WO2014022530A1 - The use of endotoxin neutralization as a biomarker for sepsis - Google Patents

Abstract

Provided herein are methods for detecting endotoxin neutralization in a subject. Also provided are methods for determining the effectiveness of a therapeutic agent for treating sepsis.

Description

THE USE OF ENDOTOXIN NEUTRALIZATION AS A BIOMARKER FOR SEPSIS

BACKGROUND

Mammalian survival is dependent on a rapid system to neutralize the potent immunostimulant effects of Gram negative bacterial endotoxin, a lipopolysaccharide found on the bacterial membrane. Acute exposure to endotoxin is responsible for many, if not all, of the toxic effects that occur during Gram-negative bacterial sepsis. A standard approach to monitor the response to this exposure or therapeutic agents to the exposure are nonexistent.

SUMMARY

Methods for detecting neutralization of endotoxin are disclosed herein. For example, provided herein are methods for determining the effectiveness of a therapeutic agent for treating sepsis. Provided herein is a method of determining the effectiveness of a therapeutic agent for treating sepsis comprising a) administering a therapeutic agent to the subject; b) adding exogenous endotoxin to a first biological sample from the subject after treatment with the therapeutic agent; c) detecting exogenous endotoxin in the sample from step b); d) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step c) from the total exogenous endotoxin added in step b); and e) calculating the percentage of total endotoxin neutralization in the first sample, wherein the percentage of total endotoxin neutralization is the percentage of undetected exogenous endotoxin of the total exogenous endotoxin, and wherein a decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

Further provided is a method of determining the effectiveness of a therapeutic agent for sepsis comprising a) administering a therapeutic agent to the subject; b) heating a first biological sample from the subject after treatment with the therapeutic agent; c) adding a selected amount of exogenous endotoxin to the first sample of step b); d) detecting exogenous endotoxin in the first sample from step c); e) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step d) from the total exogenous endotoxin added in step c); f) acidifying a second biological sample from a subject after treatment with the therapeutic agent; g) adding the selected amount of exogenous endotoxin to the second sample of step f); h) detecting exogenous endotoxin in the second sample from step g); i) determining the amount of undetected exogenous endotoxin in the second sample by subtracting the detected exogenous endotoxin from step h) from the total exogenous endotoxin added in step g); and j) calculating a percentage of protein endotoxin neutralization, utilizing the following equation:

((amount of undetected exogenous endotoxin the second sample - amount of undetected exogenous endotoxin in the first sample) / selected amount of exogenous endotoxin) X 100 wherein a decrease in the percentage of protein endotoxin neutralization after treatment as compared to a control indicates that the therapeutic agent is effective for treating sepsis.

Also provided is a method of treating sepsis in a subject comprising calculating levels of protein endotoxin neutralization, undetected exogenous endotoxin and enzymatic endotoxin neutralization in a biological sample from the subject; and calculating an endotoxin neutralization ratio using the following formula:

(protein endotoxin neutralization + undetectable exogenous endotoxin)/ enzymatic endotoxin neutralization a ratio of about 5 or greater indicating that the subject has sepsis; and administering a therapeutic agent for treating sepsis to the subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing

Figure 2 is a graph showing

Figure 3 is a graph showing

samples.

Figure 4 is a graph showing enzymatic neutralization in female control and sepsis samples.

Figure 5 is a graph showing protein neutralization in male control and sepsis samples. Figure 6 is a graph showing protein neutralization in female control and sepsis samples.

Figure 7 is a graph showing undetectable endotoxin in male control and sepsis samples.

Figure 8 is a graph showing undetectable endotoxin in female control and sepsis samples.

Figure 9 is a graph showing neutralization ratio in male control and sepsis samples. Figure 10 is a graph showing neutralization ratio in female control and sepsis samples. DETAILED DESCRIPTION

Methods for determining endotoxin neutralization in biological samples are provided herein. For example, set forth herein is a method of determining the effectiveness of a therapeutic agent for treating sepsis comprising a) administering a therapeutic agent to the subject; b) adding exogenous endotoxin to a first biological sample from the subject after treatment with the therapeutic agent; c) detecting exogenous endotoxin in the sample from step b); d) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step c) from the total exogenous endotoxin added in step b); and e) calculating the percentage of total endotoxin neutralization in the first sample, wherein the percentage of total endotoxin neutralization is the percentage of undetected exogenous endotoxin of the total exogenous endotoxin, and wherein a decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

In the methods set forth herein, sepsis is a condition in which the body has a severe response to bacteria. This response can be called systemic inflammatory response syndrome. A bacterial infection anywhere in the body can lead to sepsis. The infection can be in the bloodstream, bones, the bowel (for example, a peritoneal infection), the kidneys (for example a urinary tract infection), the lining of the brain (for example, meningitis), the liver, the gallbladder, the lungs (for example, pneumonia), the appendix, or the skin, to name a few. Other sites of infection include, but are not limited to, surgical wounds, intravenous lines, surgical drains and bedsores. In the methods set forth herein, sepsis can be gram-negative sepsis (i.e, caused by gram negative bacteria such as, for example, Hemophilus influenza, Klebsiella pneumonia, Legionella pneumophila, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcecens, Helicobacter pylori,

Salmonella enteriditis, Salmonella typhi, Neisseria meningitides and Moraxella catarrhalis, to name a few) or gram-positive sepsis (i.e., caused by gram positive bacteria such as, for example, Streptococcus, Staphyloccocus, Corynebacterium, Listeria, Bacillus and

Clostridium, to name a few).

As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with or at risk of developing sepsis. The term patient or subject includes human and veterinary subjects.

As utilized throughout, the therapeutic agent for treating sepsis can be, but is not limited to, a chemical, a small or large molecule (organic or inorganic), a drug, a protein, a peptide, a cDNA, an antibody, an aptamer, a morpholino, a triple helix molecule, an siR A, a shRNA, an miRNA, an antisense RNA, a ribozyme or any other compound now known or identified in the future that treats sepsis. Agents for treating sepsis are known in the art. For example, antibiotics (for example, and not to be limiting, ceftriaxone, cefuroxime, vancomycin, ceftazidime, tobramycin, cefotaxime, penicillin, clindamycin, ciprofloxacin, cefepime, azithromycin, ampicillin or combinations thereof), vasopressors (for example, isoproterenol, dopexamine, dobutamine, dopamine, epinephrine, norephinephrine or phenylephrine, metariminol, ephedrine or vasopressin), corticosteroids (for example, fludrocortisones, Cortisol or hydrocortisone), immunomodulators (for example, azathioprine, mercaptopurine or cyclosporine), insulin or combinations thereof can be utilized to treat sepsis.

