepsis-induced-proteolysis, and/or severity of apotosis/immunosuppression.

Changes in the scores with time, such as during treatment, can measure the response to treatments (e.g., effective, not effective, harmful).

For example, in an exemplary method, data generated by a diagnostic device may be used to provide the following information. The method may determine the phase of inflammation. The proinflammatory phase (high ratio of pro/anti-inflammatory cytokines, high CD4 count, low levels of caspases, high mitochondrial membrane potential, and high upregulation of pro- inflammatory genes), or the anti-inflammatory phase which has the opposite. Also, the method may give a quantitative score of the severity of inflammation (and prognosis). A high score (more severe and worse prognosis) occurs with high levels of inflammatory cytokines and IL- lra, oxidative stress, and caspases, low CD4 count, low levels of miRNA's and gelsolin, high levels of NFbK, actin and proteasome, high upregulation of inflammation genes and low mitochondrial membrane potential. In addition, the method may give a quantitative score of the severity of oxidative stress. A high score occurs with high GSH/GSSG ratio, high plasma levels of MDO and AOPP and intracellular levels of peroxide and superoxide. The exemplary method also may give a quantitative score to measure the severity of sepsis-induce-proteolysis. A high score occurs with low levels of Gelsolin and high levels of actin, proteasome. Further, the method may give a quantitative score of the severity of apoptosis/immunosuppression). A high score (worse apoptosis) occurs with low CD4 count, high levels of caspases and low

mitochrondrial membrane potential. The highest score could be anergy.

In an exemplary method, data generated by a diagnostic device also may be used to provide the following information. The method may give a quantitative score of the severity of sepsis. This score is formed combining data from an APACHE II score and the severity of inflammation score. The algorithm also includes an APACHE II calculator and clinical data to calculate the APACHE score. The following clinical data may be entered: fever, white cells count, SGOT/SGPT (liver damage), mean Arterial Pressure (< 60 mm Hg: septic shock), P02, P02 gradient, Ox sat (lung damage), serum creatinine, urine output <30cc per hour or <600cc during the previous 24 hrs., and Glasgow score. The method may identify a preferred modality of treatment. For example, the preferred modality may be DLT (removal of endotoxins with Toray-myxin or of inflammation mediators with high volume hemofiltration, or both, or dialysis) or non-DLT based on the presence or lack of endotoxins, the severity of inflammation, the severity of oxidative stress, the prognosis, and the severity of sepsis. In addition, the method may identify the type of DLT to be used, frequency, and/or length of treatment. Still further, the method may measure the response to treatment. Changes in scores over time can measure changes in the severity of the disease and the response to treatment.

The system may further include an apparatus to provide dialysis-like therapies created by integrating a Toray-myxin membrane to adsorb endotoxin and a filter for hemofiltration to remove mediators of inflammation.

Potential benefits of the system may include early and rapid diagnosis of endotoxemia, infection, and sepsis. This would allow initiation of treatment before the inflammatory response occurs.

The exemplary method may permit early and comprehensive assessment and monitoring of the severity of sepsis and inflammation. An algorithm may: measure changes in the severity of inflammation and sepsis and the response to treatment; identify ineffective or unnecessary treatments; make prognoses; and make rational use of dialysis-like therapies (DLT), that is, determinations whether DLT is indicated, the preferred DLT, when to start treatment, the duration and frequency of the treatment, when to stop treatment, and "adequacy" or

"sufficiency" of DLT to remove endotoxins and mediators of inflammation. The clinical parameters currently used to initiate DLT do not often justify its use.

The exemplary systems and methods will decrease the mortality of sepsis for a variety of reasons including the following.

(a) Frequent testing (e.g., 2-3 times per day in high risk patients) and fast measurement (e.g., in minutes or less) provides early and fast diagnosis of infection and endotoxemia, before signs of sepsis become clinically apparent or an excessive/overwhelming inflammatory response develops with systemic symptoms and often with organ damage. The exemplary embodiments can make early diagnosis well before the usual 1-3 days between the beginning of infection and the appearance of clinical sign of sepsis.

(b) A diagnosis of high severity of inflammation/risk of death can allow an early and more aggressive treatment, proper choice of treatment with or without DLT, as well as proper prescription of DLT (e.g., when to start, how long each treatment should last, how often treatment should occur).

(c) A diagnosis of high oxidative stress could provide an indication to use dialysis or DLT. Oxidative stress is a major cause of cell dysfunction and natural cell death or apoptosis, and stimulates the release of NFbK whose plasma levels have prognosis significance. Oxidative stress plays a key role in the severity of sepsis and outcome.

