Inventors: Daren L. Knoell, Mingjie Liu, Beth Besecker

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Ser. No.

61/265,078 filed November 30, 2009, the entire disclosure of which is expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under the National Institute of Health, National Heart, Lung and Blood Institute, Grant Nos. GRT00006348, R01 HL086981-01 and F32 HL08618602. The government has certain rights in this invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy, created on November 29, 2010, is named 604_51413_SEQLIST_OSU-10076.txt, and is 749 bytes in size.

[0004] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0005] [SEQ ID NO: 1]: 5'-GGAAGAGCCC-3' ;

[0006] [SEQ ID NO: 2]: 5 ' -AGAAGAGTTC-3 ' .

TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION

[0007] The present invention pertains to clinical and laboratory discoveries involving the

relationship of inflammatory processes and zinc processing. The relationships discovered translate into practical tools for research, diagnostics and therapeutics. Moreover, kits disclosed herein are useful for a variety of purposes. The present invention therefore applies to the fields of bioscience and medicine.

[0008] The present invention is based in part on the discovery that expression of one of the zinc transporters, known as Zip8 (SLC39A8), is significantly increased at the onset of inflammation whereas many others are not. Further, the inventors have discovered, via humans and animal studies, that Zip8 is dynamically regulated in the setting of severe infection. In particular, Zip8 levels increase with increasing infection severity and that this may further be influenced by zinc status. In view of these findings, the inventors provide practical applications associated with transcriptional regulation of the human Zip8 gene. Specifically, applications related to Zip8 gene expression as related to triggering the initial immune response in the setting of infection and inflammatory stress are herein described. Also, Zip8 as a biomarker for zinc nutritional status and host response to infection prediction is provided.

BACKGROUND

[0009] Zinc is vital to many cellular functions including protein synthesis, signal transduction, and gene transcription. Further, zinc is necessary to maintain proper immune function. Zinc deficiency is associated with immune paralysis and increased morbidity and mortality following infection. Mild to moderate zinc deficiency is not readily detectable clinically and its incidence is underappreciated. Zinc deficiency is more common in the elderly and those with chronic medical conditions, the same populations that have increased risk for sepsis mortality. Further, plasma zinc levels rapidly decline at the onset of the acute phase response, a phenomenon largely attributed to redistribution of zinc into the cellular compartment. The inventors previously reported that moderately zinc-deficient mice have an increased mortality during the early stages of polymicrobial sepsis when compared to zinc sufficient septic controls].

[0010] Further, a human pilot study showed that endotoxin administration reproducibly induced a precipitous drop in plasma zinc levels. The precipitous decline in zinc levels occurred independent of changes in urinary zinc loss or albumin binding. It has been theorized that zinc is mobilized from the vasculature into tissues, such as the liver, to support vital zinc-mediated metabolic functions.

[0011] Previous studies involving animal models have reported that zinc deficiency is associated with lower systolic arterial blood pressure, which association may be secondary to reduced activity of both serum angiotensin converting enzyme and carbonic anhydrase. In addition, zinc has been found to activate endothelin-con verting enzyme, an enzyme responsible for endothelin-1 synthesis, a central vasoconstricting factor produced by the endothelium.

[0012] A study recently conducted in a critically ill pediatric cohort proposed a relationship between zinc and pediatric infection. Researchers also reported that the extent of decline in plasma zinc concentration is associated with organ failure and mortality.

[0013] Elderly individuals may have reduced zinc absorption and lower zinc concentrations than that of a younger person. BMI may also impact plasma zinc concentration; extremely low or high BMI is associated with poor nutritional status including trace metals. Further, medications may directly or indirectly contribute to zinc status. Certain insulin preparations contain zinc, and propofol may affect zinc status. [0014] Zinc transporters comprise a family of multiple trans-membrane spanning domain proteins that are encoded by two solute-linked carrier (SLC) gene families: SLC30 (a.k.a. ZnT) and SLC39 (a.k.a. Zip). In general, ZnT and Zip family members have opposite roles in regulating cellular zinc homeostasis. ZnT transporters reduce cytosolic zinc bioavailability by promoting zinc efflux in conditions of excess, while Zip transporters function by increasing cytosolic zinc during deficient states. Members of both families exhibit tissue specific expression and possess differential responsiveness to dietary zinc as well as to physiologic stimuli including cytokines and hormones.

[0015] The present inventors recently reported that SLC39A8/Zip8 is unique relative to other

SLC39 family members in that it is induced by inflammatory stress and expressed on the cell membrane where it then facilitates zinc entry into the cytosol. Zip8-mediated zinc transport was critical for cell survival in response to TNFa. In previous animal studies, the inventors have also observed that the expression of Zipl4, the mouse orthologue to human Zip8, was significantly elevated in zinc deficient septic mice relative to normal dietary counterparts and was associated with increased mortality. In particular, the present inventors recently reported that nutritional zinc depletion resulted in an increase in plasma cytokines and chemokines associated with the innate immune response and occurred in conjunction with a significant increase in vital organ injury and mortality in a murine model.

[0016] Others reported, in a murine model, that expression of the zinc importer SLC39A14 is transcriptionally activated by IL-6 in response to LPS, thereby accounting for hypozincemia due to mobilization of zinc from the vasculature to cell compartment at the onset of systemic inflammation subsequent to infection.

[0017] Previous animal studies demonstrated that zinc deficiency results in an exaggerated

inflammatory response following endotoxin administration or cecal ligation and puncture (a method to induce polymicrobial sepsis). Previous reports describe a reduction in plasma zinc levels during the onset of the acute phase response.

[0018] Thrombocytopenia occurs in approximately 20% of medical admissions and is not

uncommon in sepsis. Further, there is an established inverse relationship between severity of sepsis and platelet count. In addition, other platelet-endothelial interactions occur in sepsis. For example, the endothelium mediates vasomotor tone and influences mean arterial blood pressure, regulates cellular and nutrient trafficking and contributes to the local balance in pro-inflammatory and antiinflammatory mediators such as cytokines and chemokines.

[0019] The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0020] The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0021] Figure 1. Plasma Zinc Concentrations. Plasma zinc concentration is significantly lower in critically ill intensive care unit patients compared to healthy non-hospitalized controls (Mean Plasma Zinc Concentration 57.2 ± 18.2 ug/dL vs. 89.6 ± 27.4 ug/dL respectively), which is statistically significant. Of clinical relevance, plasma zinc further declines in septic individuals (Mean Plasma Zinc Concentration 45.4 ± 18.1 ug/dL). Plasma zinc assessed by atomic absorption

spectrophotometry for all septic (n=22), critically ill control (n=22) and healthy (n=12) subjects.

[0022] Figure 2. Graphs and data describe the cloning and analysis of the Zip8 promoter. Figure 2 discloses SEQ ID NOs: 1-2, respectively, in order of appearance.

SUMMARY OF THE INVENTION

[0023] In a first broad aspect, there is provided herein a method for the diagnosis and prognosis of sepsis in a mammalian subject comprising the following steps: a) obtaining a biological sample from an individual suspected of having sepsis; b) determining the plasma zinc concentration in the sample using a direct or an indirect detection technique; and c) correlating the plasma zinc concentration in the sample to known standards.

