WO2008009869A1 - Prédiction de maladie par dosage des lipopolysaccharides - Google Patents

Prédiction de maladie par dosage des lipopolysaccharides Download PDF

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WO2008009869A1
WO2008009869A1 PCT/GB2006/002714 GB2006002714W WO2008009869A1 WO 2008009869 A1 WO2008009869 A1 WO 2008009869A1 GB 2006002714 W GB2006002714 W GB 2006002714W WO 2008009869 A1 WO2008009869 A1 WO 2008009869A1
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lps
disease
metabolic
individual
cardiovascular
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Philip Mcternan
Sudhesh Kumar
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University of Warwick
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • the invention relates to the field of metabolic and cardiovascular diseases, in particular the so-called metabolic syndrome, hyperinsulinaemia, insulin resistance, obesity, non-alcoholic fatty liver disease (NAFLD), atherosclerosis, coronary heart disease and type 2 diabetes (T2DM).
  • the invention also concerns methods of predicting the degree of risk in onset and development of diabetes in susceptible patient groups, in particular obese patients, and also methods of treating such individuals.
  • the invention also concerns methods of screening active agents to treat such metabolic and cardiovascular disease.
  • Diabetes mellitus is an increasing problem among Western societies and those adopting a Western lifestyle.
  • Type 2 diabetes in particular, is associated with poor diet, lack of exercise and predisposing genetic background.
  • the United States had 13.8 million diagnosed diabetics, with an estimated 5 million undiagnosed and a further 41 million pre-diabetic individuals with evidence of increasing insulin resistance (Bloomgarten, 2006, Diabetes Care 29:161-167).
  • Type 2 diabetes accounts for roughly 90% of total prevalence.
  • 'Metabolic syndrome' is a term used to describe the combination of risk factors for type 2 diabetes and cardiovascular disease (Reaven, 1988, Diabetes 37: 1595). It includes obesity (especially abdominal obesity - also known as central or visceral obesity) insulin resistance, impaired glucose metabolism, dyslipidaemia of the high triglyceride/low HDL cholesterol type and hypertension. It is estimated that as many as a quarter of the world's adult population suffer from metabolic syndrome, which carries with it three-fold increased risk of stroke or heart attack and a five-fold increased risk of developing type 2 diabetes (Dunstan etal, 2002, Diabetes Care 27: 2676; lsomaa etal, 2001, Diabetes Care 24: 683). Metabolic syndrome is linked to genetic factors, ageing, physical inactivity, dietary factors and is strongly linked to obesity and insulin resistance.
  • Obesity is known to represent one of the most important risk factors for the increased risk of type 2 diabetes and cardiovascular disease (Field et al, 2001 , Arch Intern Med 161:1581).
  • an increase in central (visceral) adiposity confers higher metabolic risk.
  • This increased metabolic risk is associated with sub-clinical inflammation, with several studies demonstrating increased proinflammatory adipocytokines in patients with obesity and type 2 diabetes (Pickup et al, 1997, Diabetologia 40: 1286; Pickup et al, 2000, Life Sci 67: 291).
  • adipose tissue secreted products such as plasminogen activator inhibitor type 1 (PAI-1), IL-6 and TNF- ⁇ are mediators of sub-clinical inflammation and potential cardiovascular risk through activation of NFKB, a key transcriptional factor in the inflammatory cascade (Pickup et al , 1997, Diabetologia 40: 1286; Festa etal, 2002, Diabetes 51:1131; Kern etal, 2002, Am J Physiol Endocrinol Metab 280: E745-51; Vozarova et al, 2001, Obes Res 9:414; Senn etal, 2002, Diabetes 51: 3391; Ahmad et al , 1997, J Cell Biochem 64:117; Uysal etal , 1997, Nature 389: 610; Collart ef a/ , 1990, MoI Cell Biol 10:1498; Baltimore etal , 1990, MoI Cell Biol 10:2327).
  • PAI-1 plasminogen activator inhibitor type
  • adipocytes In contrast to the pro-inflammatory adipoeytokines, adipocytes also secrete adiponectin, which has been shown to possess anti-inflammatory properties through its action on NFKB, and is inversely correlated with obesity and diabetes (Tomas etal, 2002, Proc Natl Acad Sci USA 99:16309; Yamauchi et al, 2002, Nat Med 8: 1288; Ouchi et al , 2001 , Circulation 103: 1057; Ouchi et al, 2000, Circulation 102:1296; Yamauchi et al, 2002, J Biol Chem 278:2461).