(d) Monitoring change in severity of inflammation and sepsis with treatment will demonstrate whether the treatments are effective or not, and can indicate discontinuation of ineffective or harmful treatments or continuation of effective treatments. For example, the dose of vasopressors would need to be titrated according to changes/severity of oxidative stress and not to an arbitrary target mean arterial pressure. In the absence of infection and high

inflammation score, antibiotics may need to be discontinued.

(e) Proper use of DLT can maximize its benefits in the treatment of sepsis.

(f) Monitoring changes in inflammation/sepsis scores and in immunological markers with DLT can demonstrate the "adequacy" or "sufficiency" of this treatment. Additional benefits of the system include tailoring and personalization of the treatment to each phase of the disease and each patient's immune response; a decrease in mortality; and monitoring of other diseases with chronic or acute inflammation, such as rheumatoid arthritis.

Two treatments that modulate the immune response in a sheep sepsis model are (1) leukapheresis of activated white cells and (2) infusion of mesenchymal stem cells, which has been done in mouse and rat models but not in large animal models such as sheep or pigs.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Even though sepsis is ultimately an inflammation disease, tools that can measure the severity of inflammation previously have not been clinically used. The severity of inflammation can be measured by the following factors: (1) the presence and severity of endotoxemia; (2) the severity of oxidative stress, i.e. levels of oxidants in plasma and/or cells stress, as oxidative stressis a major cause of cell dysfunction and apoptosis; (3) the severity of the pro-inflammation phase, i.e. the plasma levels of pro-inflammatory cytokines; (4) the severity of the anti- inflammation phase, i.e. the plasma levels of anti-inflammatory cytokines; (5) the severity of apoptosis; (6) the severity of proteolysis; (7) the magnitude of expression and upregulation of genes that influence oxidative stress, inflammation and vascular response; and (8) the magnitude of expression and upregulation of miRNA genes and miRNAs in plasma that can be used as an early marker of sepsis, as they correlate with the severity of sepsis, the SOFA score, and have prognostic significance. These tools and factors allow the implementation of specific therapies, monitoring the evolution of the disease, and predicting and monitoring the response to treatment. They also provide for personalizing the treatment to accommodate patients in different phases and varying degrees of severity of sepsis and inflammation, and to accommodate the different genetic makeup of each patient. There is currently a need for criteria to decide whether to use DLTs, to determine a modality of DLT (specifically, the removal of endotoxin or of inflammation mediators), when to start treatment, the duration of each treatment, and the frequency of treatments. The clinical parameters currently used to initiate DLT do not often justify its use. Even though DLT does not modulate the immune response, it is the only therapy currently available that can remove toxins and inflammation mediators from the blood and this benefit could be enhanced if the most appropriate modality of DLT is used at the right time. Additional benefits of DLT may include improvement to cardiovascular function, correction of fluid excess, and correction of electrolyte imbalances.

There also is a need for a tool to measure the impact of treatment on the evolution of the immune response. One methodology may address the factors mentioned above as well as additional clinical data including the phase of sepsis and liver, kidney and lung function. Such a methodology would permit the determination of whether a specific treatment would be beneficial or not.

There is a need for a therapy to modulate and prevent the hyperimmune and hypo- immune response. In exemplary embodiments, two modalities of treatment that could modulate the immune response and decrease mortality are: (1) the removal of activated white cells by leukapheresis; and (2) the infusion of mesenchymal stem cells.

An exemplary system permits early diagnosis and treatment of sepsis, comprehensive assessment of the severity of sepsis and inflammation, monitoring the evolution of sepsis and inflammation and response to treatment, and choice of appropriate treatments. Such a system may include a diagnostic apparatus, a method, and an apparatus to provide dialysis-like therapies. An exemplary diagnostic apparatus may permit rapid measurement, for example in a matter of minutes or less, of multiple analytes in a small volume of blood. The apparatus may permit rapid and frequent detection of bacteremia, endotoxemia and tissue infection (detection for example in minutes or less), and rapid and frequent measurement of multiple plasma analytes to identify the inflammation phase and the severity of inflammation. Analytes measured to determine the phase of the inflammation include, but are not limited to, the following: high levels of pro-inflammatory cytokines, intracellular IL-1 and IL-6, plasma IL-1, IL-6 , IL-6 soluble receptor gp80, IL-6 gp 130, and TNF-a (which indicate a hyperimmune phase of inflammation); and high levels of anti-inflammatory cytokines, IL-10, IL-4, ILlra (which indicate a hypoimmune phase of inflammation).