[0024] Also provided is a method where the plasma zinc concentration is detected using atomic absorption spectrophotometry.

[0025] Also provided is a method, further comprising correlating the plasma zinc concentration in the sample to known standards to predict the severity of the septic shock.

[0026] Also provided is a method further comprising correlating the plasma zinc concentration in the sample to known standards to predict whether the subject will require standard care or high risk treatment.

[0027] Also provided is a method further comprising the plasma zinc concentration in the sample to known standards to select the appropriate therapeutic treatment for the septic shock, wherein decreased plasma zinc concentration indicates that the patient is a candidate for therapies selected from antibiotics, organ support, or combinations thereof.

[0028] Also provided is a method where individuals having levels of plasma zinc concentration more than about 60 μg/dl, are considered appropriate candidates for standard care.

[0029] In another broad aspect, there is provided herein a method for the stratification of a sepsis condition in a mammalian subject for determining the effective course of treatment comprising the steps: a) obtaining a biological sample from a subject suspected of having sepsis; b) determining the plasma zinc concentration the sample using a direct or an indirect detection technique; and c) identifying the subject as appropriate for a particular treatment.

[0030] Also provided is a method where individuals having levels of plasma zinc concentration greater than about 60 μg/dl, are considered appropriate candidates for standard care.

[0031] Also provided is a method where subjects having plasma zinc concentration levels less than about 40-50 μg/dl are considered as candidates for active agents that have been identified as efficacious for the treatment of septic shock using plasma zinc concentration as a treatment criteria or for other higher risk therapies.

[0032] In another broad aspect, there is provided herein a method for the identification of a sepsis condition in a mammalian subject for determining the effective course of treatment comprising the steps: a) obtaining a biological sample from a subject suspected of having sepsis; b) determining the plasma zinc concentration in the sample using a direct or an indirect detection technique; and c) correlating the plasma zinc concentration in the sample to known standards as a biomarker in sepsis patients as an indicator of prognosis of the subject.

[0033] In yet another broad aspect, there is provided herein a method for the identification of a sepsis condition in a mammalian subject for determining the effective course of treatment comprising the steps: a) obtaining a biological sample from a subject suspected of having sepsis; b) determining the plasma zinc concentration in the sample using a direct or an indirect detection technique; c) comparing the determined plasma zinc concentration to a set of predetermined values for plasma zinc concentration; and d) categorizing the individual for purposes of determining prognosis.

[0034] Also provided is a method further comprises, selecting at least one additional sepsis markers that increase or decrease in individuals with that risk factor relative to healthy individuals.

[0035] Also provided is a method where the plasma zinc concentration is taken from the subject at different points in time.

[0036] Also provided is a method where the plasma zinc concentration is from an internal reference, an external reference, or both.

[0037] Also provided is a method further comprising the step of determining a disease state progression.

[0038] In yet another broad aspect, there is provided herein a diagnostic kit for the diagnosis and prognosis of septic shock in a mammalian subject comprising: an analyte specific for plasma zinc concentration wherein the analyte is capable of detecting a concentration of plasma zinc, such that a diagnosis or prognosis of the subject may be made; and reactants for detecting the concentration of plasma zinc.

[0039] present invention provides, inter alia, methods of predicting the clinical outcome of an ill patient, comprising detecting the level of Zip8 expression in a monocyte-containing sample obtained from an ill patient, wherein a 1.5-fold or greater increase in the level of Zip8 expression in the sample relative to a control predicts a poor clinical outcome. Preferred are those methods wherein the patient is critically ill or has been diagnosed with sepsis. However, those methods wherein the Zip8 expression is detected within 10, 15, 24, 30, 35, 40, 45, 50, 60, 70, 80, 90, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 100 hours of onset of illness are also preferred.

Preferred are methods as described, wherein the Zip8 expression is detected subsequent to the initial detection, and wherein the initial detection results and subsequent detection results are compared to each other. Also preferred are those methods as described, wherein the initial Zip8 expression is compared to control. Other preferred methods are those as described, wherein the clinical outcome is selected from the group consisting of: decreased plasma zinc concentration; increased cytosolic zinc concentration; increased SOFA score; increased SIRS score, increased severity of illness; increased plasma cytokines; increased plasma chemokines; decreased cardiovascular status; increased vasopressor use; increased mean arterial pressure; increased platelet/endothelium interactions.

Preferred are those methods wherein the control is a monocyte-containing sample obtained from a healthy subject, although those wherein the control is a standard value is also preferred. The methods herein described wherein expression of Zip8 is decreased at least 2-fold, at least 2.5-fold, at least 3- fold, at least 3.5-fold, or at least 4-fold, are preferred.

[0040] Also provided are methods of identifying a therapeutic agent for the treatment of sepsis, comprising screening candidate agents in vitro to select an agent that decreases expression of Zip 8 in monocyte cells, thereby identifying an agent for the treatment of sepsis. Preferred are those methods wherein screening comprises contacting the candidate agents with the monocyte cells. Those methods wherein expression of Zip8 in the monocyte cells is increased at least 1.5-fold, 2-fold, 2.5- fold, 3-fold, 3.5-fold, or 4-fold relative to untreated cells are preferred. Most preferred are those methods wherein the candidate agent is a cytokine or a small molecule. Preferred are any of the methods herein, wherein the monocytes are derived from a blood sample.

[0041] Also provided are methods for monitoring health risk parameters of a hospital patient, comprising: obtaining a first measurement of Zip8 expression within 24, 48, 72 hours of admission, obtaining subsequent measurement or measurements of Zip8 expression, and comparing the first measurement to subsequent measurements, wherein increases in Zip8 expression levels indicate increasing risk of poor clinical outcome and decreasing or changeless Zip8 expression levels indicate less risk of poor clinical outcome. Such methods, wherein Zip8 expression is measured by PCR are preferred.

[0042] Also provided are methods for the characterization of zinc status of a patient, comprising identifying Zip8 expression levels in a critically ill and/or septic patient, wherein at least one feature of the critically ill and/or septic state characterization is selected from one or more of the group consisting of: presence or absence of critical illness/sepsis; diagnosis of critical illness/sepsis;

prognosis of critical illness/sepsis; therapy outcome prediction of critical illness/sepsis; therapy outcome monitoring of critical illness/sepsis; suitability of critical illness/sepsis to treatment, such as suitability of critical illness/sepsis to zinc supplementation or chelation; suitability of critical illness/sepsis to Zip8 nucleic acid (DNA, RNA) or protein inhibition or associated upregulation of nucleic acid or protein expression/function; suitability of critical illness/sepsis for critical illness/sepsis therapy; suitability of critical illness/sepsis to adjuvant therapy.

[0043] Also provided are kits for the detection of critical illness and/or sepsis, the kit comprising at least one detection probe comprising one a Zip8 probe, primer, or both. Preferred are those kits wherein the kit is in the form or comprises an oligonucleotide array.

