WO2014120642A1

WO2014120642A1 - Diagnosis and treatment of hepatorenal syndrome

Info

Publication number: WO2014120642A1
Application number: PCT/US2014/013287
Authority: WO
Inventors: Ravi Thadhani; S. Ananth Karumanchi
Original assignee: The General Hospital Corporation; Beth Israel Deaconess Medical Center, Inc.
Priority date: 2013-01-30
Filing date: 2014-01-28
Publication date: 2014-08-07

Abstract

The technology described herein relates to the role of Siglec proteins (e.g. Siglec-5, Siglec-7, and/or Siglec-9) in conditions such as hepatorenal syndrome and sepsis. Described herein are method, assays, and systems relating to the diagnosis and treatment of conditions including hepatorenal syndrome and sepsis. Splanchnic vasodilation (e.g. such as occurs in subjects with HRS) can induce a form of kidney failure which has high mortality rates and a fast disease progression. Treatment is currently limited to palliative care, which merely delays the eventual need for liver transplantation.

Description

DIAGNOSIS AND TREATMENT OF HEPATORENAL SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/758,503 filed January 30, 2013, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 27, 2014, is named 030258-075901-PCT_SL.txt and is 29,585 bytes in size.

TECHNICAL FIELD

[0003] The technology described herein relates to the diagnosis and treatment of splanchnic vasodilation and/or hepatorenal syndrome (HRS).

BACKGROUND

[0004] Splanchnic vasodilation (e.g. such as occurs in subjects with HRS) can induce a form of kidney failure which has high mortality rates and a fast disease progression. Treatment is currently limited to palliative care, which merely delays the eventual need for liver transplantation. Moreover, the lack of a definitive clinical test for splanchnic vasodilation makes it difficult to distinguish subjects with splanchnic vasodilation (who do not require kidney transplantation) from subjects with other forms of kidney failure (who may require kidney transplantation). A definitive clinical test for splanchnic vasodilation and/or HRS would permit clinicians to determine whether liver

transplantation alone is indicated (e.g. as in cases of HRS) or whether the more drastic liver and kidney transplant (in instances where kidney failure is not due to HRS) is indicated.

SUMMARY

[0005] The technology described herein is generally directed to diagnostic methods, assays, and systems, as well as methods of treatment, for splanchnic vasodilation, based upon the inventors' discovery that Siglec isoforms (e.g. Siglec-7, Siglec-5, and Siglec-9) are expressed at elevated levels in subjects with splanchnic vasodilation and that Siglec stimulates the nitric oxide production which causes splanchnic vasodilation.

[0006] In one aspect, described herein is a method of reducing splanchnic vasodilation, the method comprising; administering a therapeutically effective amount of a Siglec inhibitor to a subject in need of treatment. In some embodiments, the subject can be in need of treatment for a condition selected from the group consisting of: hepatorenal syndrome (HRS) and sepsis. In some

embodiments, the Siglec inhibitor can specifically bind one or more polypeptides selected from the group consisting of: Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide. [0007] In one aspect, described herein is a method of detecting soluble Siglec, the method comprising contacting a biological sample with a reagent specific for a Siglec polypeptide, wherein reaction of soluble Siglec with said reagent indicates the presence of soluble Siglec. In some embodiments, the reagent specific for a Siglec polypeptide can be specific for a soluble Siglec polypeptide. In some embodiments, the Siglec polypeptide can be selected from the group consisting of: Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide. In some embodiments, the method can comprise a first step of removing cells from the sample. In some embodiments, the sample can comprise a urine or blood sample.

[0008] In one aspect, described herein is an assay comprising: determining the expression level of Siglec in a test sample obtained from a subject; wherein an increase in the Siglec expression level relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation. In some embodiments, the Siglec can be selected from the group consisting of: Siglec-7; Siglec-5; and Siglec-9. In some embodiments, the expression level of Siglec can be determined by measuring the level of Siglec RNA transcript. In some embodiments, the RNA transcript level can be measured using reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the expression level of Siglec can be determined by measuring the level of Siglec polypeptide. In some embodiments, the expression level of Siglec can be determined by measuring the level of soluble Siglec polypeptide. In some embodiments, the polypeptide level can be measured using immunochemistry. In some embodiments, the sample can comprise a material selected from the group consisting of a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid; ascites; and urine.

[0009] In one aspect, described herein is an assay comprising: (a) contacting a biofluid test sample obtained from a subject with a detectable anti-Siglec antibody reagent; and (b) detecting the presence or intensity of a detectable signal; wherein an increase in the level of Siglec polypeptide, indicated by the level of the detectable signal, relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation. In some embodiments, the Siglec polypeptide can be selected from the group consisting of: Siglec-7; Siglec-5; and Siglec-9. In some embodiments, the level of Siglec polypeptide can be the level of soluble Siglec polypeptide. In some embodiments, the antibody reagent can be detectably labeled or generates a detectable signal. In some embodiments, the splanchnic vasodilation can be associated with a condition selected from the group consisting of: hepatorenal syndrome (HRS) and sepsis. In some embodiments, the expression level of Siglec can be normalized relative to the expression level of one or more reference genes or reference proteins. In some embodiments, the reference expression level of Siglec can be the expression level of Siglec in a prior sample obtained from the subject. In some embodiments, an increased level of Siglec can be a level at least 25% greater than a reference level. In some embodiments, the expression level of no more than 20 other genes is determined. In some embodiments, the expression level of no more than 10 other genes is determined. In some embodiments, the subject can be a human.

[0010] In one aspect, described herein is a method of administering a treatment for splanchnic vasodilation to a subject, the method comprising: determining the expression level of Siglec in a test sample obtained from a subject; and administering a treatment for splanchnic vasodilation to the subject if the expression level of Siglec is increased relative to a reference level. In one aspect, described herein is a method of identifying a subject in need of treatment for splanchnic vasodilation, the method comprising: determining the expression level of Siglec in a test sample obtained from a subject; wherein the subject is identified as being in need of treatment for splanchnic vasodilation if the expression level of Siglec is increased relative to a reference level. In one aspect, described herein is a method of determining the efficacy of a treatment for splanchnic vasodilation, the method comprising: (a) determining the expression level of Siglec in a test sample obtained from a subject before administration of the treatment; (b) determining the expression level of Siglec in a test sample obtained from a subject after administration of the treatment; wherein the treatment is not efficacious if the expression level determined in step (b) is increased relative to the expression level determined in step (a). In some embodiments, the Siglec can be selected from the group consisting of: Siglec-7; Siglec-5; and Siglec-9. In some embodiments, the splanchnic vasodilation can be associated with a condition selected from the group consisting of: hepatorenal syndrome (HRS) and sepsis. In some embodiments, the treatment for splanchnic vasodilation is selected from the group consisting of: liver transplantation; dialysis; transjugular intrahepatic portosystemic shunt (TIPS); hemodialysis; liver dialysis; intravenous albumin infusion; administration of splanchnic vasoconstrictors; albumin-bound membrane dialysis (e.g. molecular adsorbents recirculation system (MARS)); and administration of pentoxyfylline, acetylcysteine, and/or misoprostol.

[0011] In one aspect, described herein is a method of treatment for hepatorenal syndrome (HRS) comprising; determining the expression level of Siglec in a test sample obtained from a subject; wherein if the level of Siglec is increased relative to a reference level, the subject is treated with a liver transplant; and wherein if the level of Siglec is not increased relative to a reference level, the subject is treated with a liver and kidney transplant. In some embodiments, the Siglec can be selected from the group consisting of: Siglec-7; Siglec-5; and Siglec-9. In some embodiments, an increased level of Siglec can be a level at least 25% greater than a reference level. In some embodiments, the sample can comprise a material selected from the group consisting of: a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid; ascites; and urine. In some embodiments, the expression level of Siglec can be determined by measuring the level of Siglec RNA transcript. In some embodiments, the RNA transcript level can be measured using reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the expression level of Siglec can be determined by measuring the level of Siglec polypeptide. In some embodiments, the expression level of Siglec can be determined by measuring the level of soluble Siglec polypeptide. In some embodiments, the polypeptide level can be measured using immunochemistry. In some embodiments, the

immunochemical method can comprise: (a) contacting a biofluid test sample obtained from a subject with a detectable anti-Siglec antibody reagent; and (b) detecting the presence or intensity of a detectable signal; wherein the expression level of Siglec polypeptide is indicated by the level of the detectable signal. In some embodiments, the antibody reagent can be detectably labeled or generates a detectable signal. In some embodiments, the expression level of Siglec can be normalized relative to the expression level of one or more reference genes or reference proteins. In some embodiments, the reference expression level of Siglec can be the level of Siglec in a prior sample obtained from the subject. In some embodiments, the expression level of no more than 20 other genes is determined. In some embodiments, the expression level of no more than 10 other genes is determined. In some embodiments, the subject can be a human.

[0012] In one aspect, described herein is a computer system for determining the risk of a subject having or developing splanchnic vasodilation, the system comprising: a measuring module configured to measure the expression level of Siglec in a test sample obtained from a subject; a storage module configured to store output data from the determination module; a comparison module adapted to compare the data stored on the storage module with a reference level, and to provide a retrieved content, and a display module for displaying whether the sample comprises a level of Siglec which is significantly increased relative to the reference expression level and/or displaying the relative expression level of Siglec. In some embodiments, the Siglec can be selected from the group consisting of: Siglec-7; Siglec-5; and Siglec-9. In some embodiments, the measuring module can measure the intensity of a detectable signal from an assay indicating the expression level of Siglec polypeptide in the test sample. In some embodiments, the assay can be an immunoassay. In some embodiments, the measuring module can measure the intensity of a detectable signal from a RT-PCR assay indicating the expression level of Siglec RNA transcript in the test sample. In some embodiments, if the computing module determines that the expression level of Siglec in the test sample obtained from a subject is greater by a statistically significant amount than the reference expression level, the display module can display a signal indicating that the expression levels in the sample obtained from a subject are greater than those of the reference expression level. In some embodiments, the signal can indicate that the subject has an increased likelihood of having or developing splanchnic vasodilation. In some embodiments, the signal can indicate the subject is in need of treatment for splanchnic vasodilation. In some embodiments, the signal can indicate the degree to which the expression level of Siglec in the sample obtained from a subject varies from the reference expression level. In some embodiments, the splanchnic vasodilation can be associated with a condition is selected from the group consisting of: hepatorenal syndrome (HRS) and sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 provides a table of study subject characteristics.

[0014] Figure 2 depicts a table of soluble Siglec-7 concentrations in samples obtained from the indicated subject groups.

[0015] Figure 3 depicts a table of the percent of vascular relaxation observed in response to Siglec7 administration. N=7.

[0016] Figure 4 depicts a graph of the percent of vascular relaxation observed in response to endotoxin administration.

[0017] Figure 5 depicts a table of the percent of vascular relaxation observed in response to combined endotoxin and Siglec7 administration.

[0018] Figure 6 depicts a table of endotoxin concentrations in samples obtained from the indicated subject groups.

[0019] Figure 7 depicts a table of Nitrate/Nitrite concentrations in HuVEC cells after the indicated treatments.

[0020] Figure 8 depicts a graph of Nitrate/Nitrite concentrations in HuVEC cells after the indicated treatments.

[0021] Figure 9 depicts a graph of Nitrate/Nitrite concentrations in rat SMC cells after the indicated treatments.

[0022] Figure 10 is a diagram of an exemplary embodiment of a system for performing an assay for determining the level of Siglec (e.g. Siglec 7) in sample obtained from a subject.

[0023] Figure 11 is a diagram of an exemplary embodiment of a comparison module as described herein.

[0024] Figure 12 is a diagram of an exemplary embodiment of an operating system and instructions for a computing system as described herein.