Analytes also may be measured to determine the severity of inflammation. The severity of oxidative stress may be determined by plasma measurements of plasma advanced oxidation protein (AOPP) and malondealdehyde (MDA), red cell glutathione (GSH/GSSG), and/or intracellular peroxide and superoxide. Reduced Glutathione (GSH) is the principal intracellular anti-oxidant in the body. The severity of apoptosis may be determined by the CD4 T helper cell count and CD8 T cell count, levels of caspase 8 and caspase 9, and mitochondria membrane potential. The severity of proteolysis may be determined by plasma levels of actin, gelsolin, ubiquitin, and proteasome.

Gene expression profiling is a function of the diagnostic apparatus to determine the presence and upregulation of genes that are associated with oxidative stress, inflammation, vascular response to salt intake and salt deprivation, and activation of the renal dopamine receptor that also participates in inflammation. The severity of inflammation also may be determined by miRNA genes expression profiling and plasma levels of the post-transcriptional regulators miRNA- 150, miRNA- 182, miRNA-342-5p and miR-486. The presence and plasma levels of miRNA have prognostic value.

The methodology may permit the determination of the clinical stage of sepsis and the severity of inflammation, choice of the modality of DLT, measurement of the changes in severity of the disease, and responsiveness to treatment. The method may include use of clinical data to identify the clinical stage of sepsis. The clinical data may include, but is not limited to:

temperature, white blood cell count, blood or tissue cultures, endotoxemia, and SGOT/SGPT (to detect liver damage); MAP; P0 ₂ , P0 ₂ gradient, and oxygen saturation (to detect lung damage); serum creatinine, and whether urine output is less than 30 cc per hour or less than 600 cc in the previous 24 hours; and additional components of APACHE or SOFA.

The method may include using data on the levels of cytokines, oxidants, apoptosis, proteolysis and genes expression to determine the phase of inflammation and the severity of inflammation.

The method also may include providing a recommendation concerning the modality of dialysis-like therapy to be used and when its use should begin. The dialysis-like therapies may include removal of endotoxins, removal of inflammation mediators, or both, for example according to the blood concentration of endotoxins and inflammation mediators.

The method also may provide for a comparison of changes in clinical parameters over time.

Multiple therapies to modify the excessive pro-inflammatory or anti-inflammatory phases of sepsis have been tried in humans but have either caused harm or failed to improve outcomes, including agents blocking single mediators (i.e. IL-1, TNF-a), anti-inflammatory mediators (i.e. interferon), and agents blocking mediators of the coagulation pathway.

Extracorporeal blood purification devices, also called dialysis-like therapies (DLTs), are used to remove microbial components (i.e. endotoxin) and inflammation mediators. Some devices function by adsorbing endotoxins to dialysis-like membranes, such as Polymyxin- adsorbed membranes. Others adsorb cytokines using adsorbent beads, and still others remove a broad range of small and mid-size inflammation mediators by convection, as in dialysis with either high flow or low flow hemofiltration. DLTs may be used to promote down-regulation of systemic inflammation and decreasing systemic effects of inflammation mediators. These therapies have shown promising results in small studies in patients, although no significant improvement in outcome has yet been demonstrated in large clinical trials. This may occur because of the impossibility to randomize or match patients with similar number and severity of comorbidities, small sample size, lack of standardized end points other than mortality, and lack of standardized length and frequency of treatment. Also, the clinical parameters currently used to initiate DLTs often do not justify its use. However, it is possible that DLTs do not remove enough mediators or that removal of mediators may not affect its high tissue concentration. More importantly, DLTs do not influence the activation and production of inflammation mediators by white cells, the activation of inflammation genes, the lack of down-regulation of these genes by miRNAs and apoptosis. These may be the reasons why DLTs are not very effective at decreasing the severity of illness and mortality. However, DLTs may be useful if used with well-defined indications.

An apparatus that provides dialysis-like therapies (DLT) may combine apparatuses that remove endotoxins and a broad range of inflammation mediators from the blood. See Table I below. If endotoxemia is present, the endotoxin may be removed by adsorption to Polymyxin-B that is adsorbed to a dialysis-like membrane (Toray-Myxin). If inflammation mediators are present (high levels of cytokines in the blood), they may be removed with hemofiltration which removes small and middle molecules, electrolytes and water. If both endotoxins and

inflammation mediators are present in the blood, both devices may be used. Turbulence of blood during dialysis is a problematic cause of activation of white blood cells and platelets, although extracorporeal blood tubing, needles and catheters may be designed to decrease this turbulence of the blood. The extracorporeal treatments may not use heparin or other forms of systemic anticoagulation.