[0044] Also provided are methods for the determination of suitability of a critical illness and/ or sepsis patient for treatment comprising: i) isolating at least one monocyte-containing sample from a patient suffering from critical illness and/or sepsis; ii) performing the characterization of the at least one sample according to at least one claim herein and/or utilizing detection probe or primer for Zip8, to identify at least one feature of Zip8 expression levels, including nucleic acid profiles and/or protein content; iii) based on the at least one feature identified in step ii), diagnosing the physiological status of the patient; iv) based on the diagnosis obtained in step iii), determining whether the patient would benefit from treatment of critical illness and/or sepsis.

[0045] Also provided are methods for the determination of the likely prognosis of a critical illness and/or sepsis patient comprising: i) isolating at least one monocyte-containing sample from a patient suffering from critical illness and/or sepsis; and ii) characterizing at least one sample to identify at least one feature of Zip8, including nucleic acid expression and/or protein content, wherein the feature allows for the determination of the likely prognosis of the critical illness/sepsis patient.

DETAILED DESCRIPTION

[0046] Very broadly, the present invention provides practical applications associated with the discovery that plasma zinc status correlates with severity of illness (SAPSII or SOFA Scores) in critically ill patients. Moreover, the present invention provides practical applications associated with the discovery that the extent of decline in plasma zinc levels is helpful in predicting sepsis severity and the immune response in comparison to critically ill, non-infected subjects. The invention also applies the results of experiments described herein showing that the precipitous drop in plasma zinc is associated with alteration in the expression of zinc transporters, key regulators of cellular zinc homeostasis.

[0047] The inventors compared zinc homeostasis with indices of inflammation and severity of illness between critically ill control (CIC) and septic subjects. The inventors observed that plasma zinc levels are substantially reduced in CIC adult patients during the first 24 hours of critical illness and that plasma zinc concentration declined even further in septic patients. The decrease in plasma zinc concentration inversely correlated with severity of illness scores and plasma cytokine and chemokine levels. Within the sepsis cohort the decline in zinc concentration correlated best with a decline in cardiovascular status. Furthermore, the zinc transporter SLC39A8 was the only transporter, out of 24 family members, to consistently show increased expression in conjunction with a decline in plasma zinc levels suggesting that this importer is uniquely involved in the regulation of zinc homeostasis in the critically ill.

[0048] Zinc Concentration

[0049] The plasma zinc concentration declined more in the sepsis cohort than the CIC group despite similar severity of illness scores. This distinction between septic and CIC patients did not change when other variables that could have influenced zinc status were controlled for including: age; propofol use; insulin use; and BMI. Without being bound to any particular theory, the inventors believe that zinc homeostasis is more dynamically altered in the setting of severe infection beyond that encountered during the acute phase response in non-infected, CIC subjects.

[0050] In addition to zinc levels being lower in response to infection, the zinc concentration declines predictably with increased severity of illness scores.

[0051] However, the relationship between plasma zinc concentration and severity of illness in the sepsis cohort was statistically significant for both SOFA score and SIRS criteria. The cardiovascular parameters that included vasopressor use and mean arterial pressure had the most significant impact on the association between low plasma zinc and increased SOFA scores.

[0052] Without being bound by any particular theory, the inventors believe that insufficient zinc levels or dysregulation in cellular homeostasis may contribute to hypotension in the setting of severe infection thereby leading to worse outcomes in the setting of sepsis. In order to more carefully account for changes in zinc status within study subjects the inventors controlled for factors that may alter zinc status including: age; insulin or propofol infusion; and BMI. Analysis of the data herein showed that none of these complicating factors were observed to contribute to alteration of plasma zinc levels within the study population.

[0053] Zinc Transporters

[0054] The expression of other zinc transporters was essentially unaltered in the presence of critical illness or sepsis. However, SLC39A8 expression was up-regulated in patients with low plasma zinc levels and directly correlated with severity of illness in both the CIC and sepsis groups but was most significant in the sepsis cohort irrespective of the infecting organism. Therefore, zinc homeostasis, mediated through zinc transport and the genetic and chemical markers associated with zinc transport, serves a critical role in understanding disease susceptibility and pathogenesis.

[0055] Inflammation/Immune Function

[0056] The immune response, as established by elevated cytokine and chemokine expression, inversely correlated with low plasma zinc concentration. As shown in the accompanying examples, as plasma zinc levels declined, IL-6 levels further increased. IL-6, as part of the initial proinflammatory response to infection, is increased by zinc deficiency and enhances the expression of SLC39A8. SLC39A8 plays a critical role in a positive feedback loop directed at maintaining cellular zinc content and cell viability during the early stages of sepsis. SLC39A8 as a biomarker for immune function as well as better understanding its role in monocyte behavior is therefore provided.

[0057] In the sepsis cohort, the relationship between SLC39A8 expression and severity of illness utilizing SOFA score was significant; the inventors therefore evaluated if there was a specific component comprising the SOFA score that produced that association. Platelet number was most strongly associated. Thus, the association between platelets and SLC39A8 is another way to exhibit a severity of illness relationship.

[0058] Abbreviations

[0059] BMI Body mass index

[0060] CIC Critically ill control

[0061] DNA Deoxyribonucleic acid

[0062] IFN Interferon

[0063] IL Interleukin

[0064] ISH In situ hybridization

[0065] mRNA Messenger RNA

[0066] PCR Polymerase chain reaction

[0067] qRT-PCR Quantitative reverse transcriptase polymerase chain reaction

[0068] RNA Ribonucleic acid

[0069] SAPS II Simplified acute physiology score

[0070] SIRS Systemic inflammatory response syndrome

[0071] SOFA Sequential organ failure assessment

[0072] Terms

[0073] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise.

[0074] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one. "

[0075] Also, the use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to be limiting. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y".

[0076] The term "combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.

[0077] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls.

[0078] Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0079] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0080] Adjunctive therapy: A treatment used in combination with a primary treatment to improve the effects of the primary treatment.

[0081] Candidate: As used herein, a "candidate" for therapy is a patient that is predicted to respond favorably to the treatment.

[0082] Clinical outcome: Refers to the health status of a patient following treatment for a disease or disorder, or in the absence of treatment. Clinical outcomes include, but are not limited to, an increase in the length of time until death, a decrease in the length of time until death, an increase in the chance of survival, an increase in the risk of death, survival, disease-free survival, chronic disease, metastasis, advanced or aggressive disease, disease recurrence, death, and favorable or poor response to therapy.

[0083] Control: A "control" refers to a sample or standard used for comparison with an

experimental sample. In some embodiments, the control is a sample obtained from a healthy patient or a non-diseased tissue sample obtained from a patient diagnosed with the disease. In some embodiments, the control is a historical control or standard value (i.e. a previously tested control sample or group of samples that represent baseline or normal values).

[0084] Cytokines: Proteins produced by a wide variety of hematopoietic and non-hematopoietic cells that affect the behavior of other cells. Cytokines are important for both the innate and adaptive immune responses.

[0085] Decrease in survival: As used herein, "decrease in survival" refers to a decrease in the length of time before death of a patient, or an increase in the risk of death for the patient.