DETAILED DESCRIPTION

[0025] Embodiments of the technology described herein relate to methods, assays, and systems of treating, diagnosing, and preventing, e.g. splanchnic vasodilation and/or hepatorenal syndrome (HRS). The technologies described herein are based upon the inventors' discovery that subjects with HRS exhibit increased levels of soluble Siglec, relative to healthy individuals, which levels can be detected in various test samples obtained from a subject, e.g. urine or blood samples. Further, the inventors have discovered that these increased levels of soluble Siglec contribute to vasodilation. Thus, Siglec is both a marker and a pathological factor in, e.g. splanchnic vasodilation and/or hepatorenal syndrome (HRS). In one aspect, described herein are assays, methods, and systems relating to the inventors' discovery that when a subject has or is at risk of having splanchnic vasodilation and/or hepatorenal syndrome (HRS), the subject will have increased levels of soluble Siglec as compared to a subject not experiencing splanchnic vasodilation and/or hepatorenal syndrome (HRS). The soluble Siglec is present in a number of biofluids, e.g. blood, urine, and/or ascities. In some embodiments, the Siglec one or more members of the Siglec family. In some embodiments, the Siglec can be a single Siglec, e.g. Siglec-7, Siglec-5, or Siglec-9.

[0026] As used herein, "splanchnic vasodilation" refers to a widening of the vasculature supplying the intestines, e.g. by relaxation of the smooth muscle cells lining the vasculature.

Splanchnic vasodilation can occur, e.g. in subjects with hepatorenal syndrome (HRS) and/or sepsis. In various embodiments of the aspects described herein, splanchnic vasodilation can be splanchinic vasodilation associated with and/or arising from either HRS and/or sepsis. In various embodiments of the aspects described herein, splanchnic vasodilation can be splanchinic vasodilation not associated with and/or arising from either HRS and/or sepsis. As used herein, "hepatorenal syndrome" (HRS) refers to a condition characterized by deterioration of kidney function in subjects with liver diseases (e.g. cirrhosis, alcoholic hepatitis or fulminant liver failure). HRS typically occurs when the liver suffers an acute injury, e.g. infection, bleeding in the gastrointestinal tract, and/or overuse of diuretic medications. Hepatorenal syndrome is characterized by congestion and blockage of intrahepatic microvasculature, causing portal hypertension. This in turn leads to splanchnic nitric oxide production and resultant splanchnic vasodilation. The vasodilation causes decreased effective blood volume which stimulates the renin-angiotensin system, intrarenal vasoconstriction, hypoperfusion, and the production of intrarenal prostaglandins to counteract the vasoconstriction. If the

prostaglandin production is insufficient (or is inhibited, e.g. by local ischemia and/or medications such as nonsteroidal anti-inflammatories), the kidneys will not be functional. A decrease in kidney function can be quantified, e.g. by an elevation of serum creatinine and/or decreased clearance of creatinine in the urine. Factors tending towards a diagnosis of HRS include the following: chronic or acute liver disease with advanced hepatic failure and portal hypertension; serum creatinine greater than 1.5 mg/dL; absence of shock, ongoing bacterial infection or gastrointestinal and renal fluid losses; no current or recent treatment with nephrotoxic agents; no improvement in serum creatinine after withdrawal of diuretics and fluid resuscitation; proteinuria less than 500 mg/d; and/or no ultrasonographic evidence of chronic renal disease or obstructive uropathy. Although subjects with HRS will symptomatically experience kidney failure, the kidneys are not actually damaged and can function normally in the presence of a healthy environment (e.g. if transplanted to a person with a healthy liver). Notably, the known factors associated with HRS do not provide a definitive diagnosis. As decisions concerning transplantation are influenced by a clinician's determination of whether a subject has or does not have HRS, a need is felt for methods providing a more certain diagnosis.

[0027] Two forms of HRS are currently recognized. Type I HRS involves a rapid and progressive decline in kidney function. Type I HRS subject can be treated with, e.g. inotropes or vasopressors to maintain blood pressure and heart function. Type II is characterized by ascities (e.g. fluid accumulation in the abdomen) that is resistant to treatment with standard diuretic medications. As used herein, the term HRS can refer to either or both of Type I and Type II HRS.

[0028] Treatments for HRS include liver transplantation, dialysis, insertion of a transjugular intrahepatic portosystemic shunt (TIPS) (to reduce blood pressure in the portal vein), hemodialysis, liver dialysis, intravenous albumin infusion, splanchnic vasoconstrictors (e.g. vasopressin, midodrine, somatostatin, ornipressin, terlipressin,), albumin-bound membrane dialysis (e.g. molecular adsorbents recirculation system (MARS)), pentoxyfylline, acetylcysteine, and misoprostol.

[0029] In some embodiments, the methods and assays described herein relate to diagnosis and/or treatment of cirrhosis, e.g. severe cirrhosis. As used herein, "cirrhosis" refers to replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function. Methods of grading the severity of cirrhosis are known in the art, e.g. the Child Pugh score or MELD score (see, e.g., Kim and Lee. CMH 2013 19:105-1 15; Cholongitas et al. Alimentary Pharmacology and Therapeutics 2006 24:453-465; and Francoz et al. Liver Transplantation 201 1 17: 1137-1 151 ; each of which is incorporated by reference herein in its entirety).

[0030] "Severe cirrhosis" refers to advanced cases of cirrhosis, e.g., those with short estimated lifespans and increased occurrence or risk of occurrence of complications and associated conditions (e.g. HCC). In some embodiments, severe cirrhosis can be a case of cirrhosis with a MELD score of 8 or greater. In some embodiments, severe cirrhosis can be a case of cirrhosis with a MELD score of 10 or greater. In some embodiments, severe cirrhosis can be a case of cirrhosis with a MELD score of 15 or greater. In some embodiments, severe cirrhosis can be a case of cirrhosis with a MELD score of 20 or greater.

[0031] As described herein, the inventors have demonstrated that subjects having splanchnic vasodilation and/or hepatorenal syndrome (HRS) have increased levels of Siglec, e.g. soluble Siglec. As used herein, "sialic acid-binding Ig-like lectin " or "Siglec," refers to a family of transmembrane proteins which can detect, e.g. cell-cell interaction and/or cell-pathogen interactions. The N-terminal V-like immunoglobin domain (IgV) of Siglecs binds to sialic acid. Siglecs comprise a variable number of C2-type Ig (IgC2 domains). As used herein, "Siglec" can refer to one or more members of the Siglec family, e.g. Siglec-7, Siglec-5, Siglec-9, Siglec-1, Siglec -2, Siglec-3, Siglec -4, Siglec-6, Siglec-8, Siglec-10, Siglec-11, and/or Siglec-12 and combinations thereof. In some embodiments, "Siglec" can refer to one or more of the CD33-related Siglecs, i.e. one or more of Siglec-7, Siglec-5, Siglec-9, Siglec-6, Siglec-8, Siglec-10, Siglec-11, and/or Siglec-12 and combinations thereof, which comprise two immunoreceptor tyrosine-based inhibitory motifs (ITIM)-like motifs in the cytoplasmic domain. In some embodiments, "Siglec" can refer to one or more of Siglec-7, Siglec-5, and/or Siglec- 9 and combinations thereof. In the various aspects described herein, it is contemplated that in some embodiments, detection, determination, and/or inhibition of Siglec can encompass methods which detect, determine and/or inhibit one or more Siglecs, e.g. one or more of Siglec-7, Siglec-5, and/or Siglec-9. Alternatively, in some embodiments, detection, determination, and/or inhibition of Siglec can encompass methods which are specific for a single Siglec family member, e.g. specific for Siglec- 7, specific for Siglec-5, or specific for Siglec-9. For further discussion of Siglecs, see, e.g. Crocker and Varki. Immunology 2001 103: 137-45; which is incorporated by reference herein in its entirety.

[0032] As used herein, "sialic acid-binding Ig-like lectin 7" or "Siglec7", refers to a

transmembrane protein of the Siglec family which is expressed in monocytes, macrophages, cDCs, NK cells, lung, liver, spleen, and peripheral blood leukocytes (also known as CD328). The sequence of Siglec7 for a number of species is well known in the art, e.g. human Siglec7 (e.g. SEQ ID NO: 1, NCBI Ref Seq: NP 055200; NCBI Gene ID: 27036). Siglec7 ligands include, but are not limited to ganglioside DSGb5; a2,6-linked disialic gangliosides; disialosyl Lc₄ (DSLc4); a2, 8-linked gangliosides; GD2; GD3; GTlb; and sialyl-6-sulfo lewisx. As used herein, "soluble Siglec7" refers to a non-membrane bound polypeptide comprising a portion of the extracellular domain of Siglec7, e.g. residues 19-353 of SEQ ID NO: l . As used in this context, a "portion" refers to at least 20 amino acids of the extracellular domain of Siglec7, e.g. 20 or more amino acids, 40 or more amino acids, 60 or more amino acids, 80 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, or as many as 334 amino acids of the extracellular domain. Additionally, a portion of the extracellular domain of Siglec7 can be a portion of the extracellular domain of Siglec7 which is at least 20kDa in size, e.g. at least 20 kDa in size, at least 25 kDa in size, at least 30 kDa in size or larger. In some embodiments, a portion of the extracellular domain of Siglec7 can be about 30 kDa in size. Additionally, a portion of the extracellular domain of Siglec7 can be a portion of the extracellular domain of Siglec7 which can be specifically bound by an antibody reagent specific for Siglec7, e.g. for soluble Siglec7.

[0033] As used herein, "sialic acid-binding Ig-like lectin 5" or "Siglec5", refers to a

transmembrane protein of the Siglec family which is expressed in bone marrow, spleen, neutrophils and peripheral blood leukocytes (also known as CD 170). The sequence of Siglec5 for a number of species is well known in the art, e.g. human Siglec5 (e.g. SEQ ID NO: 4, NCBI Ref Seq: NP 003821 ; NCBI Gene ID: 8778). Siglec5 ligands include, but are not limited to Factor VIII (FVIII), von Willebrand factor (VWF), N-acetylneuraminic acid, N-glycolylneuraminic acid, a2,3-linked sialic acid and a2,6-linked sialic acid. As used herein, "soluble Siglec5" refers to a non-membrane bound polypeptide comprising a portion of the extracellular domain of Siglec5, e.g. residues 17-434 of SEQ ID NO:4. As used in this context, a "portion" refers to at least 20 amino acids of the extracellular domain of Siglec5, e.g. 20 or more amino acids, 40 or more amino acids, 60 or more amino acids, 80 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, or as many as 417 amino acids of the extracellular domain. Additionally, a portion of the extracellular domain of Siglec5 can be a portion of the extracellular domain of Siglec5 which can be specifically bound by an antibody reagent specific for Siglec5, e.g. for soluble Siglec5.

[0034] As used herein, "sialic acid-binding Ig-like lectin 9" or "Siglec9", refers to a

transmembrane protein of the Siglec family which is expressed in monocytes, neutrophils, and subpopulations of lymphocytes, but not in eosinophils (also known as CD329). The sequence of Siglec9 for a number of species is well known in the art, e.g. human Siglec9 (e.g. SEQ ID NO: 5, NCBI Ref Seq: NP 001185487; and SEQ ID NO: 6, NCBI Ref Seq: NP 055256 NCBI Gene ID: 27180). Siglec9 ligands include, but are not limited to sialic acid in either the a2,3- or a2,6- glycosidic linkage to galactose, MUC16, vascular adhesion protein 1 (VAP-1). As used herein, "soluble Siglec9" refers to a non-membrane bound polypeptide comprising a portion of the extracellular domain of Siglec9, e.g. residues 18-348 of SEQ ID NO:6. As used in this context, a "portion" refers to at least 20 amino acids of the extracellular domain of Siglec9, e.g. 20 or more amino acids, 40 or more amino acids, 60 or more amino acids, 80 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, or as many as 330 amino acids of the extracellular domain. Additionally, a portion of the extracellular domain of Siglec9 can be a portion of the extracellular domain of Siglec9 which can be specifically bound by an antibody reagent specific for Siglec9, e.g. for soluble Siglec9.