Table I

NO ENDOTOXIN / HD or HF

LOW INFLAMM + AKI

NO ENDOTOXIN / HVHF

HIGH INFLAMM

NO ENDOTOXIN / HVHF

HIGH INFLAMM + AKI

HIGH OXIDATIVE STRESS HF or HD

AKI / Fluid overall / HF

LOW BP

AKI / Fluid overall / HF or HD

Normal BP

Diagnostic scores obtained before and after treatment can be used to measure response to treatment. For example, treatment can be continued if a score has improved, but re-evaluation of diagnosis and treatment is needed if a score has worsened. EXPERIMENTS

A study was conducted concerning the removal of activated white blood cells to treat sepsis. This study was conducted in a sheep sepsis model. A great number of inflammation mediators are released from activated white cells, such as monocytes, neutrophils and T cells, which can remain activated for days. Since at present it is not possible to down-regulate the genes that increase the production and release of pro-inflammation or anti-inflammation mediators, the removal of a certain number of activated white cells from the blood removes the cells releasing the predominant type of mediator at that time, i.e. pro-inflammatory or antiinflammatory, and this decreases the magnitude of the predominant phase of inflammation.

Another study was conducted that involved the infusions of mesenchymal stem cells (MSCs) to treat sepsis in a sheep sepsis model. The infusion of MSCs has beneficial effects because MSCs can differentiate into multiple adult tissues, lodge themselves in injured tissues/organs to enhance tissue repair, reduce apoptosis, modulate inflammatory response (decrease hyperimmune and hypoimmune phases) and protect endothelial integrity. MSCs also have been found to decrease mortality in mice.

The method used involves:

(a) Diagnosis of bacteremia using methods based on real-time PCR and/or DNA recognition. Devices from the Instant labs Inc., Hunter (Reston, Virginia), Illumina Inc. (San Diego, California), or similar devices can also be used to diagnose bacteremia.

(b) Diagnosis of endotoxemia using a chemiluminescent method or other methods and apparatuses.

(c) Measurement of cytokines using ELISA-based methods, flowcytometry or any other method and any of the existing apparatuses.

(d) Measurement of oxidants using fluorometric methods, Enzyme Immuno Assays, ELISA-base methods or any other method and any of the existing apparatuses.

(e) Measurement of CD4 and CD8 cells using flow cytometry or any other method and any of the existing apparatuses.

(f) Measurement of caspases 8 & 9 using ELISA or any other method and any of the existing apparatuses.

(g) Measurement of mitochondrial membrane potential could use fluorescence or any other method and any of the existing apparatuses.

These platforms also can be used to measure many other analytes.

Standard computer programming methods may be used to implement a method. In one embodiment, the therapeutic device includes a Toray-Myxin (Spectra Diagnostics, Toronto, Canada) to remove endotoxin and a Gambro's Prismaflex eXeed system, hemofilter JF1000-1400 (Gambro, Sweden), or similar devices from other manufacturers for high or low volume hemofiltration or similar apparatuses from different manufacturers; tubing and catheters with or without adsorbed heparin; and wireless connections between machines.

The protocol and methodology involves the following rationale. Early and rapid diagnosis of bacteremia, tissue infection, and endotoxemia can prevent the development of sepsis; early and rapid diagnosis, in minutes or less, of bacteremia or tissue infection (instead of the typical diagnosis occurring 12-36 hours after tissue infection), when clinical manifestations of infection and sepsis are present, allows early intervention, and prevents the development of an excessive hyperimmune inflammation response that is sepsis; and early and rapid diagnosis of endotoxemia allows the early implementation of therapeutic measures to decrease its production and to remove it from blood and prevent the development of inflammation and other deleterious effects caused by the endotoxin. Endotoxins are toxic structural components of certain bacteria which can cause organ dysfunction, shock and death.

Measuring cytokine levels allows determination of the phase of inflammation, the severity of inflammation, and choice of therapeutic interventions. In addition, high levels of IL- 6, IL-10 and IL-lra are associated with a poor outcome.

Intracellular IL-6 correlates better with the generation of cytokines than plasma levels. Membrane receptor complexes IL-6 soluble gp80 and IL-6 gpl30 mediate the activities of IL-6 and are present in the plasma.