Any appropriate route of administration may be employed to deliver the therapeutic agent. For example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration can be performed. The therapeutic agent can also be delivered intranasally, inhaled or administered with a nebulizer. Administration can be systemic or local. Therapeutic agents can be in a pharmaceutical composition that can be delivered locally to the area in need of treatment, for example by local injection or intubation. Multiple administrations and/or dosages can also be used.

After treatment with the therapeutic agent, a first biological sample is obtained from the subject. As used herein, a biological sample subjected to testing is a sample derived from a subject such as a mammal or human and includes, but is not limited to, any biological fluid, including a bodily fluid. Examples of bodily fluids include, but are not limited to, whole blood, plasma, serum, urine, saliva, ocular fluid, ascites, a stool sample, spinal fluid, tissue infiltrate, pleural effusions, lung lavage fluid, sputum, mucus and the like. The biological fluid includes a cell culture medium or supernatant of cultured cells from the subject. For example, the sample can be a blood sample or a serum sample. The sample can also comprise a citrated or EDTA-containing sample. A suitable time for obtaining the biological sample will vary depending on one or more factors, such as, but not limited to, the type of therapeutic agent, the extent of sepsis, the mode of administration, or whether single or multiple doses of the therapeutic agent must be administered to observe a therapeutic effect. The biological sample can be obtained at about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or at any time in between, after

administration of the therapeutic agent. In some cases, a control sample is collected from the subject prior to administration of the therapeutic agent. Such a control sample can be collected concurrently with administration of the therapeutic agent (so long as the agent has not had a biological effect on endotoxin) or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or at any time in between, before administration of the therapeutic agent.

Once a first biological sample is obtained from the subject, a selected amount of endogenous endotoxin is added to the sample. The selected amount of endogenous endotoxin can be added to achieve a concentration of about 50 EU/ml, 100 EU/ml, 150 EU/ml, 200 EU/ml, 250 EU/ml, 300 EU/ml, 350 EU/ml, 400 EU/ml, 450 EU/ml, 500 EU/ml, 550 EU/ml, 600 EU/ml, 650 EU/ml, 700 EU/ml, 750 EU/ml, 800 EU/ml, 850 EU/ml, 900 EU/ml, 950 EU/ml or 1000 EU/ml in the sample. The endotoxin can be obtained from commercial sources or prepared by various extraction methods that utilize chloroform, phenol, ether, acid and/or detergents. The endotoxin can also be prepared from a culture that is grown, heat- lysed and centrifuged to remove cell debris. For example, the endotoxin can be obtained from a Salmonella typhimurium LT2 stock

Prior to endotoxin detection, the methods set forth herein can further comprise heating the biological sample containing the selected amount of exogenous endotoxin; acidifying the heated sample to a pH of about 1 to 4; contacting the acidified sample with an acidic protease; and increasing the pH of the protease-treated sample to about 6 to 8. The acidic protease can be deactivated after contacting the sample with an active protease and before detecting endotoxin in the sample. Optionally, the acidic protease is inactivated by a pH of about 7.0 (e.g. 6.8-7.2).

As set forth above, prior to endotoxin detection, the sample can be heated to a temperature of about 55°C to about 70°C. Therefore, the sample can be heated to a temperature of about 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or about 70°C. The sample can be heated for about 20 to about 40 minutes. For example, the sample can be heated for about 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or about 20 minutes. The sample can be cooled after heating to about, for example, to about 18°C to about 25°C. Therefore, the sample can be cooled to about 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or to about 25°C.

The heated sample can then be acidified by adding an acid to the sample. For example, hydrochloric acid can be added to the sample to obtain a pH of about 1 to about 4. The acid can be at a normality or molarity sufficient to acidify a sample to a pH of about 1 to about 4 without unnecessary dilution of the sample. For example, the acid can be 1M HC1. Other acids include, but are not limited to, nitric acid, sulfuric acid and acetic acid.

Optionally, an alkaline phosphatase inhibitor can be included when acidifying the sample or just prior to or after acidifying the sample.

The acidified sample can be contacted with an active acidic protease at a pH of less than 4, less than 3, less than 2 or about 1. Therefore, the pH can be about 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or about 1.0. Prior to acidifying the sample, the sample can be diluted to about 1 part sample to about 10 parts diluent. For example, the sample can be diluted to about 1 part sample to about 1 part diluent, to about 1 part sample to about 2 parts diluent, to about 1 part sample to about 3 parts diluent, to about 1 part sample to about 4 parts diluent, to about 1 part sample to about 5 parts diluent, to about 1 part sample to about 6 parts diluent, to about 1 part sample to about 7 parts diluent, to about 1 part sample to about 8 parts diluent, to about 1 part sample to about 9 parts diluent or to about 1 part sample to about 10 parts diluent. The diluent can be, but is not limited to, a buffer comprising divalent cations, for example, a Tris buffer comprising MgCl₂ or a Tris buffer comprising CaCl₂.

As used herein, acid, acidic, aspartic or aspartic acid proteases refer to proteases active at low pH. For example, the protease is active at a pH from about 0.0 to about 6.0 or any pH between 0.0 and 6.0, inclusive. Such proteases are inactive at a pH of about 6.0 to about 14.0. As used herein, an inactive acidic protease refers to a protease without proteolytic activity (i.e., a protease that is unable to cleave an amino acid sequence such as a polypeptide or protein). As used herein, an active acidic protease refers to a protease with proteolytic activity (i.e., a protease that is able to cleave an amino acid sequence). By way of example, an active acidic protease can be inactivated by a pH of 6.5 or higher (i.e., the protease is inactive in a solution with a pH of 6.5 or higher). The pH of a solution can be altered by addition of chemicals to the solution. For example, hydrochloric acid can be used to reduce pH and sodium hydroxide can be used to raise pH. As discussed above, pH adjustment is performed with an acidic or a basic solution of such normality or molarity to reduce unnecessary dilution of the sample. Phosphoric acid can be used to maintain a pH of about 6.5. Optionally, a pepsin inhibitor is used to inactivate pepsin. Pepsin inhibitors include, but are not limited to, acetamidine, N-acetyl-D-phenyalanyl-L-diiodotyrosine, N- acetyl-L-phenyalanyl-D-phenylalanine, p-aminobenzamidine, benzamidine, butyamine, diazoketones, ethylamine, pepstatin, and phenylactamidine.