[0035] As demonstrated herein, soluble forms of Siglec (e.g. Siglec-7) are detectable in biofluids, e.g. blood and/or urine. Soluble Siglec forms may be generated by enzymatic cleavage and/or translation of splice variants. For further discussion of Siglec function and/or structure, see, e.g. Nicoll et al. JBC 1999 274:34089-034095 and Pialli et al. Ann Rev Immuno 2012 30:357-392; each of which is incorporated by reference herein in its entirety). In some embodiments, soluble Siglec can be detected by using a reagent which specifically binds soluble Siglec, e.g. an antibody reagent specific for an epitope of soluble Siglec that is not accessible and/or present on transmembrane versions of Siglec. In some embodiments, soluble Siglec can be detected by using a reagent which is specific for at least part of the extracellular domain of Siglec to determine the presence or absence of soluble Siglec in a cell-free sample, e.g. urine or a substantially cell-free portion of a blood sample.

[0036] As used herein, the activity of Siglec can be the ability of Siglec to bind a known ligand (examples of which are described elsewhere herein); and/or the ability of ectopic and/or elevated levels of Siglec to induce splanchnic vasodilation; and/or the ability of Siglec to inhibit NK cell cytotoxicity upon ligand binding; and/or the ability of Siglec to induce dephosphorylation of downstream targets upon ligand binding. By way of non-limiting example, Siglec7 activity can be measured by contacting, e.g. HUVEC or SMC cells with soluble Siglec7. Siglec7 activity can be measured as an increase in nitrate+nitrite concentration. In some embodiments, the activity of Siglec can be determined using an ELISA assay. Non- limiting examples of ELISA assays are described, e.g. in Levine et al. NEJM 2006 355:992-1005; which is incorporated by reference herein in its entirety. [0037] Elevated levels of Siglec, including elevated levels of soluble Siglec, are associated with splanchnic vasodilation and/or hepatorenal syndrome (HRS) as demonstrated herein. Accordingly, provided herein are methods of treating splanchnic vasodilation and/or hepatorenal syndrome (HRS). In one aspect, described herein is a method of treating splanchnic vasodilation and/or hepatorenal syndrome (HRS), the method comprising administering a therapeutically effective amount of a Siglec inhibitor to a subject in need of treatment. In some embodiments, elevated levels of Siglec, including elevated levels of soluble Siglec, are associated with complications of liver disease, e.g. HRS and/or cirrhosis as demonstrated herein.

[0038] As used herein, the term "inhibitor of Siglec" refers to an agent that can decrease the expression level and/or activity of at least one Siglec, e.g. by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%), at least 99% or more. In some embodiments, a Siglec inhibitor can decrease the level of siglec rriRNA, the level of Siglec polypeptide, and/or the level of soluble Siglec polypeptide. In some embodiments, a Siglec inhibitor can specifically bind an expression product of Siglec. In some embodiments, a Siglec inhibitor can specifically bind a Siglec polypeptide. In some embodiments, a Siglec inhibitor can specifically bind soluble Siglec. In some embodiments, a Siglec inhibitor can reduce release of soluble Siglec. In some embodiments, a Siglec inhibitor can reduce vasodilation, e.g. splanchnic vasodilation. In some embodiments, inhibitors of Siglec7 can bind to (or physically interact with) a polypeptide comprising residues 26-144 of SEQ ID NO: 1. In some embodiments, inhibitors of Siglec9 can bind to (or physically interact with) a polypeptide comprising residues 21- 140 of SEQ ID NO: 4. In some embodiments, inhibitors of Siglec9 can bind to (or physically interact with) a polypeptide comprising residues 23-140 of SEQ ID NO: 6. In this context, physical interaction can encompass steric hindrance of the interaction of one or more ligands of Siglec. In some embodiments, the Siglec inhibitor can specifically bind, e.g. Siglec7, Siglec5, and/or Siglec9 (e.g. soluble Siglec7, Siglec5, or Siglec9). In some embodiments, the Siglec inhibitor can be an agent which mimics a ligand of Siglec, e.g. a sialic acid-based inhibitor (see, e.g. Gutegemann, S.A.

"Molecular basis for the lipid raft recruitment of NK cell receptors and development of a sialic acid- based Siglec-7 inhibitor" Dissertation at Medizinische Fakultat Mannheim, 2012; which is incorporated by reference herein in its entirety).

[0039] Due to the upregulation of Siglec (e.g. Siglec7) in subjects in need of treatment for, e.g. splanchnic vasodilation and/or HRS, as described herein, the level of Siglec can be used in methods and assays relating to the prognosis, diagnosis, and/or detection of splanchnic vasodilation and/or HRS. In some embodiments, the level of Siglec can be the RNA transcript level of Siglec. In some embodiments, the level of Siglec can be the level of Siglec polypeptide. In some embodiments, the level of Siglec can be the level of soluble Siglec polypeptide. [0040] Accordingly, described herein is an assay comprising: determining the expression level of Siglec in a test sample obtained from a subject; wherein an increase in the Siglec expression level relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation and/or HRS. In some embodiments, described herein is a method of identifying a subject in need of treatment for splanchnic vasodilation, the method comprising: determining the expression level of Siglec in a test sample obtained from a subject; wherein the subject is identified as being in need of treatment for splanchnic vasodilation if the expression level of Siglec is increased relative to a reference level. In some embodiments, described herein is an assay comprising contacting a biofluid test sample obtained from a subject with a detectable anti-Siglec antibody reagent; and detecting the presence or intensity of a detectable signal; wherein an increase in the level of Siglec polypeptide, indicated by the level of the detectable signal, relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation and/or HRS.

[0041] In some embodiments, the expression level of Siglec can be measured by determining the level of an expression product of the Siglec gene, e.g. a Siglec7 RNA transcript or a Siglec7 polypeptide. Such molecules can be isolated, derived, or amplified from a biological sample, such as a biofluid.

[0042] In some embodiments, the expression product can be a Siglec niRNA transcript, e.g. when the sample comprises tissue samples, e.g. liver tissue. Assays for detecting mRNA transcripts are well known in the art and include, but are not limited to, PCR procedures, RT-PCR, Northern blot analysis, RNAse protection assay, microarray analysis, hybridization methods etc. In some embodiments, mRNA transcript expression product levels are assayed using reverse transcription polymerase chain reaction (RT-PCR).

[0043] Nucleic acid sequences encoding Siglec polypeptides have been assigned NCBI accession numbers for different species such as human, mouse and rat. In particular, the NCBI accession numbers for the nucleic acid sequences of the human Siglec7 expression products are known (e.g. SEQ ID NOs: 2 (NCBI Ref Seq: NM 014385) and 3 (NCBI Ref Seq: NM 016543) for Siglec7; SEQ ID NO: 7 (NCBI Ref Seq: NM 003830) for Siglec5); and SEQ ID NOs: 8 (NCBI Ref Seq:

NM 001198558) and 9 (NCBI Ref Seq: NM 014441) for Siglec9). Accordingly, a skilled artisan can design appropriate primers based on the known sequence for determining the mRNA level of the respective genes.

[0044] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)). [0045] In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size or for hybridization to a given probe. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.

[0046] In some embodiments, the level of Siglec can be the level of Siglec polypeptide and/or soluble Siglec polypeptide. In some embodiments, the Siglec polypeptide can comprise soluble Siglec polypeptide. In some embodiments, described herein is a method of detecting soluble Siglec-, the method comprising contacting a sample with a reagent specific for a Siglec polypeptide, wherein reaction of soluble Siglec- with said reagent indicates the presence of soluble Siglec-.

[0047] Detection of Siglec polypeptides can be according to any method known in the art. Immunological methods to detect Siglec polypeptides in accordance with the present technology include, but are not limited to antibody techniques such as immunohistochemistry,

immunocytochemistry, flow cytometry, fluorescence-activated cell sorting (FACS), immunoblotting, radioimmunoassays, western blotting, immunoprecipitation, enzyme-linked immunosorbant assays (ELISA), and derivative techniques that make use of antibody reagents as described herein.

[0048] Immunochemical methods require the use of an antibody reagent specific for the target molecule (e.g. the antigen or in the embodiments described herein, a Siglec polypeptide or fragment and/or variant thereof (e.g. Siglec7, Siglec5, Siglec9, soluble Siglec7, etc.)). In some embodiments, an antibody reagent for measuring the level of Siglec7 in a sample can be an antibody reagent specific for the extracellular domain of Siglec7, (e.g. specific for a polypeptide comprising amino acids 1 to about 19-353 of SEQ ID NO:l or a fragment thereof). In some embodiments, an antibody reagent for measuring the level of Siglec7 in a sample can be an antibody reagent specific for only the soluble form of Siglec7 (e.g. specific for a polypeptide comprising amino acids 19 to about 353 of SEQ ID NO: l or a fragment thereof and not comprising amino acids 354-376 of SEQ ID NO:l). In some embodiments, the antibody reagent can be specific for the Siglec7 and/or soluble Siglec7 in its native conformation. Antibody reagents specific for Siglecs are commercially available, e.g. Cat. Nos. AF1138 and MAB1138 for Siglec7, Cat. No. MAB1139 for Siglec9, and Cat. No. for Siglec5 MAB1072 (R&D Systems, MN).

[0049] In some embodiments, the assays, methods, and/or systems described herein can comprise: an anti-Siglec antibody reagent, e.g. an antibody reagent specific for Siglec7, Siglec5, or Siglec9 or specific for a fragment or modified version of Siglec7, Siglec5, or Siglec9 (e.g. soluble Siglec7). In some embodiments, the antibody reagent can be detectably labeled. In some

embodiments, the antibody reagent can be attached to a solid support (e.g. bound to a solid support). In some embodiments, the solid support can comprise a particle (including, but not limited to an agarose or latex bead or particle or a magnetic particle), a bead, a nanoparticle, a polymer, a substrate, a slide, a coverslip, a plate, a dish, a well, a membrane, and/or a grating. The solid support can include many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon or silica based materials, carbon, metals, inorganic glasses, and membranes.

[0050] In one embodiment, an assay, method, and/or system as described herein can comprise an ELISA. In an exemplary embodiment, a first antibody reagent can be immobilized on a solid support (usually a polystyrene micro titer plate). The solid support can be contacted with a sample obtained from a subject, and the antibody reagent will bind ("capture") antigens for which it is specific (e.g. Siglec7). The solid support can then be contacted with a second labeled antibody reagent (e.g. a detection antibody reagent). The detection antibody reagent can, e.g. comprise a detectable signal, be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. The presence of a signal indicates that both the first antibody reagent immobilized on the support and the second "detection" antibody reagent have bound to an antigen, i.e. the presence of a signal indicated the presence of a Siglec molecule. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of Siglec polypeptides in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A. Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.