[0086] Detecting level of expression: As used herein, "detecting the level of nucleic acid

expression" refers to quantifying the amount of nucleic acid present in a sample. Detecting expression of nucleic acid in a sample can be achieved using any method known in the art or described herein, such as by qRT-PCR. Detecting expression of nucleic acid includes detecting expression of either a mature form of the nucleic acid or a precursor form that is correlated with the nucleic acid expression. Typically, nucleic acid detection methods involve sequence specific detection, such as by RT-PCR. Nucleic acid-specific primers and probes can be designed using the precursor and mature nucleic acid sequences, which are known in the art and provided herein.

[0087] Patient: As used herein, the term "patient" includes human and non-human animals. The preferred patient for treatment is a human. "Patient" and "subject" are used interchangeably herein.

[0088] Pharmaceutically acceptable vehicles: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.

[0089] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0090] Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease. [0091] Screening: As used herein, "screening" refers to the process used to evaluate and identify candidate agents that increase expression of nucleic acid. In some cases, screening involves contacting a candidate agent (such as an antibody, small molecule or cytokine) with cells and testing the effect of the agent on expression of the nucleic acid. Expression of a nucleic acid can be quantified using any one of a number of techniques known in the art and described herein, such as by microarray analysis or by qRT-PCR.

[0092] Small molecule: A molecule, typically with a molecular weight less than about 1000

Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.

[0093] Therapeutic: A generic term that includes both diagnosis and treatment.

[0094] Therapeutic agent: A chemical compound, small molecule, or other composition, such as an antisense compound, antibody, protease inhibitor, hormone, chemokine or cytokine, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

"Incubating" includes a sufficient amount of time for an agent to interact with a cell or tissue.

"Contacting" includes incubating an agent in solid or in liquid form with a cell or tissue. "Treating" a cell or tissue with an agent includes contacting or incubating the agent with the cell or tissue.

[0095] Therapeutically effective amount: A quantity of a specified pharmaceutical or therapeutic agent sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.

[0096] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0097] In some cases, a method herein requires isolation of nucleic acid from a sample, such as a cell or tissue sample. Nucleic acids can be isolated using any suitable technique known in the art. For example, phenol-based extraction is a common method for isolation of RNA. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants. Phenol-based isolation procedures can recover RNA species in the 10-200-nucleotide range (e.g. , precursor and mature RNAs, 5S and 5.8S ribosomal RNA (rRNA), and Ul small nuclear RNA (snRNA)). In addition, extraction procedures such as those using TRIZOL™ or TRI REAGENT™, will purify all RNAs, large and small, and are efficient methods for isolating total RNA from biological samples that contain RNAs and small interfering RNAs (siRNAs).

[0098] Microarray: A microarray is a microscopic, ordered array of nucleic acids, proteins, small molecules, cells or other substances that enables parallel analysis of complex biochemical samples. A DNA microarray consists of different nucleic acid probes, known as capture probes that are chemically attached to a solid substrate, which can be a microchip, a glass slide or a microsphere- sized bead. Microarrays can be used, for example, to measure the expression levels of large numbers of messenger RNAs (mRNAs) and/or miRNAs simultaneously.

[0099] Microarrays can be fabricated using a variety of technologies, including printing with fine- pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.

[00100] Microarray analysis of RNAs can be accomplished according to any method known in the art (see, for example, PCT Publication No. WO 2008/054828; Ye et al, Nat. Med. 9(4):416-423, 2003; Calin et al., N. Engl. J. Med. 353(17): 1793-1801, 2005, each of which is herein incorporated by reference). In one example, RNA is extracted from a cell or tissue sample, the small RNAs (18-26- nucleotide RNAs) are size-selected from total RNA using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers are attached to the 5' and 3' ends of the small RNAs and the resulting ligation products are used as templates for an RT-PCR reaction with 10 cycles of amplification. The sense strand PCR primer has a fluorophore attached to its 5' end, thereby fluorescently labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding RNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular RNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular RNA.

[00101] In an alternative method, total RNA containing the small RNA fraction extracted from a cell or tissue sample is used directly without size-selection of small RNAs, and 3' end labeled using T4 RNA ligase and either a fluorescently-labeled short RNA linker. The RNA samples are labeled by incubation at 30°C for 2 hours followed by heat inactivation of the T4 RNA ligase at 80°C for 5 minutes. The fluorophore-labeled RNAs complementary to the corresponding RNA capture probe sequences on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The microarray scanning and data processing is carried out as described above.

[00102] There are several types of microarrays than can be employed, including spotted

oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays and spotted long oligonucleotide arrays. In spotted oligonucleotide microarrays, the capture probes are

oligonucleotides complementary to RNA sequences. This type of array is typically hybridized with amplified PCR products of size-selected small RNAs from two samples to be compared that are labeled with two different fluorophores. Alternatively, total RNA containing the small RNA fraction is extracted from the two samples and used directly without size-selection of small RNAs, and 3' end labeled using T4 RNA ligase and short RNA linkers labeled with two different fluorophores. The samples can be mixed and hybridized to one single microarray that is then scanned, allowing the visualization of up-regulated and down-regulated RNA genes in one assay.

[00103] In pre-fabricated oligonucleotide microarrays or single-channel microarrays, the probes are designed to match the sequences of known or predicted RNAs. There are commercially available designs that cover complete genomes (for example, from Affymetrix or Agilent). These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays.

[00104] Spotted long Oligonucleotide Arrays are composed of 50 to 70-mer oligonucleotide capture probes, and are produced by either ink-jet or robotic printing. Short Oligonucleotide Arrays are composed of 20-25-mer oligonucleotide probes, and are produced by photolithographic synthesis (Affymetrix) or by robotic printing.

[00105] Quantitative RT-PCR: Quantitative RT-PCR (qRT-PCR) is a modification of polymerase chain reaction used to rapidly measure the quantity of a product of polymerase chain reaction. qRT- PCR is commonly used for the purpose of determining whether a genetic sequence is present in a sample, and if it is present, the number of copies in the sample. Any method of PCR that can determine the expression of a nucleic acid molecule falls within the scope of the present disclosure. There are several variations of the qRT-PCR method known in the art, three of which are described below.

[00106] Methods for quantitative polymerase chain reaction include, but are not limited to, via

agarose gel electrophoresis, the use of SYBR Green (a double stranded DNA dye), and the use of a fluorescent reporter probe. The latter two can be analyzed in real-time.

[00107] With agarose gel electrophoresis, the unknown sample and a known sample are prepared with a known concentration of a similarly sized section of target DNA for amplification. Both reactions are run for the same length of time in identical conditions (preferably using the same primers, or at least primers of similar annealing temperatures). Agarose gel electrophoresis is used to separate the products of the reaction from their original DNA and spare primers. The relative quantities of the known and unknown samples are measured to determine the quantity of the unknown.

[00108] The use of SYBR Green dye is more accurate than the agarose gel method, and can give results in real time. A DNA binding dye binds all newly synthesized double stranded DNA and an increase in fluorescence intensity is measured, thus allowing initial concentrations to be determined. However, SYBR Green will label all double-stranded DNA, including any unexpected PCR products as well as primer dimers, leading to potential complications and artifacts. The reaction is prepared as usual, with the addition of fluorescent double-stranded DNA dye. The reaction is run, and the levels of fluorescence are monitored (the dye only fluoresces when bound to the double-stranded DNA). With reference to a standard sample or a standard curve, the double-stranded DNA concentration in the PCR can be determined.