Acid or acidic proteases, such as endopeptidases, are known and have been grouped into three families, namely, pepsin (Al), retropepsin (A2), and enzymes from

pararetroviruses (A3). The members of families Al and A2 are known to be related to each other, while those of family A3 show some relatedness to Al and A2. Microbial acid proteases exhibit specificity against aromatic or bulky amino acid residues on both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than that of pepsin. Acid proteases include microbial, fungal, viral, animal and plant acidic proteases. Microbial aspartic proteases can be broadly divided into two groups, (i) pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) rennin-like enzymes produced by Endothia and Mucor spp (Rao et al., Microbiology and Molecular Biology 62(3):597-635 (1998); Richter et al, Biochem. J. 335:481-90 (1998)). Examples of acidic proteases include, but are not limited to, pepsins, including pepsins A, B and C; rennin;

chymosin; plasmepsin; cathepsins, such as, for example, cathepsin D and cathepsin E; human urinary acid protease; and viral proteases like HIV protease. Fungal proteases include, but are not limited to, fungal proteases derived from Neurospora oryzae, Mucor pusillus, Mucor miehei, Aspergillus niger, Rhizopus chinensis, or Endothia parasitica. Microbial proteases include, but are not limited to, yeast proteinase A, aspergillopepsinogen, rhizopuspepsin, penicillopepsin, and endothiapepsin.

In the methods set forth herein, the pH of a protease-treated sample can be increased to a pH greater than 6, greater than 6.5, greater than 7, greater than 7.5 or about 8 by addition of a base to the protease-treated sample. Therefore, the pH can be about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.5, 7.7, 7.8, 7.9 or about 8. The base can be at a normality or molarity that the pH adjustment is made without unnecessary dilution of the sample. For example, 0.5 N sodium hydroxide can be used to increase pH. Other examples of bases include, but are not limited to, potassium hydroxide and ammonia. Prior to increasing the pH of the sample, from about 6 to about 8, the sample can be diluted to about 1 part sample to about 10 parts diluent. For example, the sample can be diluted to about 1 part sample to about 1 part diluent, to about 1 part sample to about 2 parts diluent, to about 1 part sample to about 3 parts diluent, to about 1 part sample to about 4 parts diluent, to about 1 part sample to about 5 parts diluent, to about 1 part sample to about 6 parts diluent, to about 1 part sample to about 7 parts diluent, to about 1 part sample to about 8 parts diluent, to about 1 part sample to about 9 parts diluent or to about 1 part sample to about 10 parts diluent. The diluent can be, but is not limited to, a buffer comprising divalent cations, for example a Tris buffer comprising MgCl₂ or a Tris buffer comprising CaCl₂.

In the methods set forth herein, endotoxin can be detected via methods standard in the art, for example, and, not to be limiting, these include gel-clot assays, turbidimetric assays, and chromogenic assays. The PyroGene^® Recombinant Factor C Endotoxin detection System (Lonza 50-658U; Allendale, NJ) is an example of a fluorescence assay that can be utilized.

Once the amount of exogenous endotoxin is detected in the first biological sample, the amount of undetected exogenous endotoxin is calculated by subtracting the amount of exogenous endotoxin detected in the first biological sample from the total exogenous endotoxin added to the first biological sample. After the amount of undetected exogenous endotoxin is calculated, the percentage of total endotoxin neutralization in the first sample is calculated. The percentage of total endotoxin neutralization is the percentage of undetected exogenous endotoxin of the total exogenous endotoxin added to the first biological sample (amount of undetected exogenous endotoxin/total exogenous endotoxin) x 100. A decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis. The control can be the percentage of total endotoxin neutralization in a sample from the same subject prior to or at about the same time as administration of the therapeutic agent, the percentage total endotoxin neutralization in a sample from the same subject after administration of a different therapeutic agent or the percentage of total endotoxin neutralization in a reference sample. An increase in total endotoxin neutralization as compared to control indicates that the therapeutic agent is not effective in treating sepsis. If an increase in total endotoxin neutralization is observed after treatment with a therapeutic agent, one of skill in the art can, for example, discontinue treatment, alter the dosage of the therapeutic agent or administer a different therapeutic agent. As utilized throughout, the reference sample can be from the same subject or a different subject before or at about the same time as administration of the therapeutic agent.

The reference sample can also be a sample obtained from a subject after the effects of the therapeutic agent have subsided. The reference sample can also be from a healthy subject. This method of determining the effectiveness of a therapeutic agent for treating sepsis can further comprise f) heating a second biological sample from the subject after treatment with the therapeutic agent; g) adding exogenous endotoxin to the second sample of step f); h) detecting exogenous endotoxin in the second sample from step g); i) determining the amount of undetected exogenous endotoxin by subtracting the detected exogenous endotoxin from step h) from the amount of added exogenous endotoxin from step g); and j) calculating the percentage of enzymatic endotoxin neutralization utilizing the following equation:

((amount of undetected exogenous endotoxin in the second sample from step i - amount of undetected exogenous endotoxin in the first sample from step b) / amount of total exogenous endotoxin added in step g) X 100;

wherein an increase in the percentage of enzymatic endotoxin neutralization in the second biological sample as compared to control indicates that the therapeutic agent is effective for treating sepsis. In this method, the second biological sample can be heated to from about 55°C to about70°C. Also, in this method, the same amount of exogenous endotoxin is added to the first and the second biological sample.

As utilized throughout, the percentage of enzymatic endotoxin neutralization is the percentage of endotoxin that is not detected due to heat inactivation of enzymatic processes in the biological sample, for example, in blood plasma. The control can be the percentage of enzymatic endotoxin neutralization in a sample from the same subject prior to or at about the same time as administration of the therapeutic agent, the percentage total enzymatic endotoxin neutralization in a sample from the same subject after administration of a different therapeutic agent or the percentage of total enzymatic endotoxin neutralization in a reference sample. A decrease in enzymatic endotoxin neutralization as compared to control indicates that the therapeutic agent is not effective in treating sepsis. If a decrease in enzymatic endotoxin neutralization is observed after treatment with a therapeutic agent, one of skill in the art can, for example, discontinue treatment, alter the dosage of the therapeutic agent or administer a different therapeutic agent.