[0051] In some embodiments, described herein is an assay to detect the amount of Siglec expression in a sample obtained from a subject, the assay comprising: (a) contacting a sample obtained from the subject with an anti-Siglec antibody reagent; (b) measuring the amount of the signal from the portion of antibody reagent bound to the sample, wherein the detection of signal from antibody reagent bound to the sample indicates the presence of Siglec; (c) comparing the amount of signal and/or expression level with a reference level, and wherein if the expression level of Siglec is increased (e.g. increased by at least 1.25-fold compared to the reference level) the subject is identified as having, at risk of having, or being in need of treatment for splanchnic vasodilation and/or HRS. [0052] In one embodiment, the assays, systems, and methods described herein can comprise a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test to measure or determine the level of Siglec polypeptide in a sample. LFIAs are a simple device intended to detect the presence (or absence) of Siglec in a sample. There are currently many LFIA tests used for medical diagnostics either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored antibody reagent which mixes with the sample, and if bound to a portion of the sample, transits the substrate encountering lines or zones which have been pretreated with a second antibody reagent. Depending upon the level of Siglec present in the sample the colored antibody reagent can become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water samples etc. Strip tests are also known as dip stick test, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip test are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be used on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibody reagents specific for a target (e.g. a Siglec7 specific antibody reagent). The test line will also contain antibody reagents (e.g. a Siglec7 specific antibody reagent). The test line will show as a colored band in positive samples. In some embodiments, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

[0053] A typical test strip consists of the following components: (1) sample application area comprising an absorbent pad (i. e. the matrix or material) onto which the test sample is applied; (2) conjugate or reagent pad- this contains antibody reagent(s) specific to the target which can be conjugated to colored particles (usually colloidal gold particles, or latex microspheres); (3) test results area comprising a reaction membrane - typically a hydrophobic nitrocellulose or cellulose acetate membrane onto which antibody reagents are immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing antibodies specific for the antibody reagents conjugated to the particles or microspheres); and (4) optional wick or waste reservoir - a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it. The components of the strip are usually fixed to an inert backing material and may be presented in a simple dipstick format or within a plastic casing with a sample port and reaction window showing the capture and control zones. While not strictly necessary, most tests will incorporate a second line which contains an antibody that picks up free latex/gold in order to confirm the test has operated correctly.

[0054] The use of "dip sticks" or LFIA test strips and other solid supports has been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos.

4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871 ; 6,565,808, U. S. patent applications Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non- limiting examples of such lateral flow test devices. Three U.S. patents (U.S. Pat. No. 4,444,880, issued to H. Tom; U.S. Pat. No. 4,305,924, issued to R. N. Piasio; and U.S. Pat. No. 4,135,884, issued to J. T. Shen) describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teaching of these "dip stick" technologies as necessary for the detection of Siglec polypeptides. In some embodiments, the dip stick (or LFIA) can be suitable for use with urine samples. In some embodiments, a dip stick can be suitable for use with blood samples.

[0055] Immunochemistry is a family of techniques based on the use of a specific antibody, wherein antibodies are used to specifically target molecules inside or on the surface of cells. In some embodiments, immunohistochemistry ("IHC") and immunocytochemistry ("ICC") techniques can be used to detect or measure the levels of Siglec polypeptide. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. In some instances, signal amplification may be integrated into the particular protocol, wherein a secondary antibody, that includes a label, follows the application of an antibody reagent specific for platelets or leukocytes. Typically, for immunohistochemistry, tissue obtained from a subject and fixed by a suitable fixing agent such as alcohol, acetone, and paraformaldehyde, is sectioned and reacted with an antibody. Conventional methods for immunohistochemistry are described in Buchwalow and Bocker (Eds) "Immunohistochemistry: Basics and Methods" Springer (2010): Lin and Prichard "Handbook of Practical Immunohistochemistry" Springer (2011); which are incorporated by reference herein in their entireties. In some embodiments, immunocytochemistry may be utilized where, in general, tissue or cells obtained from a subject are fixed by a suitable fixing agent such as alcohol, acetone, and paraformaldehyde, to which is reacted an antibody. Methods of immunocytological staining of human samples is known to those of skill in the art and described, for example, in Burry "Immunocytochemistry: A Practical Guide for Biomedical Research" Springer (2009); which is incorporated by reference herein in its entirety. [0056] In some embodiments, one or more of the antibody reagents described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing a reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into an antibody reagent are well known in the art.

[0057] In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the antibody reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the antibody reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0058] In other embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o- phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7- hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g.

Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc;

BODIPY dyes and quinoline dyes. [0059] In some embodiments, a detectable label can be a radiolabel including, but not limited to

³H, ¹²⁵1, ³⁵S, ¹⁴C, ³²P, and ³³P.

[0060] In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0061] In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0062] In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0063] In some embodiments, antibodies can also be labeled with a detectable tag, such as c- Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA.

[0064] An antibody reagent can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or

ethylenediaminetetraacetic acid (EDTA).

[0065] The assays and methods as described herein can relate to determining if a subject has an increased level of Siglec relative to a reference level. In some embodiments, the reference level of Siglec can be the level of Siglec in a healthy subject not having, or not diagnosed as having, e.g., splanchnic vasodilation and/or HRS. In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level of Siglec is to be determined. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells and/or type of sample material. Accordingly, in some embodiments, the level of Siglec which is increased can vary as demographic factors such as age, gender, genotype, environmental factors, and individual medical histories vary. In some embodiments, the reference level can comprise the level of Siglec (e.g. RNA transcript or polypeptide) in a sample of the same type taken from a subject not exhibiting any signs or symptoms of, e.g. splanchnic vasodilation and/or HRS. In some embodiments, the reference level can comprise the level of Siglec (e.g. RNA transcript or polypeptide) in a sample of the same type taken from a subject not exhibiting any signs or symptoms of, e.g. splanchnic vasodilation and/or HRS, but who is exhibiting signs or symptoms of liver failure, e.g. cirrhosis. In some embodiments, the reference expression level of Siglec can be the expression level of Siglec in a prior sample obtained from the subject. This permits a direct analysis of any change in levels in that individual.

[0066] In some embodiments, a level of Siglec can be increased relative to a reference level if the level of Siglec is at least 1.25x the reference level, e.g. at least 1.25x, at least 1.5x, at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, or greater of the reference level. In some embodiments, the expression level of Siglec can be normalized relative to the expression level of one or more reference genes or reference proteins. In some embodiments, the expression level of Siglec can be normalized relative to a reference value.

[0067] In some embodiments, an increased level of Siglec7 can be a level of Siglec7 polypeptide greater than about 2 ng/mL as measured using the Human Siglec-7/CD328 DUOSET™ ELISA kit available from R&D Systems (Cat No. DY1138).

[0068] In some embodiments, the expression level of no more than 20 other genes is determined. In some embodiments, the expression level of no more than 10 other genes is determined.

[0069] The term "sample" or "test sample" as used herein denotes a sample taken or isolated from an organism, e.g., a urine sample from a subject. Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; ascites; ascites fluid; abdominal fluid; and/or peritoneal fluid; etc. The term also includes a mixture of the above-mentioned samples. The term "test sample" also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample can comprise cells from a subject. In some embodiments, a subject with HRS may not produce sufficient quantities of urine for a clinical test. Accordingly, in some embodiments, the sample can comprise blood, serum, plasma, etc.

[0070] In some embodiments, the sample can comprise any tissue affected by symptoms of, or displaying markers of splanchnic vasodilation and/or HRS, e.g. the sample can comprise ascites. In some embodiments, the test sample can comprise or consist of urine. In some embodiments, the test sample can comprise or consist of blood and/or blood products, e.g. serum and/or plasma. In some embodiments, the sample can comprise a biofluid. As used herein, the term "biofluid" refers to any fluid obtained from a biological source and includes, but is not limited to, blood, urine, cystic fluids, and bodily secretions. [0071] The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected sample. In some embodiments, the sample can be acellular, e.g. urine. In some

embodiments, the cells present in a sample can be removed before Siglec levels in the sample are detected, e.g. at least 90% of the cells originally in the sample can be removed, e.g by centrifugation.

[0072] In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase "untreated test sample" refers to a test sample that has not had any prior sample pre- treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, prepared by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for preprocessing of biological samples required for determination of the level of Siglec as described herein.

[0073] In some embodiments, the methods, assays, and systems described herein can further comprise a step of obtaining a test sample from a subject. In some embodiments, the subject can be a human subject.

[0074] In some embodiments, the methods, assays, and systems described herein can comprise creating a report based on the level of Siglec. In some embodiments, the report denotes raw values for Siglec in the test sample (plus, optionally, the level of Siglec in a reference sample) or it indicates a percentage or fold increase in Siglec as compared to a reference level, and/or provides a signal that the subject is at risk of having, or not having splanchnic vasodilation and/or HRS.

[0075] As used herein "at risk of having" refers to at least a 2-fold greater likelihood of having a particular condition as compared to a subject that did not have an elevated and/or increased level of Siglec, e.g. a 2-fold, or 2.5-fold, or 3-fold, or 4-fold, or greater risk. [0076] In one aspect, described herein is a method of administering a treatment for splanchnic vasodilation and/or HRS to a subject, the method comprising: determining the level of Siglec expression product in a test sample obtained from a subject; and administering a treatment for splanchnic vasodilation and/or HRS to the subject if the level of Siglec is increased relative to a reference level. In one aspect, described herein is a method of administering a treatment for splanchnic vasodilation and/or HRS to a subject, the method comprising administering a treatment for splanchnic vasodilation and/or HRS to a subject determined to have an increased level of Siglec in a test sample obtained from the subject; wherein the level of Siglec is an increased level if it is increased relative to a reference level. In some embodiments, the level of Siglec can be the level of Siglec polypeptide. In some embodiments, the level of Siglec can be the level of soluble Siglec polypeptide.

[0077] The methods, assays, and systems described herein can relate to methods of treatment, methods of determining if a subject can benefit from certain therapies, and/or methods of determining if a subject should receive a kidney transplant. It is clinically important to differentiate subjects with HRS from those with other forms of kidney failure due to the fact that subjects with HRS do not typically have damage to the kidneys. Once proper blood flow and vessel tone is restored (e.g. after a successful liver transplant), the kidneys of a subject with HRS are likely to resume normal function. Therefore, a subject with HRS is not typically in need of a kidney transplant. In one aspect, described herein is a method of treatment for hepatorenal syndrome (HRS) comprising: determining the expression level of Siglec in a test sample obtained from a subject; wherein if the level of Siglec is increased relative to a reference level, the subject is treated with a liver transplant; and wherein if the level of Siglec is not increased relative to a reference level, the subject is treated with a liver and kidney transplant.

[0078] In one aspect, described herein is a method of determining the efficacy of a treatment for splanchnic vasodilation, the method comprising: (a) determining the expression level of Siglec in a test sample obtained from a subject before administration of the treatment; (b) determining the expression level of Siglec in a test sample obtained from a subject after administration of the treatment; wherein the treatment is not efficacious if the expression level determined in step (b) is increased relative to the expression level determined in step (a). In some embodiments, the step of administering a treatment for splanchnic vasodilation follows step (a) and preceeds step (b).

[0079] Treatments for splanchnic vasodilation and/or HRS are known in the art and include, but are not limited to liver transplantation, dialysis, insertion of a transjugular intrahepatic portosystemic shunt (TIPS) (to reduce blood pressure in the portal vein), hemodialysis, liver dialysis, intravenous albumin infusion, splanchnic vasoconstrictors (e.g. vasopressin, midodrine, somatostatin, ornipressin, terlipressin,), albumin-bound membrane dialysis (e.g. molecular adsorbents recirculation system (MARS)), pentoxyfylline, acetylcysteine, and misoprostol. [0080] In one aspect, described herein is a method of reducing splanchnic vasodilation, the method comprising; administering a therapeutically effective amount of a Siglec inhibitor to a subject in need of treatment. In some embodiments, a Siglec inhibitor can be administered to a subject by administering a pharmaceutical composition comprising a Siglec inhibitor, e.g. injecting an antibody reagent specific for Siglec intravenously. In some embodiments, a Siglec7 inhibitor can be administered to a subject extracorporeally, e.g. by removing a portion of a biofluid from a subject, removing and/or reducing the level of Siglec expression products in the biofluid, and returning all or a portion of the removed biofluid to the subject.