The pro-inflammatory cytokines contribute to cause a hypercatabolic syndrome that is manifested by anorexia, hypoalbuminemia (suppression of hepatic synthesis by IL-6), increased synthesis of CRP, proteolysis, muscle wasting, and enhanced leukocyte cytotoxicity and natural killer T cells which stimulate the release of other pro-inflammatory cytokines. The cytokines also stimulate endothelial cells to synthesize other cytokines, nitric oxide (NO) and endothelin-1 (ET-1), release ROS, and cause the adhesion of circulating platelets and neutrophils. IL-18 is a pro-inflammatory cytokine that acts in synergy with IL-12

MicroRNA profiling in leukocytes and plasma allows the measurement of the degree of upregulation of miRNA genes and the miRNA levels in plasma. miRNAs are post- transcriptional down-regulators of inflammation genes, which bind to targeted mRNAs; its expression is down-regulated in sepsis, and its levels (low levels) correlate with the severity of sepsis and SOFA score. The level of miRNA-150 can be used as a marker of early sepsis.

miRNAs contribute to a determination of whether the inflammation response will be weak or strong. It has been demonstrated that: (a) miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiate sepsis patients from healthy controls; (b) miR-150 levels are significantly reduced in plasma samples of sepsis patients and correlate with the level of disease severity measured by the SOFA score, but are independent of the white blood cell count; (c) plasma levels of TNF-a, IL-10, and IL-18, all genes with sequence complementarity to miR-150, are negatively correlated with the plasma levels of this miRNA; (d) miRNA-150 regulates pro- and anti-inflammatory genes involved in sepsis and its expression levels correlate with the main immune response genes, such as TNF-a and IL-18(pro-inflammatory) and IL-10 (antiinflammatory); (e) the plasma levels ratio for miR-150/IL-18 may be used for assessing the severity of the sepsis; and (f) miR-150 levels in leukocytes and plasma correlate with the aggressiveness of sepsis. Regarding gene expression profiling, in sepsis there are about 50 genes that show a unique expression profile that is pathognomonic of sepsis (BMP Tang, AS McLean, Γ\¥ Dawes, SJ Huang, RCY Lin, The use of Gene-expression profiling to identify candidate genes in human sepsis, Am J respir Crit Care Med Vol 176, pp 676-684, 2007) and that can be used for early diagnosis of inflammation. Gene expression profiling may permit the measurement of the magnitude of upregulation of inflammation genes. It indicates which genes are on and which ones are off. The magnitude of NFkB upregulation is a predictor of outcome and mortality. Changes in gene expression may help to measure the response to treatment, and may be used for early diagnosis of sepsis. In sepsis, there are three clusters of coordinately expressed genes: (1) the cluster of genes suppressed in the presence of sepsis; (2) the cluster of genes involved in immune regulation, positive regulation (less expressed in sepsis) and negative regulation (more expressed in sepsis); and (3) the cluster of genes involved in mitochondrial functions such as oxidase phosphorylation and ATP synthesis.

Proteolysis is induced by IL-1, IL-6 and TNF-a and in an exemplary embodiment, the magnitude of proteolysis may be used as a marker of severity and outcome. Plasma gelsolin (a secreted from of cytoplasmic gelsolin) binds actin released from muscle and other cells and is a marker of mortality. Plasma gelsolin also binds inflammatory mediators such as platelet activating factor and lypophosphatidic acid and its physiological function may also be to localize inflammation and blunt its systemic effects. Depletion of plasma gelsolin is associated with a poor prognosis.

Plasma actin released from necrotic cells is a marker of proteolysis and mortality in dialysis. Plasma polymerized actin is a potent cause of thrombotic microcirculatory dysfunction which is thought to play a role in sepsis. In an exemplary embodiment, plasma ubiquitin (muscle protein that "tags" proteins for degradation by proteasomes) and plasma proteasomes (intracellular proteolysis) may be markers of proteolysis & inflammation.

The magnitude of oxidative stress may be used to determine the severity of sepsis and inflammation. Oxidants may cause numerous cell dysfunctions, apoptosis, cell necrosis, stimulate the release of pro-inflammatory cytokines and endothelin-1 (ET-1) which promotes adhesion of cells to the endothelium and activation of other cells, oxidation of DNA and proteins causing cell membrane and mitochondrial membrane and function damage, modify proteins structure and functions, and activate caspases and apoptosis.

Oxidative stress also regulates the activation of transcription factors such as NFkB.

NFkB is crucial for normal immune system function, up-regulating the genes producing IL-6 and TNF-a. However, inappropriate increase and/or prolonged activation of NFkB results in a harmful overproduction of cytokines which causes oxidative stress, and this in turn causes further up-regulation of NFkB.

Apoptosis impairs the ability of the immune system to combat pathogens, and its magnitude may have prognostic significance. Caspases are the main marker of apoptosis.