Further provided is a method of determining the effectiveness of a therapeutic agent for treating sepsis comprising a) administering a therapeutic agent to the subject; b) heating a first biological sample from the subject after treatment with the therapeutic agent; c) adding a selected amount of exogenous endotoxin to the first sample of step b); d) detecting exogenous endotoxin in the first sample from step c); e) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step d) from the total exogenous endotoxin added in step c); f) acidifying a second biological sample from a subject after treatment with the therapeutic agent; g) adding the selected amount of exogenous endotoxin to the second sample of step f); h) detecting exogenous endotoxin in the second sample from step g); i) determining the amount of undetected exogenous endotoxin in the second sample by subtracting the detected exogenous endotoxin from step h) from the total exogenous endotoxin added in step g); and j) calculating a percentage of protein endotoxin neutralization, utilizing the following equation:

((amount of undetected exogenous endotoxin the second sample - amount of undetected exogenous endotoxin in the first sample) / selected amount of exogenous endotoxin) X 100 wherein a decrease in the percentage of protein endotoxin neutralization after treatment as compared to a control indicates that the therapeutic agent is effective for treating sepsis. In this method, the first biological sample can be heated to from about 55°C to about70°C.

Also, in this method, the second biological sample can be acidified to a pH of less than 4, less than 3, less than 2 or about 1. Further, in this method, the same amount of exogenous endotoxin is added to the first and the second biological sample.

As utilized throughout, the percentage of protein endotoxin neutralization is the perecentage of exogenous endotoxin not detected due to acid-inactivation via binding of blood plasma proteins, including immunoglobulins. Acid-inactivation is utilized to denature proteins the biological sample.

Further provided is a method of treating sepsis in a subject comprising calculating levels of protein endotoxin neutralization, undetected exogenous endotoxin and enzymatic endotoxin neutralization in a biological sample from the subject; calculating an endotoxin neutralization ratio using the following formula:

(protein endotoxin neutralization + undetectable exogenous endotoxin) / enzymatic endotoxin neutralization)

a ratio of about 5 or greater indicating that the subject has sepsis; and

administering a therapeutic agent for treating sepsis to the subject. This ratio is known as a neutralization ratio and is roughly equivalent to the ratio of protein inactivation to enzymatic inactivation. This ratio can also be utilized to determine the severity of the disease as lower ratios will correspond to moderate sepsis and higher ratios will correspond to more severe sepsis. For example, and not to be limiting, lower ratios correspond to moderate cases of sepsis that can be treated with one or more antibiotics. Higher ratios correspond to more severe cases of sepsis that may not respond to antibiotics alone. Therefore, one of skill in the art would know to administer a therapeutic agent for more severe cases of sepsis, such as an antibiotic in combination with a vasopressor and/or a corticosteriod. The neutralization ratio can also be used to determine the effectiveness of a therapeutic agent in treating sepsis. If there is a decrease in the neutralization ratio in a sample from the subject after administration of a therapeutic agent, as compared to control, this indicates that the therapeutic agent is effective for treating sepsis. The control can be a sample from the subject prior to

administration of the therapeutic agent or a reference sample.

In this method, protein endotoxin neutralization is calculated by a) heating a first biological sample from the subject; b) adding a selected amount of exogenous endotoxin to the first sample of step a); c) detecting exogenous endotoxin in the first sample from step b); d) determining the percentage of undetected exogenous endotoxin in the first sample using the following equation:

((total exogenous endotoxin added in step b - the detected exogenous endotoxin from step c) / the total exogenous endotoxin added in step b) X 100; e) acidifying a second biological sample from a subject; f) adding the selected amount of exogenous endotoxin to the second sample of step e); g) detecting exogenous endotoxin in the second sample from step f); h) determining the percentage of undetected exogenous endotoxin after acidification using the following equation:

((total exogenous endotoxin added in step f- the detected exogenous endotoxin from step g) / the total exogenous endotoxin added in step f) X 100; and i) calculating protein endotoxin neutralization, utilizing the following equation: percentage of undetected exogenous endotoxin in step h - percentage of undetected exogenous endotoxin in step d. In this method, undetectable endotoxin is the percentage of exogenous endotoxin that is not detected even after heat and/or acid inactivation. Undetectable endotoxin is calculated by a) acidifying a biological sample from a subject; b) adding a selected amount of exogenous endotoxin to the sample of step a); c) detecting exogenous endotoxin in the sample from step b); and d) determining the percentage of undetectable exogenous endotoxin after using the following equation:

((total exogenous endotoxin added in step b - the detected exogenous endotoxin from step c) / the total exogenous endotoxin added in step b) X 100

In this method, enzymatic endotoxin neutralization is calculated by a) adding exogenous endotoxin to a first biological sample from the subject; b) detecting exogenous endotoxin in the first sample from step a); c) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step b) from the total exogenous endotoxin added in step a); d) calculating the percentage of undetected exogenous endotoxin in the first sample using the following equation:

(the amount of undetected exogenous endotoxin in step c / total exogenous endotoxin added in step a) X 100; e) heating a second biological sample from the subject; f) adding exogenous endotoxin to the second sample of step e); g) detecting exogenous endotoxin in the second sample from step f); h) determining the amount of undetected exogenous endotoxin in the second sample by subtracting the detected exogenous endotoxin from step g) from the amount of added exogenous endotoxin from step f); i) calculating the percentage of undetected exogenous endotoxin in the second sample using the following equation:

(the amount of undetected exogenous endotoxin in step h / total exogenous endotoxin added in step f) X 100; and

j) calculating the percentage of enzymatic endotoxin neutralization utilizing the following equation:

((percentage of undetected exogenous endotoxin in the second sample - percentage of undetected exogenous endotoxin in the first sample) / amount of total exogenous endotoxin added in step a) X 100. If a subject has a neutralization ratio of about 5 or greater, a therapeutic agent can be administered to treat sepsis. The ratio can be about 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45 or greater. As set forth above, the therapeutic agent can be, but is not limited to, a chemical, a small or large molecule (organic or inorganic), a drug, a protein, a peptide, a cDNA, an antibody, an aptamer, a morpholino, a triple helix molecule, an siR A, a shR A, an miRNA, an antisense RNA, a ribozyme or any other compound now known or identified in the future that decreases sepsis.

By treat, treating, or treatment is meant a method of reducing or slowing sepsis.