[0081] In some embodiments, described herein is a method of reducing splanchnic vasodilation, the method comprising administering an extracorporeal treatment which comprises removing (and/or reducing the amount or concentration of) Siglec expression products in a biofluid obtained from a subject and returning at least a portion of the biofluid to the subject. In some embodiments, the biofluid can be blood. Extracorporeal treatment can comprise, e.g. affinity columns; non-specific columns; antibody columns; specific columns; and/or apheresis. In some embodiments, removal of Siglec can be specific, e.g. a Siglec7 inhibitor which binds specifically to Siglec7 can be used to remove Siglec7. In some embodiments, removal of Siglec can be non-specific, e.g. soluble proteins can be removed from the biofluid. In some embodime ts, extracorporeal treatment can comprise an ex vivo or in vitro method. In some embodiments, the method further comprises, prior to the step of removing Siglec expression products, collecting a biofluid, e.g. peripheral blood, from the subject. In some embodiments, the biofluid can be continuously collected from the patient. In some

embodiments, the biofluid can be continuously returned to the subject, e.g. by use of a pump. In some embodiments, the extracorporeal treatment can additionally comprise dialysis. In some embodiments, the administration comprises extracorporeal apheresis.

[0082] The term "apheresis" as used herein refers to the process or procedure in which fluid, e.g. blood, is drawn from a donor subject and separated into two or more components, some of which are retained, and the remainder returned to the same general fluid compartment of the subject from which the fluid was removed. This process can also be referred to in the art as hemapheresis or pheresis. In some embodiments, the fluid can be separated into a component comprising Siglec polypeptide, e.g. soluble Siglec polypeptide, and a component not comprising Sigle7 polypeptide. In some embodiments, the fluid can be separated into a component comprising Siglec polypeptide, e.g. soluble Siglec polypeptide, and a component comprising a reduced amount and/or concentration of Siglec polypeptide as compared to the original fluid withdrawn from the subject.

[0083] In some embodiments, extracorporeal treatment can comprise extracorporeal adsorption. In extracorporeal adsorption, the Siglec expression product is adsorbed by a surface, thereby reducing the level of Siglec expression product in the biofluid. In some embodiments, the surface can comprise an adsorption agent, e.g. an agent that can bind to Siglec, either specifically or nonspecifically. In some embodiments, the adsorption agent can comprise a Siglec inhibitor which can specifically bind to a Siglec expression product, (e.g. an anti-Siglec7 antibody reagent and/or a ligand-derived binding agent). The substrate or matrix to be used in practicing extracorporeal treatment as described herein needs to allow sufficient permeation of flow so that biofiuid components are distributed throughout the substrate or matrix material, so that substantial contact is made between the biofiuid components and the adsorption agent comprised by the substrate or matrix. Suitable substrates or matrices are known to one skilled in the art. Substrates or matrices can include silica gel, dextran, agarose, nylon polymers, polymers of acrylic acid, co-polymers of ethylene and maleic acid anhydride,

aminopropylsilica, ammocelite, glass beads, silicate containing diatomaceous earth or other substrates or matrices known in the art. Examples of such are described in the following patents, each of which are incorporated by reference herein in their entirety: Lentz U.S. Pat. No. 4,708,713, Motomura U.S. Pat. No. 5,667,684, Takashirna et al U.S. Pat. No. 5,041 ,079, and Porath and Janson U.S. Pat. No. 3,925, 152. Substrates can be selected from known substrates previously used in the art, including, by way of non-limiting example SEPHAROSE™ made by Amersham-Biosciences, Upsala, Sweden, as well as acrylamide and agarose particles or beads. The substrates used can have the properties of being able to tightly bind an absorption agent if desired, the ability to be produced in a sterile means, and be compatible with standard dialysis/extracorporeal tubings. In some embodiments, the substrate can be one that does not cause blood to coagulate, e.g. a heparinized substrate. Conjugation of an adsorption agent to said substrate can be accomplished by numerous methods known in the art. Said methods include avidin-streptavidm, cynanogen bromide coupling, covalentJy, by cross-Jinking, inclusion, encapsulation, and/or by the use of a linker such as a polyethylene glycol or another linker.

[0084] In some embodiments, extracorporeal treatment can comprise the use of a surface which filters Siglec from the biofiuid on the basis of size, charge, etc. Non-limiting examples include filters, capillary dialysis tubing, porous beads, and/or porous membranes which permit Siglec to transit, but which restrict the transit of cells and/or larger proteins, e.g. filters which can separate molecules of less than 60 kDa from those of more than 60 kDa and/or filters which can separate molecules of less than 45 kDa from those of more than 45 kDa. In some embodiments, filters, capillary dialysis tubing, porous beads, and/or porous membranes which permit soluble Siglec to transit, but which restrict the transit of cells and/or larger proteins can be used, e.g. filters which can separate molecules of less than 20 kDa from those of more than 20 kDa and/or filters which can separate molecules of less than 40 kDa from those of more than 40 kDa. In some embodiments, filters, capillary dialysis tubing, porous beads, and/or porous membranes which permit soluble Siglec to transit, but which restrict the transit of cells and/or larger proteins can be used, e.g. filters which can separate molecules of less than 25 kDa from those of more than 25 kDa and/or filters which can separate molecules of less than 35 kDa from those of more than 35 kDa. [0085] Non-specific means of apheresis are commercially available, see e.g. the "PLASMAFLUX P2™" hollow fiber filter cartridge (sold by Fresenius); PLASMART™ PS60 cartridges (sold by Medical srf) and are known in the art, e.g. U.S. Pat. No. 4,714,556; 4,714,556; 4,787,974; and 6,528,057; each of which is incorporated herein by reference in its entirety.

[0086] Any of the foregoing embodiments of extracorporeal treatment can be combined, e.g. specific and non-specific adsorption and/or filtration can be combined. Apheresis systems for the removal of specific proteins from biofluids are known and one of skill can adapt such systems for use in the methods described herein, see, e.g. U.S. Patent 6,231 ,536 Bi and U.S. Patent 6,231,536 each of which is incorporated by reference herein in their entirety).

[0087] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a Siglec inhibitor as described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a Siglec inhibitor as described herein.

[0088] In some embodiments, the pharmaceutical composition comprising a Siglec inhibitor as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration to a patient, including, but not limited to, administration of DUROS^®-type dosage forms, and dose-dumping.

[0089] Suitable vehicles that can be used to provide parenteral dosage forms of a Siglec inhibitor as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a Siglec inhibitor as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0090] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the Siglec inhibitor can be administered in a sustained release formulation.

[0091] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0092] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0093] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example,

hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS^® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

[0094] In some embodiments, a Siglec inhibitor as described herein can be administered in a liposome formulation. As used herein, "lipid vesicle" or "liposome" refers to vesicles surrounded by a bilayer formed of lipid components usually including lipids optionally in combination with non- lipidic components. The interior of a vesicle is generally aqueous. One major type of liposomal composition not generally found in nature includes phospholipids other than naturally-derived phosphatidylcholine. Neutral lipid vesicle compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic lipid vesicle compositions generally are formed from dimyristoyl phosphatidylglycerol. Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Lipids for lipid vesicle or liposome formation are known in the art or described herein below.

Liposomes are formed by the self-assembly of phospholipid molecules in an aqueous environment. The amphipathic phospholipid molecules form a closed bilayer sphere in an attempt to shield their hydrophilic groups from the aqueous environment, while still maintaining contact with the aqueous phase via the hydrophilic head group. The resulting closed sphere can encapsulate aqueous soluble drugs or agents within the bilayer membrane. Non- limiting examples of liposome compositions include those described U.S. Pat. Nos. 4,983,397; 6,476,068; 5,834,012; 5,756,069; 6,387,397; 5,534,241 ; 4,789,633; 4,925,661 ; 6, 153,596; 6,057,299; 5,648,478; 6,723,338; 6,627218; U.S. Pat. App. Publication Nos: 2003/0224037; 2004/0022842; 2001/0033860; 2003/0072794; 2003/0082228; 2003/0212031 ; 2003/0203865; 2004/0142025; 2004/0071768; International Patent Applications WO 00/74646; WO 96/13250; WO 98/33481 ; Papahadjopolulos D, Allen T M, Gbizon A, et al. "Sterically stabilized liposomes. Improvements in pharmacokinetics and antitumor therapeutic efficacy" Proc Natl Acad Sci U.S.A. (1991) 88: 1 1460-1 1464; Allen T M, Martin F J. "Advantages of liposomal delivery systems for anthracyclines" Semin Oncol (2004) 31 : 5-15 (suppl 13). Weissig et al. Pharm. Res. (1998) 15: 1552-1556 each of which is incorporated herein by reference in its entirety.

[0095] The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of additional agents and/or therapies which can be used to treat splanchnic vasodilation and/or HRS can include liver transplantation, insertion of a transjugular intrahepatic portosystemic shunt (TIPS) (to reduce blood pressure in the portal vein), hemodialysis, liver dialysis, intravenous albumin infusion, splanchnic vasoconstrictors (e.g. vasopressin, midodrine, somatostatin, ornipressin, terlipressin,), albumin-bound membrane dialysis (e.g. molecular adsorbents recirculation system (MARS)), pentoxyfylline, acetylcysteine, and misoprostol.

[0096] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having splanchnic vasodilation and/or HRS. In some embodiments, subjects having, e.g. HRS can be identified by a physician using current methods of diagnosing HRS. By way of non- limiting example, symptoms and/or complications of HRS which characterize this condition and aid in diagnosis are well known in the art and include but are not limited to, chronic or acute liver disease with advanced hepatic failure and portal hypertension; serum creatinine greater than 1.5 mg/dL;

absence of shock, ongoing bacterial infection or gastrointestinal and renal fluid losses; no current or recent treatment with nephrotoxic agents; no improvement in serum creatinine after withdrawal of diuretics and fluid resuscitation; proteinuria less than 500 mg/dL; and/or no ultrasonographic evidence of chronic renal disease or obstructive uropathy. Tests that may aid in a diagnosis of, e.g. HRS include, but are not limited to, ultrasound of the kidneys and/or testing creatinine levels in the blood and/or urine. A family history of, or the presence of risk factors for, HRS and/or liver disease can also aid in determining if a subject is likely to have HRS or in making a diagnosis.

[0097] The compositions and methods described herein can be administered to a subject having or diagnosed as having splanchnic vasodilation and/or HRS. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a Siglec inhibitor to a subject in order to alleviate a symptom of, e.g. HRS. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with a condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%), 95%), 99% or more as measured by any standard technique. A variety of approaches for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), and pulmonary administration. Administration can be local or systemic.

[0098] The term "effective amount" as used herein refers to the amount of a therapy needed to alleviate at least one or more symptoms of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a therapy that is sufficient to cause a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using no more than routine experimentation.

[0099] In some embodiments, an effective amount can be an amount which causes the extent and/or degree of splanchnic vasodilation and/or kidney dysfunction (e.g. as measured by creatinine levels as described elsewhere herein) to decrease or, at least, to increase at a lower rate than they would be expected to increase in a subject not receiving a composition as described herein. In some embodiments, an effective amount can be an amount that decreases the amount of Siglec polypeptide, e.g. soluble Siglec7 present in a biofluid of the subject by a statistically significant amount. The amount of Siglec polypeptide can be measured by methods known in the art and those described herein, e.g. the level of Siglec polypeptide in a sample obtained from a subject can be determined by a immunoassay as described elsewhere herein.

[00100] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50%> of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a therapeutic agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[00101] In certain embodiments, an effective dose of a composition comprising a Siglec inhibitor as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a Siglec inhibitor can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a Siglec inhibitor therapy such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. A composition comprising a Siglec inhibitor can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration can be repeated, for example, on a regular basis, such as hourly for 3 hours, 6 hours, 12 hours or longer or such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer.

[00102] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. HRS by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[00103] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the therapeutic. The desired dose for, e.g. decrease of vasodilation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.

[00104] In some aspects, the invention described herein is directed to systems (and computer readable media for causing computer systems) for obtaining data from at least one sample obtained from at least one subject, the system comprising 1) a measuring module configured to measure the level of Siglec in a test sample obtained from a subject, 2) a storage module configured to store output data from the measuring module, 3) a comparison module adapted to compare the data stored on the storage module with a reference level, and to provide a retrieved content, and 4) a display module for displaying whether the sample comprises a level of Siglec which is significantly increased relative to the reference expression level and/or displaying the relative expression level of Siglec.