  • TLR Toll-like receptor
  • LPS lipopolysaccharide
  • cytokines such as IL- 6, IL-12, IL-15, IL-18, TNF- ⁇ , macrophage migration inhibitory factor (MlF), and cytokine-like molecules such as high mobility group B1 (HMGB1), which, in turn activate neutrophils, lymphocytes and vascular endothelium, up-regulate cell adhesion molecules, and induce prostaglandins, nitric oxide synthase and acute- phase proteins.
  • PAF platelet activating factor
  • prostaglandins prostaglandins
  • leukotrienes and thromboxane activates vascular endothelium, regulates vascular tone and activates the extrinsic coagulation cascade.
  • Dysregulation of these responses results in the complications of sepsis and septic shock in terms of peripheral vasodilation leading to hypotension, and abnormal clotting and fibrinolysis producing thrombosis and intravascular coagulation (Cohen, 2002, Nature 420: 885-891).
  • the best characterised high-affinity LPS receptor on myeloid cells comprises the LPS receptor CD 14, together with TLR4 and the leucine-repeat rich molecule
  • TLR2 Another TLR, TLR2, also appears to be involved in a functional LPS receptor on, for instance, pancreatic islet ⁇ cells (Vives-Pi et al, 2003, Clin Exp Immunol 133: 208). Activation of TLRs leads to translocation of NFKB to the nucleus and transcription of IL-6, IL-1 and TNF- ⁇ to initiate an acute phase response ( Muzio et al , 2000, Biochem Soc Trans
  • TLR 2 and 4 remain the most studied of these receptors, and have been found to be expressed in 3T3-L1 adipocytes, with LPS being shown to stimulate TLR 2 expression and induce IL-6 and TNF- ⁇ from these cells (Lin et al, 2000, J Biol Chem 275: 24255).
  • CD14 is a glycosyl phosphatidylinositol (GPI) -anchored membrane protein capable of binding LPS complexed with the acute phase serum glycoprotein, LPS-binding protein (LBP).
  • GPI glycosyl phosphatidylinositol
  • LBP LPS-binding protein
  • 0027308 discloses a method of measuring cell-surface CD14 receptor clusters comprising the integrin subunit CD11b for the diagnosis of systemic inflammation including arteriosclerosis.
  • European patent application EP 1571160 and Japanese application JP 2005/106694 propose determining the concentration of soluble CD14 isoforms by use of specific antibodies as an indicator of sepsis.
  • WO 94/21280 describes the use of an assay for neutrophil bactericidal/ permeability increasing protein (BPI), a protein with 44% identity with LBP and which also binds the lipid A portion of LPS.
  • BPI neutrophil bactericidal/ permeability increasing protein
  • US 5,618,675 describes a method blocking LPS-meditated activation of the inflammatory response by use of CAP18 protein (obtained from mammalian granulocytes) or peptides derived therefrom. CAP18 is capable of binding LPS and so compete with functional LPS receptors.
  • TLRs Although the main function of TLRs is to form the basis of the innate immune response to infection, there is increasing evidence of their involvement in inappropriate inflammatory reactions contributing to a number of disease states. For instance, CD14 and TLR4 are implicated in the development of atherosclerosis (Arroyo-Espliguero et al, 2006, Heart 90: 983) and inflammatory bowel disease. Blocking of TLR4 has a beneficial effect in murine models of inflammatory bowel disease (Fort et al, 2005, J Immunol 174: 6416). However, attempts to establish a link between TLR4 polymorphisms and diabetes or metabolic syndrome have been unsuccessful (lllig et al, 2003, Diabetes 52: 2861).
  • LPS Long et al, 2003, Cardiovasc Toxicol 3: 363; Plesner et al, 2002, Scand J Immunol 56: 522), although in these cases type 1 diabetes was the model. It has been suggested that high levels of glucose itself might have pro-inflammatory effects (Das, 2002, Critical Care 6: 389). It is also known that LPS is able to stimulate IL-6 production by adipocytes and this may be blocked by thizolidinediones such as pioglitazone (Yamaguchi et al, 2005, J Dent Res 84: 240).
  • Periodontal disease which provides a means for LPS to enter the circulation
  • coronary heart disease Periodontal disease
  • Periodontal disease is also associated with diabetes and it has been suggested that thiazolidinedione might suppress this by an action on diabetic macrophages, which over-produce TNF- ⁇ and have up-regulated expression of activation markers such as CD14 and CD18 (Salvi et al, 1997, J Clin Periodontol 24: 8; Fogelstrand et al, 2004, Diabetologia 47: 1948).
  • activation markers such as CD14 and CD18
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • both steatosis and type 2 diabetes are associated with insulin resistance/hyperinsulinaemia and chronic sub-clinical inflammation (Yki-Jarvinen and Westerbacka, 2005, Curr MoI Med 5:287).