[00109] The fluorescent reporter probe method uses a sequence-specific nucleic acid based probe so as to only quantify the probe sequence and not all double stranded DNA. It is commonly carried out with DNA based probes with a fluorescent reporter and a quencher held in adjacent positions (so- called dual-labeled probes). The close proximity of the reporter to the quencher prevents its fluorescence; it is only on the breakdown of the probe that the fluorescence is detected. This process depends on the 5' to 3' exonuclease activity of the polymerase involved.

[00110] The real-time quantitative PCR reaction is prepared with the addition of the dual-labeled probe. On denaturation of the double-stranded DNA template, the probe is able to bind to its complementary sequence in the region of interest of the template DNA. When the PCR reaction mixture is heated to activate the polymerase, the polymerase starts synthesizing the complementary strand to the primed single stranded template DNA. As the polymerization continues, it reaches the probe bound to its complementary sequence, which is then hydrolyzed due to the 5'-3' exonuclease activity of the polymerase, thereby separating the fluorescent reporter and the quencher molecules. This results in an increase in fluorescence, which is detected. During thermal cycling of the real-time PCR reaction, the increase in fluorescence, as released from the hydrolyzed dual-labeled probe in each PCR cycle is monitored, which allows accurate determination of the final, and so initial, quantities of DNA.

[00111] In Situ Hybridization: In situ hybridization (ISH) applies and extrapolates the technology of nucleic acid hybridization to the single cell level, and, in combination with the art of cytochemistry, immunocytochemistry and immunohistochemistry, permits the maintenance of morphology and the identification of cellular markers to be maintained and identified, and allows the localization of sequences to specific cells within populations, such as tissues and blood samples. ISH is a type of hybridization that uses a complementary nucleic acid to localize one or more specific nucleic acid sequences in a portion or section of tissue {in situ), or, if the tissue is small enough, in the entire tissue (whole mount ISH). RNA ISH can be used to assay expression patterns in a tissue, such as the expression of RNAs.

[00112] Sample cells or tissues are treated to increase their permeability to allow a probe, such as a RNA-specific probe, to enter the cells. The probe is added to the treated cells, allowed to hybridize at pertinent temperature, and excess probe is washed away. A complementary probe is labeled with a radioactive, fluorescent or antigenic tag, so that the probe's location and quantity in the tissue can be determined using autoradiography, fluorescence microscopy or immunoassay. The sample may be any sample as herein described.

[00113] In Situ PCR: In situ PCR is the PCR-based amplification of the target nucleic acid sequences prior to ISH. For detection of RNA, an intracellular reverse transcription step is introduced to generate complementary DNA from RNA templates prior to in situ PCR. This enables detection of low copy RNA sequences.

[00114] Prior to in situ PCR, cells or tissue samples are fixed and permeabilized to preserve

morphology and permit access of the PCR reagents to the intracellular sequences to be amplified. PCR amplification of target sequences is next performed either in intact cells held in suspension or directly in cytocentrifuge preparations or tissue sections on glass slides. In the former approach, fixed cells suspended in the PCR reaction mixture are thermally cycled using conventional thermal cyclers. After PCR, the cells are cytocentrifuged onto glass slides with visualization of intracellular PCR products by ISH or immunohistochemistry. In situ PCR on glass slides is performed by overlaying the samples with the PCR mixture under a coverslip which is then sealed to prevent evaporation of the reaction mixture. Thermal cycling is achieved by placing the glass slides either directly on top of the heating block of a conventional or specially designed thermal cycler or by using thermal cycling ovens.

[00115] Detection of intracellular PCR products is generally achieved by one of two different

techniques, indirect in situ PCR by ISH with PCR-product specific probes, or direct in situ PCR without ISH through direct detection of labeled nucleotides (such as digoxigenin-11-dUTP, fluorescein-dUTP, 3H-CTP or biotin-16-dUTP), which have been incorporated into the PCR products during thermal cycling.

[00116] As used herein, "poor prognosis" generally refers to a decrease in survival, or in other words, an increase in risk of death or a decrease in the time until death. Poor prognosis can also refer to an increase in severity of the disease.

[00117] Methods of screening candidate agents to identify therapeutic agents for the treatment of disease are well known in the art. Methods of detecting expression levels of nucleic acids are known in the art and are described herein, such as, but not limited to, microarray analysis, RT-PCR

(including qRT-PCR), in situ hybridization, in situ PCR, and Northern blot analysis. In one embodiment, screening comprises a high-throughput screen. In another embodiment, candidate agents are screened individually.

[00118] The candidate agents can be any type of molecule, such as, but not limited to nucleic acid molecules, proteins, peptides, antibodies, lipids, small molecules, chemicals, cytokines, chemokines, hormones, or any other type of molecule. In some embodiments, the candidate agents are molecules that play a role in the signaling pathway described herein. In one embodiment, the candidate agents are cytokines. In another embodiment, the candidate agents are small molecules.

[00119] Also described herein is a method for the characterization of disease, wherein at least one feature of the disease is selected from one or more of the group consisting of: presence or absence of disease; diagnosis of disease; prognosis of disease; therapy outcome prediction; therapy outcome monitoring; suitability of disease to treatment, such as suitability of disease to chemotherapy treatment and/or radiotherapy treatment; suitability of disease to hormone treatment; suitability of disease for removal by invasive surgery; suitability of disease to combined adjuvant therapy.

[00120] Also described herein are kits comprising at least one detection probe comprising one or more nucleic acid herein. The kit can be in the form of or comprise an oligonucleotide array.

[00121] EXAMPLES

[00122] Example I: Plasma Zinc Concentrations in Critically III Adult Sepsis Patients

[00123] MATERIALS AND METHODS for Example I

[00124] Patients

[00125] Patients in this prospective study were admitted to the MICU at The Ohio State University

Medical Center (OSUMC), an academic tertiary care hospital. 296 patients were screened over a four month period from August through December 2007. Inclusion was determined within 24 hours of admission by chart review; males and females greater than 18 years of age, who spent at least one day in the MICU were eligible. Patients admitted to the MICU without criteria for sepsis were eligible for the CIC Arm (Table 1 Inclusion and Exclusion Criteria). Blood was also collected from twelve healthy donors during the same time period for comparison to the sepsis and CIC patient groups.