Caspase-9 mediates the loss of B cells and CD4 T helper cells in sepsis, when massive lymphocyte clonal expansion is needed the most. Caspases are responsible for the cleavage of the key cellular proteins, such as cytoskeletal proteins, that lead to the typical morphological changes observed in cells undergoing apoptosis.

Mitochrondrial membrane potential (delta psi) is an important parameter of mitochondrial function used as an indicator of cell health. In apoptosis, when energy metabolism is disrupted, delta psi is reduced, and when the membrane is ruptured by chemical or physical agents, delta psi is zero.

In sepsis there is loss of B cells and CD4T helper cells, and normal CD8T and NK cells. The loss of CD4 helper T cells by apoptosis decreases the number of circulating CD4 helper T cells which decreases the maturation of B cells into plasma cells, memory B cells, the activation of cytotoxic T cells and macrophages and also secretes and produces several cytokines.

DIALYSIS LIKE THERAPIES (OUT)

Removal of endotoxins from the blood improves overall organ function. In particular, it improves hemodynamics, reduces the relative risk of all-cause mortality and organ failure, and improves hospital survival. Improvements may be measured by changes in the SOFA score.

Removal of cytokines favorably modulates the host inflammatory response, decreases the magnitude of the pro-inflammatory response in the early phase of sepsis and the antiinflammatory response in late sepsis. It limits organ damage, and decreases cytokine

concentrations at the tissue level. It also normalizes the function of monocytes, neutrophils and lymphocytes.

Several devices remove inflammation mediators from the blood. They include high volume hemofiltration (HVHF), low volume hemofiltration (LVHF), hemoadsorption, coupled plasma filtration and adsorption, and high adsorption hemofiltration.

HVHF and LVHF are the most widely used methods to remove inflammatory mediators. Low volume hemofiltration (LVHF) and high volume hemofiltration (HVHF) use a flow rate of less than 35 milliliters per hour and greater than 70 milliliters per hour, respectively.

Hemofiltration with a low blood flow rate and continuous dialysis has been used with great success in acute kidney failure and fluid overload in critically ill patients for over 20 years. Hemofiltration (HF) removes significant amounts of inflammatory mediators from plasma.

Because they are water-soluble, convection carries both plasma water and solutes across a semipermeable membrane along a hydrostatic pressure gradient. However, many cytokines are also adsorbed to the filter's membrane. Most inflammatory mediators are so-called middle- molecular-weight molecules with a mass of 5-60 kDa and convection is far more effective than diffusion in removing these molecules. HF removes eicosanoids, leukotrienes, complement, cytokines, chemokines, coagulation factors and other potentially important small peptides and vasogenic substances. It also removes oxidants and cellular fragments. HVHF has beneficial hemodynamic effects such as less norepinephrine required and improved P02/FI02.

With hemoadsorption, sorbents in direct contact with blood via an extracorporeal circuit attract solutes through hydrophobic interactions, ionic attraction, hydrogen bonding, and van der Waals interactions.

Polymyxin B-immobilized polystyrene-derived fibers in a cartridge of a Toray-myxin device adsorb endotoxins from the blood. Investigators have found hemodynamic improvement and oxygen transport function, along with less need for renal replacement therapy, overall improvement of organ function in particular hemodynamics, reduced organ failure, and improved hospital survival as measured by changes in the SOFA score. However, it has been highlighted that many of these studies were of suboptimal quality. Toray-myxin has been used extensively in Japan and there is a clinical method available to measure the blood concentration of endotoxin.

CytoSorb are cartridges containing biocompatible polystyrene divinyl benzene copolymer beads which remove cytokines. This has been used in rats and in a few small clinical studies. CTTR resin-Kaneka, porous cellulose beads, effectively adsorb small to middle-sized proteins such as cytokines enterotoxins, and toxic shock syndrome toxin-2 in vitro and in an endotoxemic rat model and reduced mortality.

With coupled plasma filtration and adsorption, plasma is separated and run through hemoadsorption device and then the blood runs through a hemofilter. For high adsorption hemofiltration, a hemofilter membrane was modified to adsorb endotoxin.

HVHF may be chosen in place of hemoadsorption to remove inflammation mediators because it has not been reported that hemoadsorption removes more mediators than HVHF. HVHF removes more substances including oxidants, lactic acid, polypeptides, and likely many of the over 200 putative mediators participating in the inflammatory response. It also allows correction of fluid overload and plasma abnormalities caused by kidney failure and electrolyte imbalance. It is used with citrate regional anticoagulants with systemic heparinization of the patient. In some embodiments, heparin may be adsorbed to the dialysis membrane.