[00105] In one embodiment, provided herein is a system comprising: (a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes 1) a measuring module configured to measure the expression level of Siglec in a test sample obtained from a subject, 2) a storage module configured to store output data from the measuring module, 3) a computing module adapted to identify from the output data whether the level of Siglec in a sample obtained from a subject is statistically significantly greater than a reference level, and 4) a display module for displaying a content based in part on the data output from the measuring module, wherein the content comprises a signal indicative of the level of Siglec and (b) at least one processor for executing the computer program (see Figure 10).

[00106] In some embodiments, the measuring module can measure the presence and/or intensity of a detectable signal from an assay indicating the presence and/or level of Siglec RNA in the test sample, e.g. from a quantitative RT-PCR assay or a next-generation sequencing assay. In some embodiments, the measuring module can measure the presence and/or intensity of a detectable signal from an immunoassay indicating the presence and/or level of Siglec in the test sample. Exemplary embodiments of a measuring module can include an automated immunoassay, etc.

[00107] The measuring module can comprise any system for detecting a signal elicited from an assay to determine the level of Siglec as described above herein. In some embodiments, such systems can include an instrument, e.g. an automated blood analyzer (e.g. iSTAT™) or a qRT-PCR instrument (e.g. CFX96 TOUCH™ Real-Time PCR Detection System). In another embodiment, the measuring module can comprise multiple units for different functions, such as measurement of Siglec polypeptide levels (and/or detectable signals from Siglec-specific antibody reagents) and

measurement of another gene or metabolite level (e.g. creatinine levels). In one embodiment, the measuring module can be configured to perform the methods described elsewhere herein, e.g.

immunoassay, or detection of any detectable label or signal.

[00108] In some embodiments, the measuring system or a further module can be configured to process whole blood samples, e.g. to separate cells or portions of cells from whole blood for use in the assays described herein. In some embodiments, the measuring system or a further module can be configured to process urine samples.

[00109] The term "computer" can refer to any non-human apparatus that is capable of accepting a structured input, processing the structured input according to prescribed rules, and producing results of the processing as output. Examples of a computer include: a computer; a general purpose computer; a supercomputer; a mainframe; a super mini-computer; a mini-computer; a workstation; a micro-computer; a server; an interactive television; a hybrid combination of a computer and an interactive television; and application-specific hardware to emulate a computer and/or software. A computer can have a single processor or multiple processors, which can operate in parallel and/or not in parallel. A computer also refers to two or more computers connected together via a network for transmitting or receiving information between the computers. An example of such a computer includes a distributed computer system for processing information via computers linked by a network.

[00110] The term "computer-readable medium" can refer to any tangible storage device used for storing data accessible by a computer, as well as any other means for providing access to data by a computer. Examples of a storage-device-type computer-readable medium include: a magnetic hard disk; a floppy disk; an optical disk, such as a CD-ROM and a DVD; a magnetic tape; a memory chip. A transient signal or carrier wave is not a computer-readable medium as the term is used herein. The term a "computer system" can refer to a system having a computer, where the computer comprises a computer-readable medium embodying software to operate the computer. The term "software" is used interchangeably herein with "program" and refers to prescribed rules to operate a computer. Examples of software include: software; code segments; instructions; computer programs; and programmed logic.

[00111] The computer readable storage media can be any available tangible media that can be accessed by a computer. Computer readable storage media includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer readable storage media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM

(electrically erasable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non- volatile memory, and any other tangible medium which can be used to store the desired information and which can be accessed by a computer including any suitable combination of the foregoing.

[00112] Computer-readable data embodied on one or more computer-readable media can define instructions, for example, as part of one or more programs that, as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations and combinations thereof. Such instructions can be written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof. The computer-readable media on which such instructions are embodied can reside on one or more of the components of a system, or a computer readable storage medium described herein can be distributed across one or more of such components.

[00113] The computer-readable media can be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein. In addition, it should be appreciated that the instructions stored on the computer-readable medium, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions can be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the present invention. The computer executable instructions can be written in a suitable computer language or combination of several languages. Basic computational biology methods are known to those of ordinary skill in the art and are described in, for example, Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997);

Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001).

[00114] Embodiments of the invention can be described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed. The modules are segregated by function for the sake of clarity. However, it should be understood that the modules/systems need not correspond to discreet blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times. Furthermore, it should be appreciated that the modules can perform other functions, thus the modules are not limited to having any particular functions or set of functions.

[00115] The functional modules of certain embodiments of the invention include at minimum a measuring module, a storage module, a computing module, and a display module. The functional modules can be executed on one, or multiple, computers, or by using one, or multiple, computer networks. The measuring module has computer executable instructions to provide e.g., levels of Siglec7 etc. in computer readable form.

[00116] The information determined in the measuring system can be read by the storage module. As used herein the "storage module" is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and local and distributed computer processing systems. Storage modules also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as magnetic/optical storage media. The storage module is adapted or configured for having recorded thereon, for example, sample name, biomolecule assayed and the level of said biomolecule. Such information may be provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.

[00117] As used herein, "stored" refers to a process for encoding information on the storage module. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising expression level information.

[00118] In some embodiments of any of the systems described herein, the storage module stores the output data from the measuring module. In additional embodiments, the storage module stores reference information such as levels of Siglec in healthy subjects, and/or subjects not having splanchnic vasodilation and/or HRS.

[00119] The "computing module" can use a variety of available software programs and formats for computing the level of Siglec. Such algorithms are well established in the art. A skilled artisan is readily able to determine the appropriate algorithms based on the size and quality of the sample and type of data. The data analysis tools and equations described herein can be implemented in the computing module of the invention. In some embodiments, the computing module can comprise a computer processor and/or a computer system. In one embodiment, the computing module further comprises a comparison module, which compares the level of Siglec in a sample obtained from a subject as described herein with a reference level as described herein (see, e.g. Figure 11). By way of an example, when the level of Siglec in a sample obtained from a subject is measured, a comparison module can compare or match the output data with the mean level of Siglec in a population of subjects not having signs or symptoms of splanchnic vasodilation and/or HRS (i.e. a reference level). In certain embodiments, the mean level of Siglec in a population of subjects not having signs or symptoms of splanchnic vasodilation and/or HRS can be pre-stored in the storage module. During the comparison or matching process, the comparison module can determine whether the level of Siglec in a sample obtained from a subject is statistically significantly greater than the reference level. In various embodiments, the comparison module can be configured using existing commercially- available or freely-available software for comparison purpose, and may be optimized for particular data comparisons that are conducted.

[00120] The computing and/or comparison module, or any other module of the invention, can include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server. World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements). Generally, the executables will include embedded SQL statements. In addition, the World Wide Web application can include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The configuration file also directs requests for server resources to the appropriate hardware-as may be necessary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as

"Intranets." An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site). In some embodiments users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers (see, e.g. Figure 12).

[00121] The computing and/or comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide content based in part on the comparison result that can be stored and output as requested by a user using an output module, e.g., a display module.

[00122] In some embodiments, the content displayed on the display module can be a report, e.g. the level of Siglec in the sample obtained from a subject. In some embodiments, a report can denote the level of Siglec, e.g. Siglec7 RNA transcript levels and/or Siglec7 polypeptide levels. In some embodiments, a report can denote the degree to which the expression level of Siglec in the sample obtained from the subject varies from the reference level.

[00123] In some embodiments, if the computing module determines that the level of Siglec in the sample obtained from a subject is greater by a statistically significant amount than the reference level, the display module provides a report displaying a signal indicating that the level in the sample obtained from a subject is greater than that of the reference level. In some embodiments, the content displayed on the display module or report can be the relative level of Siglec in the sample obtained from a subject as compared to the reference level. In some embodiments, the signal can indicate the degree to which the level of Siglec in the sample obtained from the subject varies from the reference level. In some embodiments, the signal can indicate that the subject is at increased risk of having, e.g. splanchnic vasodilation and/or HRS. In some embodiments, the splanchnic vasodilation can be associated with HRS and/or sepsis. In some embodiments, the signal can indicate the subject can benefit from treatment with a therapy for splanchnic vasodilation and/or HRS. In some embodiments, the content displayed on the display module or report can be a numerical value indicating one of these risks or probabilities. In such embodiments, the probability can be expressed in percentages or a fraction. For example, higher percentage or a fraction closer to 1 indicates a higher likelihood of a subject having splanchnic vasodilation and/or HRS. In some embodiments, the content displayed on the display module or report can be single word or phrases to qualitatively indicate a risk or probability. For example, a word "unlikely" can be used to indicate a lower risk for having or developing splanchnic vasodilation and/or HRS, while "likely" can be used to indicate a high risk for having or developing splanchnic vasodilation and/or HRS.

[00124] In one embodiment of the invention, the content based on the computing and/or comparison result is displayed on a computer monitor. In one embodiment of the invention, the content based on the computing and/or comparison result is displayed through printable media. The display module can be any suitable device configured to receive from a computer and display computer readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM -type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett- Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.

[00125] In one embodiment, a World Wide Web browser is used for providing a user interface for display of the content based on the computing/comparison result. It should be understood that other modules of the invention can be adapted to have a Web browser interface. Through the Web browser, a user can construct requests for retrieving data from the computing/comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.

[00126] Systems and computer readable media described herein are merely illustrative embodiments of the invention for determining the level of Siglec in a sample obtained from a subject, and therefore are not intended to limit the scope of the invention. Variations of the systems and computer readable media described herein are possible and are intended to fall within the scope of the invention. The modules of the machine, or those used in the computer readable medium, may assume numerous configurations. For example, function may be provided on a single machine or distributed over multiple machines.

[00127] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. [00128] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[00129] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100%) inhibition as compared to a reference level. In the context of a marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[00130] The terms "increased" /'increase", "enhance", or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of doubt, the terms "increased", "increase", "enhance", or "activate" mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%), or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100%) increase or any increase between 10-100%) as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4- fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom is meant a statistically significant increase in such level.

[00131] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "individual," "patient" and "subject" are used interchangeably herein.

[00132] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of, e.g. endometriosis. A subject can be male or female. [00133] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. splanchnic vasodilation and/or HRS) or one or more complications related to such a condition, and optionally, have already undergone treatment for, e.g., splanchnic vasodilation and/or HRS or the one or more complications related to splanchnic vasodilation and/or HRS. Symptoms of splanchnic vasodilation and/or HRS can include, but are not limited to, chronic or acute liver disease with advanced hepatic failure and portal hypertension;

increased serum creatinine; ongoing bacterial infection or gastrointestinal and renal fluid losses; and/or kidney failure. Alternatively, a subject can also be one who has not been previously diagnosed as having, e.g., splanchnic vasodilation and/or HRS or one or more complications related to splanchnic vasodilation and/or HRS. For example, a subject can be one who exhibits one or more risk factors for, e.g., splanchnic vasodilation and/or HRS or one or more complications related to splanchnic vasodilation and/or HRS or a subject who does not exhibit risk factors.

[00134] A "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[00135] As used herein, the term "antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen (e.g. Siglec7). An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[00136] The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

[00137] The terms "antigen-binding fragment" or "antigen-binding domain", which are used interchangeably herein refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding

functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those of skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.

[00138] As used herein, the term "antibody" refers to immunoglobulin molecules and

immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen. The terms also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms besides antibodies; including, for example, Fv, Fab, and F(ab)'2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). In some embodiments, antibody reagents, e.g. antibodies, monoclonal and chimeric antibodies useful in the methods as disclosed herein can be manufactured using well-known methods, e. g., as described in Howard and Kaser "Marking and Using Antibodies: A Practical Handbook" CRC Press (2006); which is incorporated by reference herein in its entirety.

[00139] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.

[00140] Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an antibody reagent described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule.