Treatment can also refer to a method of reducing the disease or condition associated with sepsis or reducing or slowing one or more of the symptoms. The treatment or slowing can be any reduction or slowing from native levels and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. Treatment can range from a positive change in a symptom or symptoms to complete amelioration as detected by art-known techniques. For example, a disclosed method is considered to be a treatment if there is about a 10% reduction in sepsis in a subject when compared to native levels in the same subject or control subjects or a 10% increase in weight gain. Thus, the reduction or improvement can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction or improvement in between as compared to native or control levels.

The choice of therapeutic agent will depend on the symptoms and medical history of the subject. One of skill in the art can administer one or more therapeutic agents suitable for treating sepsis, depending on the severity or the stage of a disease, as determined by the neutralization ratio. For example, if the subject shows signs or symptoms of sepsis, one of skill in the art can administer antibiotics (for example, and not to be limiting, ceftriaxone, cefuroxime, vancomycin, ceftazidime, tobramycin, cefotaxime, penicillin, clindamycin, ciprofloxacin, cefepime, azithromycin, ampicillin or combinations thereof), vasopressors (for example, isoproterenol, dopexamine, dobutamine, dopamine, epinephrine, norephinephrine or phenylephrine, metariminol, ephedrine or vasopressin), corticosteroids (for example, fludrocortisones, Cortisol or hydrocortisone), immunomodulators (for example, azathioprine, mercaptopurine or cyclosporine), insulin or combinations thereof to treat sepsis. In another example, if the subject exhibits signs or symptoms of viral, fungal bacterial or parasitic infection, an appropriate antiviral, antibacterial, antifungal or antiparasitic agent can be administered. Antibacterial agents include, but are not limited to, antibiotics (for example, penicillin and ampicillin), sulfa drugs and folic acid analogs, Beta-lactams, aminoglycosides, tetracyclines, macrolides, lincosamides, streptogramins, fluoroquinolones, rifampin, mupirocin, cycloserine, aminocyclitol and oxazolidinones.

Antiparasitic agents include, but are not limited to, antihelmintics, antinematodal agents, antiplatyhelmintic agents, antiprotozoal agents, amebicides, antimalarials,

antitrichomonal agents, aoccidiostats and trypanocidal agents.

Antifungal agents include, but are not limited to, polyenes, imidazoles, triazoles, thiazoles and allylamines, echinocandins,

Any of the therapeutic agents set forth herein can be combined immunotherapy, antiinflammatory agents, dialysis, or surgery.

The agents described herein can be provided in a pharmaceutical composition.

Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the agent described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

Compositions containing the agent(s) described herein suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol,

polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens,

chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin,

polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,

dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

Administration can be carried out using therapeutically effective amounts of the agents described herein for periods of time effective to treat sepsis. The effective amount may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150mg/kg of body weight of active compound per day, about 0.5 to lOOmg/kg of body weight of active compound per day, about 0.5 to about 75mg/kg of body weight of active compound per day, about 0.5 to about 50mg/kg of body weight of active compound per day, about 0.5 to about 25mg/kg of body weight of active compound per day, about 1 to about

20mg/kg of body weight of active compound per day, about 1 to about lOmg/kg of body weight of active compound per day, about 20mg/kg of body weight of active compound per day, about lOmg/kg of body weight of active compound per day, or about 5mg/kg of body weight of active compound per day.

According to the methods taught herein, the subject is administered an effective amount of the agent. The terms effective amount and effective dosage are used

interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration. Administration can be systemic or local. Pharmaceutical compositions can be delivered locally to the area in need of treatment, for example by topical application or local injection. Multiple administrations and/or dosages can also be used. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In an example in which a nucleic acid is employed, such as an antisense or an siR A molecule, the nucleic acid can be delivered intracellularly (for example by expression from a nucleic acid vector or by receptor-mediated mechanisms), or by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, for example by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (such as a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (for example Jo Hot et al. , Proc. Natl. Acad. Sci. USA 1991, 88: 1864-8). siR A carriers also include, polyethylene glycol (PEG), PEG- liposomes, branched carriers composed of histidine and lysine (HK polymers), chitosan- thiamine pyrophosphate carriers, surfactants (for example, Survanta and Infasurf), nanochitosan carriers, and D5W solution. The present disclosure includes all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral delivery, whether integrated into the genome or not.

Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al, Blood 87:472-478, 1996) to name a few examples. These methods can be used in conjunction with any of these or other commonly used gene transfer methods.

All of the calculations set forth herein can be performed by a computer. For example, a computer-readable medium, on which are stored executable instructions that, when executed by a computer processor, perform any of the methods or calculations set forth herein is provided. In another example, provided herein is a computer system comprising software for effecting the following steps: a) receiving a set of detected exogenous endotoxin values for at least one sample from a subject after treatment with a therapeutic agent for treating sepsis; b)determining the amount of undetected exogenous endotoxin in the sample; and c) calculating the percentage of total endotoxin neutralization in the sample; wherein a decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

Also provided is a computer system comprising software for effecting the following steps: a) receiving a set of detected exogenous endotoxin values for at least one sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is heated; b) determining the amount of undetected exogenous endotoxin in the heated sample; and c) calculating the percentage of enzymatic endotoxin neutralization in the sample;

wherein an increase in the percentage of enzymatic endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

Further provided is a computer system comprising software for effecting the following steps: a) receiving a set of detected exogenous endotoxin values for at least one first sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is heated; b) receiving a set of detected exogenous endotoxin values for at least one second sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is acidified; c) determining the amount of undetected exogenous endotoxin in the heated sample; d) determining the amount of undetected exogenous endotoxin in the acidified sample; and e) calculating the percentage of protein endotoxin neutralization in the sample; wherein a decrease in the percentage of enzymatic endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

Also provided is a computer system comprising software for effecting the following steps: a) receiving a set of protein endotoxin neutralization values, undetected exogenous endotoxin values and enzymatic endotoxin neutralization values from at least one biological sample from a subject; and b) calculating an endotoxin neutralization ratio, wherein a ratio of about 5 or greater indicates that the subject has sepsis. The computer system can further comprise a processor, configured to execute instructions and memory on which are stored executable instructions, wherein the instructions are configured to perform any of the methods or calculations set forth herein.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

As used throughout, ranges can be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as about that particular value in addition to the value itself. For example, if the value 10 is disclosed, then "about 10" is also disclosed. It is also understood that throughout the application data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

In this study the extent and nature of endotoxin neutralization was measured in blood plasma samples to develop a system for using endotoxin as a biomarker for both gram- negative and gram-positive sepsis. The data shows that both male and female septic patients have elevated levels of endotoxin neutralization. This is achieved by a decreased capacity of enzymatic endotoxin neutralization and an increased capacity of endotoxin neutralization via protein binding.