Hemoperfusion with polymyxin- adsorbed membrane (Toray-myxin) may be chosen to remove endotoxins because Toray-myxin has been used extensively in Japan; Toray-myxin removes oxidants and cytokines that are adsorbed to the membrane, and endotoxin levels in the blood can be measured.

As discussed below, two studies would be instructive concerning the exemplary embodiments.

STUDY 1 - REMOVAL OF ACTIVATED WHITE CELLS WITH LEUKAPHERESIS One proposed study involves a sheep sepsis model using the intraperitoneal implantation of a fibrin clot with E. coli. The animals would be divided into a control group and a treatment group. Leukapheresis of activated monocytes or neutrophils would be performed using the method and device of IBD Column Therapies International AB, Karolinske Hospital, Stockholm. Removal of 20% of the activated white cells in blood would be performed in the treatment group on days 2, 4, 6, 8, or until death. Measurements would be taken included daily blood pressure, temperature, and complete blood count (CBC). Pre-leukapheresis measurements would be taken including IL-6, IL-1, and IL-10, oxidative stress, endotoxin, blood cultures, intracellular oxidants, CD4, CD4, caspases 8 and 9, actin, gelsolin, proteasome and gene expression profiling.

Activated white blood cells could be eliminated by a method including identification of cell surface markers of activated leukocytes selected from T lymphocytes, neutrophil

granulocytes, and eosinophil granulocytes in the suspension; raising antibodies against one of more of the activated cells and immobilizing them on a support; and providing a column loaded with the support; diverting a portion of the patient's peripheral blood to make it pass through the column before re-infusing it to the patient to make the activated leukocytes couple with antibodies on the support, thereby eliminating them from the blood stream. Although not described here, exemplary embodiments may include corresponding columns and supports.

STUDY 2 - MESENCHYMAL STEM CELLS STUDY

Another proposed study involves mesenchemyl stem cells and would be performed with a sheep sepsis model using the intraperitoneal implantation of a fibrin clot with E. coli.

Mesenchymal stem cells (MSCs) would be isolated from the same animal species or from humans. The sheep would be divided into a control group and a treatment group. The treatment group would receive approximately 2.0 million MSCs on days 2, 4, 6, 8 or until death.

Measurements would be taken including daily BP, temp, and CBC. Pre-leukapheresis measurements would be taken including IL-6, IL-1, IL-10, oxidative stress, endotoxin, blood cultures, intracellular oxidants, CD4, CD4, caspase 8 and 9, actin, gelsolin, proteasome and gene expression profiling.

LABORATORY METHODS AND MATERIALS

By making use of various measurement technologies, an exemplary embodiment permits early diagnosis, monitoring, and treatment of sepsis. An exemplary embodiment may involve determining the phase and severity of inflammation, the clinical phase of sepsis, and which treatments to provide, to reduce the high incidence of mortality in patients with sepsis.

In vitro testing has determined that it is possible to detect the presence of bacteria, endotoxin, cytokines, CD4 and CD8 cells, mitochondrial membrane potential, and caspases 8 and 9. Software implementing the method may be used to choose a therapeutic modality based on data sent to it by one or more diagnostic devices. In an exemplary method which may be implemented through software, it is possible to identify change(s) in the form of improvement or worsening of patients' conditions. Additionally, in vitro testing, done with a closed circuit using blood, demonstrated safety in the form of no hemolysis and also demonstrated effectiveness by removing endotoxins, urea, and other markers. In vivo testing also was done in a clinical trial using sheep without kidney failure or sepsis.

SHEEP SEPSIS MODEL

Sheep (ewes) of 30-40 kg were chosen for the sepsis model because they have a hyperdynamic response compared to humans. Peritonitis was induced by implantation of an organism-impregnated fibrin clot (e. coli 011.B4) into the peritoneum. This model imitates human sepsis because: it creates a persistent nidus of infection; it causes systemic bacteremia over several days; survival of the animal is dose-dependent; the animals are conscious; it allows extraction of serial blood samples and continuous monitoring; cardiovascular responses mimic multiple aspects of human sepsis; and the variability among animals with peritonitis is avoided by standardization of the model. Bacteria at a concentration of lxlO ⁹ cfu/kg were incorporated into a fibrin clot and the bacteria-laden fibrin clot was implanted into the peritoneum under brief general anesthesia through a small midline abdominal incision of about 5 cm to 7 cm. The concentration of bacteria ranged from 1 x 10 ⁹ to 1 x 10 ¹¹ colony forming units per kilogram (cfu/kg) and produced a mortality rate of 70% to 85% in untreated animals. Sixty percent of survivors and eighty percent of no survivors can be demonstrated to have bacteremia at 30 minutes after implantation, with that number decreasing to 25% in survivors and increasing to 100% in no survivors at 2 hours. With fluid resuscitation, a hypodynamic state accompanied by hypotension, low cardiac output, and high systemic vascular resistance (SVR) is decreased or avoided. Criteria for euthanasia, heart rate greater than 200/min. This method was described by Zanotti-Cavazzoni SL, et al, Crit Care Clin 25 (2009)703-719, and was found to be applicable to the studies supporting the exemplary method, the supportive studies including the removal of activated white blood cells with leukapheresis and the mesenchymal stem cells study.