Table 1 - Inclusion and Exclusion Criteria

Critically 111 Sepsis Arm

Inclusion Criteria:*

Knows or suspected infection and

> 2/4 SIRS Criteria

1) Temperature < 36C or > 38C

2) Heart Rate > 90

3) Respiratory Rate > 20 or PA CQ ₂ < 32

4) White blood cell count < 4,000 or > 12,000 or > 10% bands

Exclusion Criteria:

1) Consent not available or patient declined

2) Prisoner

3) Patient died before blood was collected

4) Patient met criteria for sepsis onset at an outside hospital > 24 hours prior to transfer

* Blood was collected within 24 hours of sepsis onset (regardless of patient location

when inclusion criteria were first met

Critically 111 Control Arm Inclusion Criteria

1) < 24 hours since medical intensive care unit admission

2) No know or suspected infection based on review of treating physicians' assessment 3) Lack of antibiotic therapy (except for prophylactic therapy)

4) No known microbiologic and radiographic signs of infection

Exclusion Criteria

1) Consent not available or patient declined

2) Prisoner

3) Patient died before blood was collected

4) Patient critically ill in a medical intensive case unit > 24 hours

[00126] Procedures

[00127] After informed consent was obtained, baseline demographic data were recorded and 20

milliliters of blood was drawn. Fifteen milliliters of blood was collected in EDTA vacuum tubes for RNA analysis and five milliliters was collected in heparin containing vacuum tubes for zinc and cytokine analysis. All tubes were immediately placed on ice at the time of collection.

[00128] Monocyte purification

[00129] Monocytes were isolated from fresh donor blood by Histopaque-1077 (Sigma-Aldrich, St.

Louis, MO) density gradient centrifugation at 2000 RPM for 20 minutes at room temperature. The mononuclear layer was removed and washed twice in RPMI 1640 (BioWhittaker, WalkersviUe, MD). Monocytes were isolated by positive selection with anti-CD 14-coated magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. This method consistently yields an equal to or greater than 98% pure population of CD 14+ monocytes as previously confirmed by flow cytometry analysis (data not shown). Zinc contamination is minimized by utilizing conical tubes and isolation medium without the detectable presence of zinc (per manufacturer reporting).

Monocytes were then immediately re-suspended in TRIzol reagent (Invitrogen, Carlsbad, CA), for RNA related studies, within four hours of blood collection and then frozen at -80°C.

[00130] RNA isolation

[00131] RNA was extracted using Trizol reagent as per standard operating procedure and then 1 μg of total RNA was reverse transcribed to generate cDNA by ThermoScript RNase H- Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA) followed by dilution (1 : 10) using RNase free water. The cDNA was then subject to quantitative polymerase chain reaction (PCR) analysis using SYBR Green PCR Master Mix and a PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). ACt values were first used for all statistical analysis and then transformed to a relative copy number (RCN) value to facilitate ease of data interpretation. RCN of selected genes were determined by normalization to the expression of two housekeeping genes, GAPDH and cyclic AMP-accessory protein (CAP-1), and calculated with the equation: RCN = E ^{" ACt} x 100, where E = efficiency of PCR, and ACt = Ct target - Ct reference (average of two housekeeping genes) [10]. All PCR primer pairs were previously validated and reported [9].

[00132] Plasma cytokine measurements

[00133] Heparinized blood was centrifuged at 2000 RPM for 10 minutes at 4°C and then the plasma was carefully removed. Fifty micro liters of plasma was analyzed to simultaneously quantify cytokine or chemokine protein levels utilizing a Bio-Plex Multiplex Cytokine Assay (BioRad, Hercules, CA) per manufacturer' s instructions.

[00134] Plasma zinc measurements

[00135] Separated plasma (as described in plasma cytokine measurement) was also assessed for total zinc content using atomic absorption spectrophotometry (Analyst 400, PerkinElmer, Waltham, MA). Two hundred micro liters of plasma was diluted 1 : 15 with de- ionized water and compared to zinc standards (PerkinElmer, Waltham, MA). All patient samples were analyzed in triplicate.

[00136] Statistical Analysis

[00137] Patient information at the time of blood collection was used to calculate the Simplified Acute Physiology Score (SAPS II) and Sequential Organ Failure Assessment (SOFA) if available. If not available, the patients' worst reported values within the 12 hours before or following the blood draw were used. Patient disposition at the time of hospital discharge and in-hospital mortality were also recorded. Once collected, the data was de-identified and entered into a secure data management program. Statistical analysis was performed using JMP 6.0 (SAS institute, Cary, NC) and STATA 6.0 (Stata Corp., College Station, TX) in collaboration with a biostatistician at The Ohio State University Center for Biostatistics.

[00138] RESULTS for Example I

[00139] Patients

[00140] A total of 23 CIC, 22 septic, and 12 normal control subjects were enrolled and compared in this study. Overall, the CIC and sepsis cohorts were well matched based on demographic and laboratory values (Table 2 below). Comparison of the SAPS II was also similar between the two groups whereas the septic group had a higher SOFA score (p=.02). Most patients survived to hospital discharge and a majority of patients in both groups were discharged to home or an acute rehabilitation facility. Two septic patients died before hospital discharge, one from an abdominal infection and the other of pneumonia. The source of infection was identified by culture data in 16 of 22 septic patients and included: C. glabratta, C. albicans, methicillin sensitive and methicillin resistant Staph areus, E. coli, C. difficile, Alpha-hemolytic strep, P. aeruginosa, and E.fecalis. Cultures were negative in the CIC patients and one died following a sub-arachnoid hemorrhage. Table 2 Demographics and Diagnoses

[00141] Zinc Analysis

[00142] Plasma zinc levels were determined by atomic absorption in 22 CIC and all septic patients (n=22). One CIC patient did not yield an adequate volume of plasma for zinc analysis. All septic patients and 21 out of 22 CIC patients had below normal plasma zinc levels recorded within the first 24 hours of sepsis onset or admission to the MICU. Pre-ICU admission zinc levels were not obtainable. The majority of patients in both groups had zinc levels greater than two standard deviations below normal values. Ten of twelve healthy controls also had decreased zinc levels but the majority of those were within one standard deviation of normal. Further analysis revealed a statistically significant decrease in plasma zinc in the CIC cohort (p= .0002) compared to healthy controls and a further decline in the septic group compared to the CICs (p= .038) (Figure 1).

[00143] Using a multivariate linear regression model, plasma zinc concentration was decreased in the septic population when compared to the CIC group, even when adjusted for age, gender, use of an insulin drip, or use of propofol infusion (p= .030). Using univariate linear regression, plasma zinc concentrations were evaluated in association with five additional parameters including age, SAPS II score, diagnosis of diabetes mellitus, BMI, and concomitant propofol infusion for all MICU patients enrolled (CIC and sepsis patients combined). An inverse correlation occurred with age, SAPS II, and diagnosis of diabetes mellitus, suggesting that zinc levels fall as age or SAPS II rises whereas a direct correlation was found with body mass index and propofol infusion suggesting that zinc levels are higher in patients with more body mass or those that received propofol infusion. However, none of these achieved statistical significance. Further analysis of the sepsis cohort revealed an inverse correlation between plasma zinc concentration and sepsis severity as determined by Systemic Inflammatory Response Syndrome (SIRS) criteria (p= .02) and SOFA scores (p=.05).