Protocols

ESP - Endotoxin Sample Preparation Protocol

1. Pipette citrated plasma into glass tube and cover with parafilm.

2. Heat tube in 55-60°C water bath for 25 minutes.

3. Mix 30 μΐ of heat-inactivated citrated plasma with 270 μΐ ESP Buffer #1. a. ESP Buffer #1 - 10 mM Tris-HCl pH 1.5

b. This step lowers the sample pH which prepares the sample for ESP enzymatic digestion. In addition, the pH shift further inactivates blood plasma enzymes.

Add 30 μΐ of ESP Protease Solution.

a. ESP Protease Solution - 5% Pepsin in 10 mM Tris-HCl pH 1.5

Incubate tube in a 37°C shaking water bath for 120 minutes.

Mix 50 μΐ sample with 450 μΐ ESP Buffer #2.

a. ESP Buffer #2 - 10 mM Tris-HCl pH 8.5

b. This step neutralizes the sample for recombinant Factor C testing.

Test each sample with and without PPC in the Lonza PyroGene® assay (AUentown, NJ).

Endotoxin Neutralization Protocol

Each sample is tested after 4 treatments:

Endogenous

1. Citrated plasma is treated with the ESP™ protocol and tested with the Lonza PyroGene® assay.

Spiked

1. 90 μΐ citrated plasma is mixed with 10 μΐ endotoxin- free water.

2. 72 μΐ of this is mixed with 8 μΐ endotoxin stock solution.

3. Treat with the ESP™ protocol and test with the Lonza PyroGene® assay.

Heated

1. 90 μΐ citrated plasma is mixed with 10 μΐ endotoxin- free water.

2. Sample is heated in a 55-60°C water bath for 25 minutes.

3. 72 μΐ of this is mixed with 8 μΐ endotoxin stock solution.

4. Treat with the ESP™ protocol and test with the Lonza PyroGene® assay.

Acidified

1. 90 μΐ citrated plasma is mixed with 10 μΐ 2 M hydrochloric acid.

2. 72 μΐ of this is mixed with 8 μΐ endotoxin stock solution.

3. Treat with the ESP™ protocol and test with the Lonza PyroGene® assay.

RESULTS

A total of 84 patient samples were tested:

• 30 - control patients (10 males, 20 females)

• 8 - patients with Gram-Negative Sepsis (4 males, 4 females)

• 16 - patients with Gram-Negative Sepsis (7 males, 9 females)

Each patient sample was tested with each of the 4 neutralization protocols with and without PPC controls according to the ESP™ protocol. Endotoxin was measured using the Lonza PyroGene® assay according to manufacturer's specifications. Total Endotoxin Neutralization

There were significant differences in "Total Endotoxin Neutralization" between control and septic patients in males. In females, the data for disease samples is similar to the males but the level of "Total Endotoxin Neutralization" in the control samples is much higher, making discrimination between control and disease samples difficult. For all samples, the "Total Endotoxin Neutralization" was 82.7% in control patients compared with 92.3%) (p = 0.00392) for gram-negative sepsis and 96.1% (p = 0.0000002) for gram-positive sepsis. In males, "Total Endotoxin Neutralization" was 74.6% in control samples compared to 94.8%) in gram-negative sepsis (p = 0.00014) and 97.6% in gram-positive sepsis (p = 0.0000005) (Fig. 1). Control females had a "Total Endotoxin Neutralization" of 86.7% compared to 89.9%> for gram-negative sepsis (p = 0.33269) and 95.0% for gram-positive sepsis (p = 0.00041) (Fig. 2). These results show that "Total Endotoxin Neutralization" can be a marker for infection in male patient populations.

Enzymatic Endotoxin Neutralization

There was no statistically significant difference in "Enzymatic Endotoxin

Neutralization" between the males and females of any of the groups. For all patients, control samples had an average "Enzymatic Endotoxin Neutralization" of 19.9% that was reduced to 6.8%) in gram-positive sepsis (p = 0.000005). Oddly, at 12.6% gram-negative septic patients had a significantly (p = 0.02882) higher value than gram-positive patients though still over 6% lower than control (p = 0.0587). In males, "Enzymatic Endotoxin Neutralization" was 22.7%) in control samples compared to 13.2% in gram-negative sepsis (p=0.10448) and 6.8%> in gram-positive sepsis (p=0.00281) (Fig. 3). Control females had an "Enzymatic Endotoxin Neutralization" of 18.5% compared to 12.1% for gram-negative sepsis (p=0.12584) and 6.7% for gram-positive sepsis (p=0.00549)(Fig. 4). These results show that even though "Total Endotoxin Neutralization" is elevated in diseased patients, the enzymatic component is decreased. This suggests that "Protein Endotoxin Neutralization" should be considerably higher in disease samples.

Protein Endotoxin Neutralization

As predicted above, "Total Endotoxin Neutralization" is significantly elevated in disease states in both males and females. However, the extent is much more dramatic in males. In males, the average control was 51.2% compared to 71.0% (p = 0.1 1579) for gram- negative sepsis and 84.9% (p = 0.00279) for gram-positive sepsis (Fig. 5). In females, the average control was 45.1%> compared to 42.2% (p = 0.67647) for gram negative sepsis and 68.8% (p = 0.00009) for gram-positive sepsis (Fig. 6). It is of note that gram-positive infections cause a much greater protein neutralization reaction than gram-negative infections. Undetectable Endotoxin

Gram-negative septic males show an increase in "Undetectable Endotoxin" to 14.4% and gram-positives show an increase to 12.3% (Fig. 7). In female sepsis patients, there is little significant change in "Undetectable Endotoxin" in any of the sample populations.

Gram-positives samples have a value of 21.6%, almost identical to the 23.2% in controls (Fig. 8). Gram-negative samples show an average elevation to 35.6%> (p = 0.03035), however, most of these samples are in a range overlapping the control population.

Summarily, these results show that both gram-negative and gram-positive septic patients have "Total Endotoxin Neutralization" higher than control patients. The internals of this value have the same pattern in both males and females, but is much more pronounced in males. For gram-negative sepsis, males have a 20.2% increase in "Total Endotoxin

Neutralization" compared to control. This is a result of a 9.5% decrease in enzymatic activity and a 29.1% increase in protein activity ("Protein Endotoxin Neutralization" plus

"Undetectable Endotoxin"). In females the total is only 3.2% which is the result of a 6.4% decrease in enzyme activity and a 9.5% increase in protein activity. For gram-positive sepsis, the male total is 23.0% higher than control due to a 15.9% decrease in enzyme activity and a 40.9% increase in protein. In females the total is 8.3% higher due to an 1 1.8% decrease in enzyme and 22.1 % increase in protein.