  • the role of the gastrointestinal tract as a source of endotoxin and sub-clinical inflammation in chronic diseases such as fatty liver disease and type 2 diabetes has received limited attention to date, with portal vein circulating endotoxin levels implicated as a consequence of disease rather than the potential mediator. Given the growing significance of type 2 diabetes and the long sub-clinical prediabetic period associated with it, attempts have been made to identify reliable diagnostic markers.
  • One of the defining features of the pre-diabetic period is the development of impaired glucose tolerance and insulin resistance, that is, the failure of target tissues to respond to elevated levels of insulin by effectively lowering blood glucose levels.
  • Current knowledge suggests that development of glucose intolerance or diabetes is initiated by insulin resistance and is worsened by the compensatory hyperinsulinaemia.
  • insulin resistance is difficult to measure directly (by the complex and invasive 'hyperinsulinaemic euglycaemic clamp' method) and is usually inferred from measuring fasting glucose levels and glucose tolerance testing.
  • WO 2005/017532 describes oral administration of a controlled quantity of glycaemic carbohydrate of known composition.
  • a number of markers for frank diabetes or pre-diabetic insulin resistance have been developed as an alternative. It is known that chronic hyperglycaemia leads to abnormal non-enzymatic glycosylation ('glycation') of serum proteins such as albumin. US2006121532 discloses the use of glycated insulin as a biomarker for diabetes. US 5,183,764 discloses an indirect test for insulin resistance based on a quantitative assay for c ⁇ /ro-inositol and reports an inverse relationship between serum levels of this and insulin resistance.
  • IMM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • E-selectin Wexler et al, 2005, Obesity Research 1_3: 1772.
  • adipose tissue secreted products such as plasminogen activator inhibitor type 1 (PAI-1) and TNF- ⁇ are mediators of sub-clinical inflammation and potential cardiovascular risk through activation of NFKB, a key transcriptional factor in the inflammatory cascade, as is IL-6 ((Pickup et al , 1997, Diabetologia 40: 1286; Festa et al, 2002, Diabetes 51; 1131; Kern et al, 2002, Am J Physiol Endocrinol Metab 280: E745-51 ; Vozarova et al, 2001 , Obes Res 9:414; Senn et al, 2002, Diabetes 51: 3391; Ahmad etal , 1997, J Cell Biochem 64:117; Uysal et al , 1997, Nature 389: 610; Collart et al , 1990, MoI Cell Biol 10:1498; Baltimore et al , 1990, MoI Cell Biol 10:2327).
  • PAI-1
  • RBP4 retinol-binding protein 4
  • WO 2005/059564 discloses methods of diagnosing insulin resistance by detecting modulation of RBP4 activity.
  • US 2005/0244892 discloses the monitoring of serum levels of resistin, a product of human monocytes and macrophages and, to a lesser extent, adipocytes.
  • Levels of resistin correlate with chronic inflammation and may be used as a biomarker for cardiovascular disease.
  • WO 2005/028509 discloses the use of a cross-reacting anti-TLR2 antibody to block TLR-2-mediated immune ceil interaction, particularly in the case of septic shock.
  • LAL Limulus amoebocyte lysate
  • WO 00/53165 discloses the blocking of the production and absorption of LPS from the gut in individuals suffering from cachexia and wasting syndromes. Many chronic diseases are linked to cachexia and weight loss linked to an inflammatory response and this application proposes blocking LPS from the gut as a source of this inflammation.
  • the methods disclosed include administration of bile acids, but also blocking the action of LPS by anti-LPS antibodies, LPS binding protein, soluble CD14 and TLR 2- and TLR4-blocking agents. The method is suggested as suitable for those suffering from cachexia, including cachexia resulting from diabetes.
  • Thiozolidinediones are a class of insulin-sensitising compounds, commonly used as an adjunct treatment for the insulin resistance characteristic of type 2 diabetes. Thiozolidinediones act by binding to nuclear peroxisome proliferator- activated receptors (PPARs), primarily PPARy, which are normally activated by free fatty acids and eicosanoids. Methods of treatment of diabetes-related conditions with such compounds are well-known in the art (see, for instance, EP 1671637).
  • PPARs nuclear peroxisome proliferator- activated receptors
  • LPS derived from commensal gut bacteria might contribute to sub-clinical inflammation and perhaps, in part, to the pathogenesis of type 2 diabetes, with elevated levels of LPS being demonstrated in type 2 diabetes patients (McTeman et al, 21 January 2006, Keystone Conference Symposia Proceedings. Diabetes Mellitus and the Control of Cellular Energy Metabolism. Abstract No. 235: Sub-clinical Inflammation in Type 2 Diabetes: Effect of Endotoxemia on Initiating the Inflammatory Cascade in Human Adipose Tissue. pp168).