[00144] The same trend was observed between plasma zinc concentration and SAPS II but did not achieve statistical significance (p= .17). These findings show that plasma zinc levels decline with increasing sepsis severity and organ failure. Since an inverse relationship was observed between plasma zinc concentration and SOFA score the inventors then determined whether specific components of the SOFA score (vasopressor use, mean arterial pressure, Pa0 ₂ /Fi0 ₂ , platelet count and serum creatinine) were more predictive of plasma zinc levels. The association between plasma zinc and Glasgow Coma Score or bilirubin was not evaluated. Strikingly, plasma zinc concentration was (inversely) related to the cardiovascular parameters: vasopressor use (yes or no) (p=.008) and mean arterial pressure / vasopressor need (as demonstrated by the SOFA Cardiovascular category rank 0-4) (p=.039) (Table 3), whereas no significant correlation was observed with the other three parameters.

Table 3, Plasma Zinc Concentrations in Septic Patients

Vasopressors Mean ° SD Median

No 57.2 20.2 54.5 p= .002

Yes 35.6 8.03 36

Twelve patient: in the septic group required vasopressors

a Mean plasma zinc concentration for each group in ug/dL

SD= Standard Deviation

0= No vasopressors; 1 = mean arterial pressure <70 mm Hg;

2 - Dopamine <= 5 mcg/kg/minute or Dobutamine {any dose)

3- Dopamine >5 or Norepinephrine <— 0.1 meg/minute

4— Dopamine > 1 5 or Epinephrine/Norepinephrine > .01

[00145] Cytokine measurements

[00146] Of the cytokines evaluated IL-Ιβ, IL-6, IL-8, MCP-1, MIP-la, and IFNy, were consistently elevated in sepsis patients when compared to critically ill controls and all eight, which also included (IL-10 and TNF-a), were elevated when compared to healthy controls (Table 4). The inventors then determined by multivariate analysis whether there was a relationship between cytokine levels and plasma zinc concentration in the combined CIC and sepsis groups. As shown, all cytokines that were evaluated simultaneously increased as plasma zinc levels decreased (p=.05) (Table 4). Multivariate regression revealed a statistically significant correlation between zinc concentration and IL-6 (p=.033) or IL-8 plasma levels (p=.002).

Table 4. Relationship Between Cytokines and Plasma Zinc

Cytokine Septic ⁰ CIC° Healthy ⁰

IL-10 80 4 ± 261 .3 142.7 ± 594.6 1 0.0 ± 20.3

IL-6 287.0 ± 665.9 131 .8 ± 202.9 21 .9 ± 30.8

!L-8 55.8 ± 1 04.6 32.7 ± 27.0 33.2 ± 69.1

Interferon 215.5 ± 622.7 61 .0 ± 166.6 85.7 ± 1 38.6

IL-Ιβ 10.1 ± 31 .6 9.6 + 33.1 4.1 ± 9.8

MCP-1 214.8 ± 265.5 148.7 ± 125.8 154.8 ± 148.8

MIP-1 a. 141 .2 ± 162.6 1 16.3 ± 48.0 1 30.3 ± 103. 1

TNF-o 76.4 ± 267.1 88.3 ± 340.2 1 .6 ± 1 9. 1

Correlation between plasma zinc and cytokine concentrations for all critically ill patients

Cytokine^ Slope

In IL-10 -0.167 0.263

In IL-6 -0.293 0.033

In IL-8 -0.200 0.002

In Interferon -0.312 0.079

In 1L-1 I'. -0.14 0.273

In mcp-1 -0.105 0.082

In mip-1< ' -0.033 0.368

In Tnf-« -0.073 0.599

a Mean plasma cytokine protein concentration +/- standard deviation

CIC= Critically III Control

b Cytokine values have been log transformed for this mul tivariate regression analysis

c A negative slope represents an inverse correlation and is shown for a 10 unit change

in plasma zinc.

[00147] Zinc Transporters

[00148] Total RNA obtained from peripheral blood monocytes was initially screened to quantitatively determine the expression level of each of the 24 known human zinc transporters for each study subject. The inventors then determined whether there was any correlation between plasma zinc levels and the expression of specific zinc importers. Strikingly, there was a strong inverse correlation between low plasma zinc concentration and elevated SLC39A8 (Zip8) expression (p< .001) within all groups including healthy controls. In contrast, the opposite effect was observed between plasma zinc and three other transporters examined including: SLC39A1 (p=.999), SLC39A4 (p=.288), and

SLC39A14 (p=.015). When the same evaluation was conducted for each patient group individually a statistically significant relationship was observed between plasma zinc and SLC39A8 in the CIC group (p=.048) and between zinc concentration and SLC39A4 in the healthy patient group (p=.001). There is an inverse relationship between plasma zinc concentration and SLC39A8 (low plasma zinc concentration and elevated SLC39A8 expression) in the CIC group and a direct relationship between plasma zinc concentration and SLC39A4 (low plasma zinc and suppressed SLC39A4 expression) in the healthy group. SLC39A8 was the only transporter consistently shown to have increased

expression in conjunction with a decline in plasma zinc levels. Based on this, the inventors then evaluated SLC39A8 expression in the context of severity of illness scores in sepsis and CIC patients. SLC39A8 expression was directly related with SAPS II for both the sepsis and CIC cohorts but did not achieve statistical significance (p= .674 and p= .613 respectively). In addition, SLC39A8

expression directly correlated with SOFA scores in the sepsis cohort and achieved statistical

significance (p= .027). Further evaluation was then conducted to analyze the association, if any, between SLC39A8 expression and the SOFA parameters [acute lung injury score (Pa02/Fi02),

platelet count, vasopressor use, Mean Arterial Pressure (MAP), or creatinine] (Table 5C below). The only association to achieve statistical significance in the septic cohort was that observed between

SLC39A8 expression and platelet count (p< 0.001).

Table 5. Zinc Transporter Data

A. RNA expression by Real-time PGR for each for the four zinc transporters analyzed

A "Septic: ACt Septic RCN ° CIC: ACt CIC RCN "Healthy: ACt Healthy RCN

SLC39A1 8.2 ± 3.7 0.76 6.9 ± 0.9 0.97 7.0 ± 0.9 0.90

SLC39A4 20.3 ± 2.2 0.00 20.3 ± 2.0 0.00 20.3 ± 1 .9 0.00

SLC39A8 6.1 ± 1.4 2.23 7.4 ± 3.1 1.17 8.1 ± 1.3 0.61

SLC39A14 12.8 ± 1.5 0.02 12.9 ± 2.8 0.02 1 1.8 ± 0.4 0.03

B. Correlation between plasma zinc concentration and the four importers of interest

B. All Groups Septic CIC Healthy

bRho p-value I " Rho p-value I ^b Rho p-value I °R o p-value

SLC39A1 < -0.01 > 0.99 0.09 0.68 0.10 0.66 < 0.01 0.99

SLC39A4 -0.14 0.29 -0.24 0.27 0.06 0.79 -0.S2 < 0.01

SLC39A8 0.45 < 0.01 0.29 0.20 0.43 0.05 -0.04 0.90

SLC39A14 -0.32 0.02 -0.13 0.58 -0.22 0.33 0.16 0.62

C. Association between SLC39A8 expression and five SOFA categories in the

septic cohort

C. Septic Cohort p-value ACt= Cycle threshold standardized to housekeeping genes

'' Mean cycle threshold values +/- standard deviation

Vasopressors 0.60 RCN= Relative Copy Number

MAP 0.49 CIC= Critically III Control

Pa02/Fi02 0.67 ^Analysis compares plasma zinc values with the ACt value

Platelets <.01 using Spearman Correlation, negative "rho" (indirect) and

Creatinine 0.85 positive "rho" (direct).