The major difference between the sexes in endotoxin neutralization is the amount of protein activity. Due to an increased susceptibility of infection, females have a broader and higher-affinity repertoire of immunoglobulins to bind and neutralize endotoxin. Because of this, dramatic changes in endotoxin neutralization seen in males are not seen in females. In control males, the level, and possibly affinity of immunoglobulins, is much lower and therefore the response to infection is more pronounced.

Given the above results, "Total Endotoxin Neutralization" can be used as an accurate biomarker for bacterial infection even though distinct differences exist in the individual components of endotoxin neutralization. Therefore, a formula was developed to measure the ratio of enzymatic neutralization to protein neutralization. For this formula, "Undetectable Endotoxin" is caused by an unidentified protein and thus is included in protein neutralization. The result of this formula is the "Endotoxin Neutralization Ratio" and it is defined by the following formula: "Protein Endotoxin Neutralization " + "Undetectable Endotoxin "

"Enzymatic Endotoxin Neutralization"

Endotoxin Neutralization Ratio

The control male "Endotoxin Neutralization Ratio" samples clustered in an area between 0.85 to 5.11, with one exception at 12.76. The average was 3.6. There was an extensive difference in the ratio between gram-negative and gram-positive samples. The gram-negative samples clustered from 4.3 to 8.3 with an average of 6.9. The gram-positives ranged from 4.6 to 249.0 with an average of 59.0 (Fig. 9). Though the diseased samples are diverse in their values, comparison with the control groups can predict infection. A horizontal line at a value of 5.3 demarcates control from infected patients with great accuracy. For controls 9/10 samples fall below this line. For the diseased samples, 3/4 gram- negative samples and 6/7 gram-positive samples are above this line. This indicates that the line correctly predicts infection in 18 of the 21 samples, an accuracy of over 85%.

The female samples are similar. The control female "Endotoxin Neutralization Ratio" samples clustered in an area between 1.4 to 6.9 with three exceptions over 10. The average was 5.1 (Fig. 10). As with the males there was a correlation between ratio value and treatment and/or disease severity. The gram-negative samples ranged from 3.7 to 11.5 with an average of 7.1. The gram-positives ranged from 1.3 to 250.0 with an average of 117.0. When the same line is applied at a value of 5.3 in the females, it also shows great predictive power. For controls 16/20 samples are below the line. For the diseased samples, 3/4 gram- negative samples and 8/9 gram-positive samples are above this line. In females the line correctly predicts 27 of the 33 samples, an accuracy of about 82%>.

Described herein is the utility of using endotoxin neutralization as a biomarker in sepsis. Differences in "Total Endotoxin Neutralization", "Enzymatic Endotoxin

Neutralization", "Protein Endotoxin Neutralization" and "Undetectable Endotoxin" between sexes and/or between control and disease groups were observed. "Total Endotoxin

Neutralization" is a powerful of indication of infection in males. As shown in Table 1, if an arbitrary horizontal line is applied at 83% on the male graph, infection is predicted 100% of the time. Table 1 -Total Endotoxin Neutralization (line of demarcation at 83%)

The more valuable tool for detecting infection among both sexes was "Endotoxin Neutralization Ratio". As discussed above, a horizontal line at a value of 5.3 is used to demarcate the values shown in Table 2.

Table 2-Endotoxin Neutralization Ratio (line of demarcation at 5.3)

Claims

What is claimed is:

1. A method of determining the effectiveness of a therapeutic agent for treating sepsis: a) administering a therapeutic agent to the subject;

b) adding exogenous endotoxin to a first biological sample from the subject after treatment with the therapeutic agent;

c) detecting exogenous endotoxin in the sample from step b);

d) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step c) from the total exogenous endotoxin added in step b); and

e) calculating the percentage of total endotoxin neutralization in the first sample, wherein the percentage of total endotoxin neutralization is the percentage of undetected exogenous endotoxin of the total exogenous endotoxin, and wherein a decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

2. The method of claim 1 further comprising:

f) heating a second biological sample from the subject after treatment with the therapeutic agent;

g) adding exogenous endotoxin to the second sample of step f);

h) detecting exogenous endotoxin in the second sample from step g);

i) determining the amount of undetected exogenous endotoxin by subtracting the detected exogenous endotoxin from step h) from the amount of added exogenous endotoxin from step g); and

j) calculating the percentage of enzymatic endotoxin neutralization utilizing the following equation:

((amount of undetected exogenous endotoxin in the second sample from step i - amount of undetected exogenous endotoxin in the first sample) / amount of total exogenous endotoxin added in step g) X 100; wherein an increase in the percentage of enzymatic endotoxin neutralization in the second biological sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

3. A method of determining the effectiveness of a therapeutic agent for treating sepsis comprising:

a) administering a therapeutic agent to the subject; b) heating a first biological sample from the subject after treatment with the therapeutic agent;

c) adding a selected amount of exogenous endotoxin to the first sample of step b); d) detecting exogenous endotoxin in the first sample from step c);

e) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step d) from the total exogenous endotoxin added in step c);

f) acidifying a second biological sample from a subject after treatment with the therapeutic agent;

g) adding the selected amount of exogenous endotoxin to the second sample of step f);

h) detecting exogenous endotoxin in the second sample from step g);

i) determining the amount of undetected exogenous endotoxin in the second

sample by subtracting the detected exogenous endotoxin from step h) from the total exogenous endotoxin added in step g); and

j) calculating a percentage of protein endotoxin neutralization, utilizing the

following equation:

((amount of undetected exogenous endotoxin the second sample - amount of undetected exogenous endotoxin in the first sample) / selected amount of exogenous endotoxin) X 100

wherein a decrease in the percentage of protein endotoxin neutralization after treatment as compared to a control indicates that the therapeutic agent is effective for treating sepsis.

4. A method of treating sepsis in a subject comprising:

a) calculating levels of protein endotoxin neutralization, undetected exogenous endotoxin and enzymatic endotoxin neutralization in a biological sample from the subject; and

b) calculating an endotoxin neutralization ratio using the following formula:

(protein endotoxin neutralization + undetectable exogenous endotoxin) / enzymatic endotoxin neutralization

a ratio of about 5 or greater indicating that the subject has sepsis; and c) administering a therapeutic agent for treating sepsis to the subject.