Typically, antigen-binding proteins (such as an antibody reagent described herein) will bind to their cognate or specific antigen with a dissociation constant (K_D of 10^"5 to 10^{" 12} moles/liter or less, and

1 12 8 12

preferably 10^" to 10^" moles/liter or less and more preferably 10^" to 10^" moles/liter (i.e. with an association constant (K_A) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10^s to 10¹² liter/moles). Any K_D value greater than 10^"4 mol/liter (or any K_A value lower than 10⁴ M^"1) is generally considered to indicate non-specific binding. The K_D for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10^" ¹⁰ M (0.1 nM) to 10^"5 M (10000 nM). The stronger an interaction is, the lower is its K_D. Preferably, a binding site on an antibody reagent described herein will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antibody reagent to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.

[00141] Accordingly, as used herein, "selectively binds" or "specifically binds" refers to the ability of an agent (e.g. an antibody reagent) described herein to bind to a target, such as a molecule present on the cell-surface, with a K_D 10^"5 M (10000 nM) or less, e.g. , 10^"6 M or less, 10^"7 M or less, 10^"8 M or less, 10^"9 M or less, 10^"10 M or less, lO^"11 M or less, or 10^"12 M or less. For example, if an agent described herein binds to Siglec7 with a K_D of 10^"5 M or lower, but not to another Siglec, or a related homologue, then the agent is said to specifically bind Siglec7. Specific binding can be influenced by, for example, the affinity and avidity of the agent and the concentration of the agent. The person of ordinary skill in the art can determine appropriate conditions under which the agents described herein selectively bind the targets using any suitable methods, such as titration of an agent in a suitable cell and/or peptide binding assay.

[00142] As used herein, the term "reagent specific for a Siglec polypeptide" refers to a reagent which can bind specifically to Siglec, including soluble Siglec and/or a portion of the extracellular domain of Siglec. In some embodiments, a reagent can be specific for a particular Siglec polypeptide, e.g. for Siglec7, and does not specifically bind other Siglec polypeptides, e.g. Siglec5 or Siglec9. For example, an exemplary reagent specific for a Siglec7 polypeptide can be an antibody specific for a Siglec7 polypeptide, examples of which are described elsewhere herein.

[00143] The term "label" refers to a composition capable of producing a detectable signal indicative of the presence of an antibody reagent (e.g. a bound antibody reagent). Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[00144] As used herein, the terms "proteins" and "polypeptides" are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated,

glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and

"polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[00145] The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell. An agent can be selected from a group comprising: polynucleotides; polypeptides; small molecules; antibodies; or functional fragments thereof.

[00146] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. splanchnic vasodilation and/or HRS. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with, e.g. splanchnic vasodilation. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced.

Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[00147] As used herein, the term "pharmaceutical composition" refers to the one or more active agents in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00148] As used herein, the term "administering," refers to the placement of an agent or a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent or compound at a desired site. Pharmaceutical compositions comprising the compounds or agents disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject, e.g. oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, injection, administration.

[00149] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) difference.

[00150] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[00151] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[00152] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00153] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment. [00154] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00155] Definitions of common terms in cell biology and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy", 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Immunology by Werner

Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1 -56081 -569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[00156] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

[00157] Other terms are defined herein within the description of the various aspects of the invention.

[00158] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00159] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00160] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00161] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. A method of reducing splanchnic vasodilation, the method comprising;

administering a therapeutically effective amount of a Siglec inhibitor to a subject in need of treatment.

2. The method of paragraph 1, wherein the subject is in need of treatment for a condition

selected from the group consisting of:

hepatorenal syndrome (HRS); severe cirrhosis; and sepsis.

3. The method of any of paragraphs 1-2, wherein the Siglec inhibitor specifically binds one or more polypeptides selected from the group consisting of:

Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide.

4. The method of any of paragraphs 1-3, wherein the inhibitor is administered in vivo.

5. The method of any of paragraphs 1-3, wherein the inhibitor is administered ex vivo.

6. The method of any of paragraphs 1-3, wherein the ex vivo administration comprises

extracorporeal apheresis. The method of paragraph 6, wherein the extracorporeal aphereis comprises:

removing a portion of the subject's blood;

contacting all or a portion of the blood obtained from the subject with a siglec inhibitor that specifically binds one or more polypeptides selected from the group consisting of:

Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide, whereby the level of one or more of Siglec polypeptides in the the blood obtained from the subject is reduced;

and returning all or a portion of the subject's blood to the subject.

The method of paragraph 7, wherein the Siglec inhibitor is an antibody reagent.

The method of paragraph 8, wherein a column comprises the antibody reagent.

A method of detecting soluble Siglec, the method comprising contacting a biological sample with a reagent specific for a Siglec polypeptide, wherein reaction of soluble Siglec with said reagent indicates the presence of soluble Siglec.

The method of paragraph 10, wherein the reagent specific for a Siglec polypeptide is specific for a soluble Siglec polypeptide.

The method of any of paragraphs 10-11, wherein the Siglec polypeptide is selected from the group consisting of:

Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide.

The method of any of paragraphs 10-12, wherein the method comprises a first step of removing cells from the sample.

The method of any of paragraphs 10-13, wherein the sample comprises a urine or blood sample.

An assay comprising:

determining the expression level of Siglec in a test sample obtained from a subject; wherein an increase in the Siglec expression level relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation.

The assay of paragraph 15, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

The assay of any of paragraphs 15-16, wherein the expression level of Siglec is determined by measuring the level of Siglec RNA transcript.

The assay of paragraph 17, wherein the RNA transcript level is measured using reverse transcription polymerase chain reaction (RT-PCR).

The assay of any of paragraphs 15-18, wherein the expression level of Siglec is determined by measuring the level of Siglec polypeptide. The assay of paragraph 19, wherein the expression level of Siglec is determined by measuring the level of soluble Siglec polypeptide.

The assay of any of paragraphs 19-20, wherein the polypeptide level is measured using immunochemistry.

The assay of paragraph 21, wherein the polypeptide level is measured using enzyme-linked immunosorbent assay (ELISA).

The assay of any of paragraphs 15-22, wherein the sample comprises a material selected from the group consisting of:

a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid;

ascites; and urine.

An assay comprising:

(a) contacting a biofluid test sample obtained from a subject with a detectable anti- Siglec antibody reagent; and

(b) detecting the presence or intensity of a detectable signal;

wherein an increase in the level of Siglec polypeptide, indicated by the level of the detectable signal, relative to a reference level indicates the subject has a higher risk of having or developing splanchnic vasodilation.

The assay of paragraph 24, wherein the Siglec polypeptide is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

The assay of any of paragraphs 24-25, wherein the level of Siglec polypeptide is the level of soluble Siglec polypeptide.

The assay of any of paragraphs 24-26, wherein the antibody reagent is detectably labeled or generates a detectable signal.

The assay of any of paragraphs 24-27, wherein the splanchnic vasodilation is associated with a condition selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis.

The assay of any of paragraphs 24-28, wherein the expression level of Siglec is normalized relative to the expression level of one or more reference genes or reference proteins.

The assay of any of paragraphs 24-29, wherein the reference expression level of Siglec is the expression level of Siglec in a prior sample obtained from the subject.

The assay of any of paragraphs 24-30, wherein an increased level of Siglec is a level at least 25% greater than a reference level.

The assay of any of paragraphs 24-31, wherein the expression level of no more than 20 other genes is determined. The assay of any of paragraphs 24-32, wherein the expression level of no more than 10 other genes is determined.

The assay of any of paragraphs 24-33, wherein the subject is a human.

The assay of any of paragraphs 24-34, wherein the sample comprises a material selected from the group consisting of:

a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid;

ascites; and urine.

The assay of any of paragraphs 15-35, wherein the subject is indicated as having a higher risk of having or developing cirrhosis of the liver.

The assay of any of paragraphs 15-36, wherein the subject is indicated as having a higher risk of having or developing severe cirrhosis of the liver.

A method of administering a treatment for splanchnic vasodilation to a subject, the method comprising:

determining the expression level of Siglec in a test sample obtained from a subject; and

administering a treatment for splanchnic vasodilation to the subject if the expression level of Siglec is increased relative to a reference level.

A method of identifying a subject in need of treatment for splanchnic vasodilation, the method comprising:

determining the expression level of Siglec in a test sample obtained from a subject; wherein the subject is identified as being in need of treatment for splanchnic vasodilation if the expression level of Siglec is increased relative to a reference level. A method of determining the efficacy of a treatment for splanchnic vasodilation, the method comprising:

(a) determining the expression level of Siglec in a test sample obtained from a

subject before administration of the treatment;

(b) determining the expression level of Siglec in a test sample obtained from a

subject after administration of the treatment;

wherein the treatment is not efficacious if the expression level determined in step (b) is increased relative to the expression level determined in step (a).

The method of any of paragraphs 38-40, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

The method of any of paragraphs 38-41, wherein the splanchnic vasodilation is associated with a condition selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis. The method of any of paragraphs 38-42, wherein the treatment for splanchnic vasodilation is selected from the group consisting of:

liver transplantation; dialysis; transjugular intrahepatic portosystemic shunt (TIPS); hemodialysis; liver dialysis; intravenous albumin infusion; administration of splanchnic vasoconstrictors; albumin-bound membrane dialysis (e.g. molecular adsorbents recirculation system (MARS)); and administration of pentoxyfylline, acetylcysteine, and/or misoprostol.

A method of treatment for hepatorenal syndrome (HRS) comprising;

determining the expression level of Siglec in a test sample obtained from a subject; wherein if the level of Siglec is increased relative to a reference level, the subject is treated with a liver transplant; and

wherein if the level of Siglec is not increased relative to a reference level, the subject is treated with a liver and kidney transplant.

The method of paragraph 44, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

The method of any of paragraphs 38-45, wherein an increased level of Siglec is a level at least 25% greater than a reference level.

The method of any of paragraphs 38-46, wherein the sample comprises a material selected from the group consisting of:

a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid;

ascites; and urine.

The method of any of paragraphs 38-47, wherein the expression level of Siglec is determined by measuring the level of Siglec RNA transcript.

The method of paragraph 48, wherein the RNA transcript level is measured using reverse transcription polymerase chain reaction (RT-PCR).

The method of any of paragraphs 38-47, wherein the expression level of Siglec is determined by measuring the level of Siglec polypeptide.

The method of paragraph 50, wherein the expression level of Siglec is determined by measuring the level of soluble Siglec polypeptide.

The method of any of paragraphs 50-51, wherein the polypeptide level is measured using immunochemistry.

The method of paragraph 52, wherein the immunochemical method comprises:

(b) detecting the presence or intensity of a detectable signal; wherein the expression level of Siglec polypeptide is indicated by the level of the detectable signal.

The method of paragraph 53, wherein the antibody reagent is detectably labeled or generates a detectable signal.

The method of any of paragraphs 38-54, wherein the expression level of Siglec is normalized relative to the expression level of one or more reference genes or reference proteins.

The method of any of paragraphs 38-55, wherein the reference expression level of Siglec is the level of Siglec in a prior sample obtained from the subject.

The method of any of paragraphs 38-56, wherein the expression level of no more than 20 other genes is determined.

The method of any of paragraphs 38-57, wherein the expression level of no more than 10 other genes is determined.

The method of any of paragraphs 38-58, wherein the subject is a human.

The method of any of paragraphs 38-59, wherein the inhibitor is administered in vivo.

The method of any of paragraphs 38-59, wherein the inhibitor is administered ex vivo.

The method of any of paragraphs 38-61, wherein the ex vivo administration comprises extracorporeal apheresis.

The method of paragraph 62, wherein the extracorporeal aphereis comprises:

removing a portion of the subject's blood;

and returning all or a portion of the subject's blood to the subject.

The method of paragraph 63, wherein the Siglec inhibitor is an antibody reagent.

The method of paragraph 63, wherein a column comprises the antibody reagent.