Laboratory methods used to measure endotoxins in the blood included an endotoxin activity assay (EAA), Spectral Diagnostics, Toronto, Canada, whole-blood chemiluminescent test.

Gene-expression profiling was performed in peripheral neutrophils using oligonucleotide microarrays (Adelaide Microarray Facility, Adelaide, Australia). See M P Tang, AS McLean, I W Dawes, SJ Huang, RBYLin, Am J Respir Crit Care Med Vol 176, pp 676-684, 2007, incorporated by reference in its entirety.

Regarding miRNA profiling in blood leukocytes and plasma, microarray technology and serial analysis of gene expression (SAGE, SuperSAGE), and quantitative reverse transcription- polymerase chain reaction in plasma were used. The All-in-One™ miRNA qRT-PCR Detection Kit Gene Copoeia, from Rockville MD, was used.

Intracellular and supernatant cytokines IL-1, IL-6, TNF-a, IL-10, IL-4, IL-lra were measured by ELISA kits from R & D system, Minneapolis, MN. A FACScan flow cytometer from Becton Dickinson, Bedford, MA, was used. Data was acquired and analyzed using Cell Quest software from Becton Dickinson, Bedford, MA.

Intracellular Superoxide measurements are preferably taken by measuring

dihydroethidium using an HPLC-based assay. See Am J Phys cell Physiol, 287: C895-C902, 2004, and Hydrogen Peroxide: fluorometric method (Cells Biolab Inc, San Diego, CA), which is incorporated herein by reference in its entirety.

In one preferred, exemplary embodiment, plasma AOPP measurements may be taken by performing a radioimmunossay. For example, the radioimmunoassay may be AOPP from Alpco Diagnostics, Salem, NH.

In one preferred, exemplary embodiment, red cell glutathione, GSH and GSSG measurements may be taken by EIA and may be from Cayman Chemical, Ann Arbor, MI.

Mandenylaldehyde (MDA) measurements were taken by Immunoblot/ELISA test for example from Cells Biolab Inc., San Diego, CA.

Gelsolin measurements were taken from a western blot using stained monoclonal antibody to human plasma gelsolin (for example, GS-2C4, Sigma, St Louis, MO), peroxidase- conjugated monoclonal antibody against mouse IgG (Sigma), and enhanced chemi-luminescence (for example, from Amersham, Arlington Heights, IL) used to detect the gelsolin band and purified human plasma gelsolin (Cytoskeleton, Denver, Co). Actin was measured by Western blot, with a monoclonal antibody against a synthetic actin C-terminal peptide (clone AC-40; Sigma). BUN was measured with a urease method. Plasma proteasome was also measured with a Western blot, using polyclonal antibody anti- human recombinant proteasome 19S8 from rabbit, by Boston Biochem, Cambridge MA, red cells glutathione, GSH and GSSG by EIA (Cayman chemical Ann Arbor MI). See Jarorek M and Glickman MH (2004) Cell Mol Life Sci 61: 1579-1588, which is incorporated herein by reference in its entirety.

Caspases 8 and 9 were measured with ELISA, in a preferred, exemplary embodiment from eBioscience San Diego, CA.

The mitochondrial membrane potential was measured with fluorescence, in a preferred, exemplary embodiment from Cayman Chemical, Ann Arbor, Michigan.

While various descriptions of the embodiments are described above, it should be understood that the various features can be used singly or in any combination thereof. Therefore, the inventions are not to be limited to only the specifically preferred embodiments depicted herein.

Further, it should be understood that variations and modifications within the spirit and scope of the inventions may occur to those skilled in the art to which the inventions pertain. Accordingly, all expedient modifications readily attainable by one versed in the art from the disclosure set forth herein that are within the scope and spirit of the inventions are to be included as further embodiments of the inventions. The scope of the inventions is accordingly defined as set forth in the appended claims.