5. The method of claim 4, wherein protein endotoxin neutralization is calculated by: a) heating a first biological sample from the subject; b) adding a selected amount of exogenous endotoxin to the first sample of step a); c) detecting exogenous endotoxin in the first sample from step b);

d) determining the percentage of undetected exogenous endotoxin in the first

sample using the following equation:

((total exogenous endotoxin added in step b - the detected exogenous endotoxin from step c) / the total exogenous endotoxin added in step b) X 100;

e) acidifying a second biological sample from a subject;

f) adding the selected amount of exogenous endotoxin to the second sample of step e);

g) detecting exogenous endotoxin in the second sample from step f);

h) determining the percentage of undetected exogenous endotoxin after

acidification using the following equation:

((total exogenous endotoxin added in step f- the detected exogenous endotoxin from step g) / the total exogenous endotoxin added in step f) X 100;

and

i) calculating protein endotoxin neutralization, utilizing the following equation: percentage of undetected exogenous endotoxin in step h - percentage of undetected exogenous endotoxin in step d.

6. The method of claim 4, wherein undetectable endotoxin is calculated by:

a) acidifying a biological sample from a subject;

b) adding a selected amount of exogenous endotoxin to sample of step a);

c) detecting exogenous endotoxin in the sample from step b); and

d) determining the percentage of undetectable exogenous endotoxin after using the following equation:

((total exogenous endotoxin added in step b - the detected exogenous endotoxin from step c) / the total exogenous endotoxin added in step b) X 100;

7. The method of claim 4, wherein enzymatic endotoxin neutralization is calculated by: a) adding exogenous endotoxin to a first biological sample from the subject;

b) detecting exogenous endotoxin in the first sample from step a);

c) determining the amount of undetected exogenous endotoxin in the first sample by subtracting the detected exogenous endotoxin from step b) from the total exogenous endotoxin added in step a); d) calculating the percentage of undetected exogenous endotoxin in the first sample using the following equation:

(the amount of undetected exogenous endotoxin in step c / total exogenous endotoxin added in step a) X 100;

e) heating a second biological sample from the subject;

f) adding exogenous endotoxin to the second sample of step e);

g) detecting exogenous endotoxin in the second sample from step f);

h) determining the amount of undetected exogenous endotoxin in the second

sample by subtracting the detected exogenous endotoxin from step g) from the amount of added exogenous endotoxin from step f);

i) calculating the percentage of undetected exogenous endotoxin in the second sample using the following equation:

(the amount of undetected exogenous endotoxin in step h / total exogenous endotoxin added in step f) X 100;

j) calculating the percentage of enzymatic endotoxin neutralization utilizing the following equation:

((percentage of undetected exogenous endotoxin in the second sample - percentage of undetected exogenous endotoxin in the first sample) / amount of total exogenous endotoxin added in step a) X 100.

8. The method of claim 3 or claim 5 wherein the acidification step comprises

acidification to a pH of amount 1 to 4.

9. The method of claim 8 further comprising:

a) contacting the acidified sample with an acidic protease; and

b) increasing the pH of the protease-treated sample to a pH of about 6 to 8.

10. The method of any of claims 1-9, wherein the sample is selected from the group consisting of plasma, blood, serum, ascites, pleural fluid, ocular fluid and spinal fluid.

11. The method of claim 10, wherein the plasma is citrated plasma or EDTA collected plasma.

12. The method of claim 10, wherein the acidic protease is a pepsin.

13. The method of claim 2-12, wherein the biological sample is heated to a temperature of about 55°C to about 70°C.

14. The method of claim 8, wherein the biological sample is diluted to about 1 part sample to about 10 parts diluent prior to acidification.

15. The method of claim 14, wherein the diluent is a Tris solution comprising MgCl₂.

16. The method of claim 9, wherein the biological sample is diluted to about 1 part sample to about 10 parts diluent prior to acidification of the protease-treated sample.

17. The method of claim 16, wherein the diluent is a Tris solution.

18. The method of claim 17, wherein the Tris solution is at a pH of about 6 to about 8.5.

19. The method of any of claims 8-18, further comprising inactivating the acidic

protease.

20. The method of claim 1 or 2, further comprising mixing about 9 parts biological sample with about 1 part endotoxin free water prior to step a).

21. The method of claim 2, further comprising mixing about 9 parts biological sample with about 1 part endotoxin free water prior to step a).

22. The method of claim 3, wherein the biological sample is acidified by adding 1 part acidic solution to about 9 parts biological sample.

23. The method of any of claims 1-3, wherein 1 part exogenous endotoxin is added to about 9 parts biological sample.

24. The method of any of claims 1-23, wherein the sepsis is gram-negative sepsis or gram-positive sepsis.

25. A computer system comprising software for effecting the following steps:

a) receiving a set of detected exogenous endotoxin values for at least one sample from a subject after treatment with a therapeutic agent for treating sepsis;

b) determining the amount of undetected exogenous endotoxin in the sample; and c) calculating the percentage of total endotoxin neutralization in the sample;

wherein a decrease in total endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

26. A computer system comprising software for effecting the following steps:

a) receiving a set of detected exogenous endotoxin values for at least one sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is heated;

b) determining the amount of undetected exogenous endotoxin in the heated

sample;

c) calculating the percentage of enzymatic endotoxin neutralization in the sample; and wherein an increase in the percentage of enzymatic endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

27. A computer system comprising software for effecting the following steps:

a) receiving a set of detected exogenous endotoxin values for at least one first sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is heated;

b) receiving a set of detected exogenous endotoxin values for at least one

second sample from a subject after treatment with a therapeutic agent for treating sepsis, wherein the sample is acidified;

c) determining the amount of undetected exogenous endotoxin in the heated sample;

d) determining the amount of undetected exogenous endotoxin in the acidified sample; and

e) calculating the percentage of protein endotoxin neutralization in the sample; wherein a decrease in the percentage of enzymatic endotoxin neutralization in the first sample as compared to control indicates that the therapeutic agent is effective for treating sepsis.

28. A computer system comprising software for effecting the following steps:

a) receiving a set of protein endotoxin neutralization values, undetected

exogenous endotoxin values and enzymatic endotoxin neutralization values from at least one biological sample from a subject; and

b) calculating an endotoxin neutralization ratio, wherein a ratio of about 5 or greater indicates that the subject has sepsis.