A computer system for determining the risk of a subject having or developing splanchnic vasodilation, the system comprising:

a measuring module configured to measure the expression level of Siglec in a test sample obtained from a subject;

a storage module configured to store output data from the determination module;

a comparison module adapted to compare the data stored on the storage module with a reference level, and to provide a retrieved content, and a display module for displaying whether the sample comprises a level of Siglec which is significantly increased relative to the reference expression level and/or displaying the relative expression level of Siglec.

67. The system of paragraph 66, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

68. The system of any of paragraphs 66-67, wherein the measuring module measures the intensity of a detectable signal from an assay indicating the expression level of Siglec polypeptide in the test sample.

69. The system of paragraph 68, wherein the assay is an immunoassay.

70. The system of any of paragraphs 66-69, wherein the measuring module measures the intensity of a detectable signal from a RT-PCR assay indicating the expression level of Siglec RNA transcript in the test sample.

71. The system of any of paragraphs 66-70, wherein if the computing module determines that the expression level of Siglec in the test sample obtained from a subject is greater by a statistically significant amount than the reference expression level, the display module displays a signal indicating that the expression levels in the sample obtained from a subject are greater than those of the reference expression level.

72. The system of any of paragraphs 66-71, wherein the signal indicates that the subject has an increased likelihood of having or developing splanchnic vasodilation.

73. The system of any of paragraphs 66-72, wherein the signal indicates the subject is in need of treatment for splanchnic vasodilation.

74. The system of any of paragraphs 66-73, wherein the signal indicates the degree to which the expression level of Siglec in the sample obtained from a subject varies from the reference expression level.

75. The system of any of paragraphs 66-74, wherein the splanchnic vasodilation is associated with a condition is selected from the group consisting of:

hepatorenal syndrome (HRS); severe cirrhosis; and sepsis.

EXAMPLES

EXAMPLE 1

[00162] The level of soluble Siglec-7 in plasma samples obtained from subjects with, e.g. hepatic conditions (Figure 1) was determined (Figure 2). Increased soluble Siglec-7 levels were observed in subjects with HRS. These results demonstrate that soluble Siglec7 is detectable in urine at levels of 10-20s. Additionally, after liver transplantation in cirrhotic patients, Siglec7 levels drop

precipitously, e.g. Cirrhotic pt #1, pretransplant Siglec-7: 1765 -> 745 ( 1 week post liver transplant) and Cirrhotic pt #2, pretransplant Siglec-7: 1794 273 (2 months post liver transplant). In contrast, non-cirrhotic patients undergoing liver surgery develop elevated Siglec7 levels, e.g. Pt #1 Small hepatic resection for hemangioma, post op Siglec-7 level : 600s and Pt #2 Partial hepatectomy for adenoma, post op Siglec-7 Level: 1781. These results indicate that siglec7 is located in the liver and when the liver gets injured/inflammed, more soluble siglec-7 is produced and released into circulation, (s = pg/mL)

[00163] Siglec7 administration was demonstrated to increase vascular relaxation (Figure 3). A similar response was observed in response to administration of LPS (Figure 4). Administration of both Siglec7 and LPS demonstrated an additive effect on vascular relaxation (Figure 5) as measured using standard microvascular reactivity.

[00164] The samples obtained from the subjects of Figure 1 were examined for endotoxin concentration (Figure 6).

[00165] LPS and Siglec7 were administered to HuVEC and rat SMC cells and were observed to increase the nitrate+nitrite concentrations (Figures 6-9). As rats do not produce Siglec7, rat cells provide a background-free system for studying the effects of Siglec 7.

[00166] The data described herein demonstrate that Siglec7 induces nitric oxide, leading to mesenteric vasodilation which is the hallmark of hepatorenal syndrome.

EXAMPLE 2

[00167] Siglec 9, a member of the siglec subgroup of immunoglobulin superfamily that is expressed in the liver (Zhang JQ et al, J Biol Chem 2000, 275:22121-22126) was examined. To measure Aiglec 9 levels in plasma a commercially available ELISA kit (Catalog # DY1139, R&D systems, MN) was used. It was noted that soluble siglec 9 levels were significantly elevated in samples obtained from human cirrhosis complicated by hepatorenal syndrome (Table 1).

[00168] The results described herein indicate that circulating levels of Siglec 9 in the blood are diagnostic of hepatorenal syndrome, a complication of liver disease/cirrhosis. Moreover, soluble siglec 9 levels can be used in combination with Aiglec 7 levels to diagnose cirrohosis with hepatorenal syndrome.

Table 1

Claims

What is claimed herein is:

1. A method of reducing splanchnic vasodilation, the method comprising;

2. The method of claim 1, wherein the subject is in need of treatment for a condition selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis.

3. The method of any of claims 1-2, wherein the Siglec inhibitor specifically binds one or more polypeptides selected from the group consisting of:

Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide.

4. A method of detecting soluble Siglec, the method comprising contacting a biological sample with a reagent specific for a Siglec polypeptide, wherein reaction of soluble Siglec with said reagent indicates the presence of soluble Siglec.

5. The method of claim 4, wherein the reagent specific for a Siglec polypeptide is specific for a soluble Siglec polypeptide.

6. The method of any of claims 4-5, wherein the Siglec polypeptide is selected from the group consisting of:

Siglec-7 polypeptide; Siglec-5 polypeptide; and Siglec-9 polypeptide.

7. The method of any of claims 4-6, wherein the method comprises a first step of removing cells from the sample.

8. The method of any of claims 4-7, wherein the sample comprises a urine or blood sample.

9. An assay comprising:

10. The assay of claim 9, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

11. The assay of any of claims 9-10, wherein the expression level of Siglec is determined by measuring the level of Siglec RNA transcript.

12. The assay of claim 11, wherein the RNA transcript level is measured using reverse

transcription polymerase chain reaction (RT-PCR).

13. The assay of any of claims 9-10, wherein the expression level of Siglec is determined by measuring the level of Siglec polypeptide.

14. The assay of claim 13, wherein the expression level of Siglec is determined by measuring the level of soluble Siglec polypeptide.

15. The assay of any of claims 13-14, wherein the polypeptide level is measured using

immunochemistry.

16. The assay of any of claims 1-15, wherein the sample comprises a material selected from the group consisting of:

a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid;

ascites; and urine.

17. An assay comprising:

(b) detecting the presence or intensity of a detectable signal;

18. The assay of claim 17, wherein the Siglec polypeptide is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

19. The assay of claim 15, wherein the level of Siglec polypeptide is the level of soluble Siglec polypeptide.

20. The assay of any of claims 18-19, wherein the antibody reagent is detectably labeled or

generates a detectable signal.

21. The assay of any of claims 18-20, wherein the splanchnic vasodilation is associated with a condition selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis.

22. The assay of any of claims 18-21, wherein the expression level of Siglec is normalized

relative to the expression level of one or more reference genes or reference proteins.

23. The assay of any of claims 18-22, wherein the reference expression level of Siglec is the expression level of Siglec in a prior sample obtained from the subject.

24. The assay of any of claims 18-23, wherein an increased level of Siglec is a level at least 25% greater than a reference level.

25. The assay of any of claims 18-24, wherein the expression level of no more than 20 other genes is determined.

26. The assay of any of claims 18-24, wherein the expression level of no more than 10 other genes is determined.

27. The assay of any of claims 18-26, wherein the subject is a human.

28. A method of administering a treatment for splanchnic vasodilation to a subject, the method comprising:

29. A method of identifying a subject in need of treatment for splanchnic vasodilation, the

method comprising:

determining the expression level of Siglec in a test sample obtained from a subject; wherein the subject is identified as being in need of treatment for splanchnic vasodilation if the expression level of Siglec is increased relative to a reference level.

30. A method of determining the efficacy of a treatment for splanchnic vasodilation, the method comprising:

(c) determining the expression level of Siglec in a test sample obtained from a

subject before administration of the treatment;

(d) determining the expression level of Siglec in a test sample obtained from a

subject after administration of the treatment;

31. The method of any of claims 28-30, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

32. The method of any of claims 28-31, wherein the splanchnic vasodilation is associated with a condition selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis.

33. The method of any of claims 28-32, wherein the treatment for splanchnic vasodilation is selected from the group consisting of:

34. A method of treatment for hepatorenal syndrome (HRS) comprising;

determining the expression level of Siglec in a test sample obtained from a subject; wherein if the level of Siglec is increased relative to a reference level, the subject is treated with a liver transplant; and wherein if the level of Siglec is not increased relative to a reference level, the subject is treated with a liver and kidney transplant.

35. The method of claim 34, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

36. The method of any of claims 28-35, wherein an increased level of Siglec is a level at least 25% greater than a reference level.

37. The method of any of claims 28-36, wherein the sample comprises a material selected from the group consisting of:

a biofluid sample; blood or a product thereof; serum; plasma; abdominal fluid; ascites; and urine.

38. The method of any of claims 28-37, wherein the expression level of Siglec is determined by measuring the level of Siglec RNA transcript.

39. The method of claim 38, wherein the RNA transcript level is measured using reverse

transcription polymerase chain reaction (RT-PCR).

40. The method of any of claims 28-37, wherein the expression level of Siglec is determined by measuring the level of Siglec polypeptide.

41. The method of claim 40, wherein the expression level of Siglec is determined by measuring the level of soluble Siglec polypeptide.

42. The method of any of claims 40-41, wherein the polypeptide level is measured using

immunochemistry.

43. The method of claim 42, wherein the immunochemical method comprises:

(c) contacting a biofluid test sample obtained from a subject with a detectable anti- Siglec antibody reagent; and

(d) detecting the presence or intensity of a detectable signal;

wherein the expression level of Siglec polypeptide is indicated by the level of the detectable signal.

44. The method of claim 43, wherein the antibody reagent is detectably labeled or generates a detectable signal.

45. The method of any of claims 28-44, wherein the expression level of Siglec is normalized relative to the expression level of one or more reference genes or reference proteins.

46. The method of any of claims 28-45, wherein the reference expression level of Siglec is the level of Siglec in a prior sample obtained from the subject.

47. The method of any of claims 28-46, wherein the expression level of no more than 20 other genes is determined.

48. The method of any of claims 28-46, wherein the expression level of no more than 10 other genes is determined.

49. The method of any of claims 28-48, wherein the subject is a human.

50. A computer system for determining the risk of a subject having or developing splanchnic vasodilation, the system comprising:

a storage module configured to store output data from the determination module;

a comparison module adapted to compare the data stored on the storage module with a reference level, and to provide a retrieved content, and

a display module for displaying whether the sample comprises a level of Siglec which is significantly increased relative to the reference expression level and/or displaying the relative expression level of Siglec.

51. The system of claim 50, wherein the Siglec is selected from the group consisting of:

Siglec-7; Siglec-5; and Siglec-9.

52. The system of any of claims 50-51, wherein the measuring module measures the intensity of a detectable signal from an assay indicating the expression level of Siglec polypeptide in the test sample.

53. The system of claim 52, wherein the assay is an immunoassay.

54. The system of any of claims 50-51, wherein the measuring module measures the intensity of a detectable signal from a RT-PCR assay indicating the expression level of Siglec RNA transcript in the test sample.

55. The system of any of claims 50-54, wherein if the computing module determines that the expression level of Siglec in the test sample obtained from a subject is greater by a statistically significant amount than the reference expression level, the display module displays a signal indicating that the expression levels in the sample obtained from a subject are greater than those of the reference expression level.

56. The system of any of claims 50-55, wherein the signal indicates that the subject has an

increased likelihood of having or developing splanchnic vasodilation.

57. The system of any of claims 50-56, wherein the signal indicates the subject is in need of treatment for splanchnic vasodilation.

58. The system of any of claims 50-57, wherein the signal indicates the degree to which the expression level of Siglec in the sample obtained from a subject varies from the reference expression level.

59. The system of any of claims 50-58, wherein the splanchnic vasodilation is associated with a condition is selected from the group consisting of:

hepatorenal syndrome (HRS) and sepsis.