[00149] Example 11. The transcriptional regulation of human SLC39A8 by nuclear factor κ B

[00150] Methods

[00151] Human cell lines and primary cultures were treated with TNFaLPS, or Francislla tularensis

in conjunction with the NF-κΒ inhibitor BAY11-7082. Promoter cloning and characterization was performed using multiple strategies including RACE, a luciferase reporter assay, site-directed

mutagenesis, and CHIP analysis.

[00152] Results for Example II

[00153] The expression of Zip8 was induced by TNFa (in A549 and primary lung epithelia) and LPS

(in THP1) or Francisella tularensis (in primary monocytes). Treatment with the NF-κΒ inhibitor,

BAY11-7082, inhibited Zip8 induction. RACE analysis identified the transcription start sites (TSS). A 2kb 5' flanking region of zip8 gene was then cloned and corresponding serial deletion constructs were placed upstream of the luciferase reporter gene in a pGL3-Basic vector. Promoter activity was determined by luciferase assay following transfection of the constructs. A 120bp proximal region upstream of the TSS was found to be essential for promoter activity. The promoter activity was enhanced by either TNFa treatment or co-transfection with a p65 plasmid that was further confirmed by inhibition with an ΙκΒα super repressor. In silico analysis using the Match® program identified four potential NF-κΒ binding sites. Generation of serial deletion constructs identified that one binding site (κΒ2) is critical for promoter activity. Site-directed mutagenesis of the κΒ2 site resulted in a significant decrease in activity. CHIP analysis demonstrated the in vivo binding of NF-κΒ p65 with the Zip8 promoter region.

[00154] Example II demonstrates that human Zip8 expression is directly regulated by NF-κΒ at the transcriptional level.

[00155] EXAMPLE III. NF- KB regulates the zinc transporter Zip8 in lung and contributes to zinc homeostasis in the setting of infection.

[00156] Methods

[00157] Human cell lines (A549, HEK293, HepG ₂ , and THP-1) and primary cultures (lung epithelia and monocytes) were treated with TNFa, LPS, or Francislla tularensis, in combination with the NF- KB inhibitor BAY 11-7082 and RELA siRNAs. Promoter cloning and characterization were performed using RACE assay, luciferase reporter assay, site-directed mutagenesis, and CHIP.

C57BL/6J mice were randomized into 3 groups and subjected to CLP (cecal ligation and puncture). Mice were sacrificed at 2 and 6hrs and their lungs were collected after lavage and perfusion. Total RNA from lung and liver was extracted and analyzed by real-time PCR.

[00158] Results for Example III

[00159] The expression of Zip8 was induced by TNFa (lung epithelia) and LPS (THP1) or

Francisella tularensis (primary monocytes) but not in HEK293 and HepG ₂ cells. Treatment with BAY11-7082 and RELA siRNAs inhibited Zip8 induction. Analysis of mouse lung tissue revealed that Zip8 was induced as early as 2hrs after CLP treatment, in conjunction with other NF-κΒ target genes such as SAA1/2 and ΙκΒα. Inspection of Zip8 transcription by RACE identified the transcription start sites (TSS). A 2kb 5' flanking region was cloned and its serial deletion constructs were placed in a luciferase vector (pGL3-Basic). A 120bp proximal region upstream of the TSS, induced by either TNFa treatment or co-transfection of p65, but not p50, was essential for promoter activity. Co-transfection with an ΙκΒα super repressor inhibited the induction effect by p65. In silico analysis identified four potential NF-κΒ binding sites. Serial deletion of these sites identified that one binding site is critical for promoter activity, as confirmed by site-directed mutagenesis. CHIP analysis demonstrated the binding of RELA with the Zip8 promoter region.

[00160] Example III demonstrates that Zip8 expression is directly regulated by NF-κΒ at the

transcriptional level, making it unique relative to most other zinc transporters. The inventors contend that Zip8 contributes to the hypozincemia encountered during sepsis and at the cellular level, serves a vital role in facilitating host immune defense against infection.

[00161] Example IV: Kits

[00162] Any of the compositions described herein may be comprised in a kit. In a non-limiting

example, reagents for isolating RNA, labeling RNA, and/or evaluating an RNA population using an array are included in a kit. The kit may further include reagents for creating or synthesizing RNA probes. The kits will thus comprise, in suitable container means, an enzyme for labeling the RNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the RNA probes, and components for isolating RNA. Other kits may include components for making a nucleic acid array comprising oligonucleotides complementary to RNAs, and thus, may include, for example, a solid support.

[00163] For any kit embodiment, including an array, there can be nucleic acid molecules that contain a sequence that is identical or complementary to all or part of any of SEQ ID NOS: 1- 2.

[00164] The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[00165] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being one preferred solution. Other solutions that may be included in a kit are those solutions involved in isolating and/or enriching RNA from a mixed sample.

[00166] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also include components that facilitate isolation of the labeled RNA. It may also include components that preserve or maintain the RNA or that protect against its degradation. The components may be RNAse-free or protect against RNAses.

[00167] Also, the kits can generally comprise, in suitable means, distinct containers for each

individual reagent or solution. The kit can also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the invention. Also, the kits are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of RNA.

[00168] It is also contemplated that any embodiment discussed in the context of an RNA array may be employed more generally in screening or profiling methods or kits of the invention. In other words, any embodiments describing what may be included in a particular array can be practiced in the context of RNA profiling more generally and need not involve an array per se.

[00169] It is also contemplated that any kit, array or other detection technique or tool, or any method can involve profiling for any of these RNAs. Also, it is contemplated that any embodiment discussed in the context of an RNA array can be implemented with or without the array format in methods of the invention; in other words, any RNA in an RNA array may be screened or evaluated in any method of the invention according to any techniques known to those of skill in the art. The array format is not required for the screening and diagnostic methods to be implemented.

[00170] The kits for using RNA arrays for therapeutic, prognostic, or diagnostic applications and such uses are contemplated by the inventors herein. The kits can include an RNA array, as well as information regarding a standard or normalized RNA profile for the RNAs on the array. Also, in certain embodiments, control RNA or DNA can be included in the kit. The control RNA can be RNA that can be used as a positive control for labeling and/or array analysis.

[00171] The methods and kits of the current teachings have been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the current teachings. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00172] Example V: Array Preparation and Screening

[00173] Also provided herein are the preparation and use of RNA arrays, which are ordered

macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of RNA molecules or precursor RNA molecules and that are positioned on a support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted. Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.

[00174] Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes,

oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of RNA- complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.

[00175] A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art. Useful substrates for arrays include nylon, glass and silicon. The arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like. The labeling and screening methods described herein and the arrays are not limited in its utility with respect to any parameter except that the probes detect RNA; consequently, methods and compositions may be used with a variety of different types of RNA arrays.

[00176] In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. The inventors therefore claim as our invention all that comes within the scope and spirit of these claims.