  • the present invention provides a method of determining the risk of metabolic or cardiovascular condition or disease in an individual comprising the steps of: (a) obtaining a biological sample from the individual, and
  • the invention also provides a method for monitoring an individual for the onset or stage of metabolic or cardiovascular condition or disease, comprising the steps of:
  • An elevated concentration or amount of LPS may usually be indicative of the onset or progression of said metabolic or cardiovascular condition or disease, the concentration or amount of LPS being measured:
  • the standard value may be one which has previously been established through clinical studies. The value may be for patients in general regardless of sex, age, BMI or ethnic origin. For example, for non-diabetic individuals in a population, the mean serum level of LPS is about 3.1 +/- 1.7 EU ml "1 .
  • the level of LPS in the sample from the individual may be combined with other data taken from the individual. Such data may include one or more of gender, age, fat mass, body mass index (BMI) and/or ethnicity.
  • BMI body mass index
  • the individual selected for testing is obese or alternatively or additionally is a member of a defined ethnic origin, for example South Asian, Indian, Black African or Caucasian. Most preferably, the subject is of South Asian or Indian origin.
  • onset of T2DM in any given individual is indicated by a mean serum level in the range of 3.1 to 5.5 EU ml "1 .
  • T2DM An individual who has developed T2DM is indicated by an LPS serum level of at least 5.5 +/- 1.6 EU mI "1 .
  • the biological sample used for the method is blood or plasma, preferably serum.
  • the invention also provides a method of diagnosing a metabolic or cardiovascular condition or disease in an individual comprising the steps of
  • the metabolic or cardiovascular condition or disease may be one selected from obesity, polycystic ovary syndrome (PCOS), non-alcoholic fatty liver disease (NAFLD), atherosclerosis, coronary artery disease, metabolic syndrome, hyperinsulinaemia, insulin resistance and type 2 diabetes mellitus (T2DM).
  • PCOS polycystic ovary syndrome
  • NAFLD non-alcoholic fatty liver disease
  • atherosclerosis CAD
  • coronary artery disease CAD
  • metabolic syndrome hyperinsulinaemia
  • insulin resistance type 2 diabetes mellitus
  • the metabolic condition or disease is type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver degeneration or insulin resistance.
  • T2DM type 2 diabetes mellitus
  • non-alcoholic fatty liver degeneration or insulin resistance.
  • An elevated concentration or amount of LPS may indicate that the individual has the metabolic or cardiovascular disease or condition. In preferred embodiments it is the elevated concentration or amount of LPS which is determined:
  • An LPS serum value of 4 to 20 EU ml "1 , and more preferably 5 to 15 EU ml "1 may be indicative of the presence of type 2 diabetes mellitus (T2DM).
  • the subject of any of the methods of the invention is preferably a member of a high risk group, more preferably the subject is obese or of a defined ethnic origin, for example South Asian or Indian, Black African or Caucasian. More preferably they are of South Asian or Indian origin.
  • a defined ethnic origin for example South Asian or Indian, Black African or Caucasian. More preferably they are of South Asian or Indian origin.
  • LPS serum level When employing methods of the invention for assessment of risk, onset or stage of non-alcoholic fatty liver disease, this is indicated by an LPS serum level of about 5.5 EU ml "1 and above.
  • the presence of NAFLD in an individual is indicated by LPS serum levels in the range 5.5 - 12 EU ml "1 .
  • the invention also provides the use of LPS as a marker in an assay for determining the risk of metabolic or cardiovascular condition or disease in an individual, wherein the assay is carried out on a biological sample from the individual as hereinbefore described.
  • LPS is used as a marker in an assay for monitoring an individual for the onset or stage of metabolic or cardiovascular condition or disease, wherein the assay is carried out on a biological sample from the individual as hereinbefore described.
  • the invention further provides a method of treating an individual at risk of developing a metabolic or cardiovascular disease or condition comprising
  • the anti-inflammatory agent may be a thiazolidinedione or a pharmaceutically acceptable salt thereof, preferably rosiglitazone.
  • the anti-inflammatory agent may be an anti-LPS antibody, an anti- CD 14 antibody, an anti-TLR 2 antibody or a TLR 2 antagonist.
  • the invention further provides a method of identifying an agent for the prevention or treatment of metabolic or cardiovascular disease comprising:
  • LPS endotoxin
  • TLR Toll-like receptor
  • the at least one Toll-like receptor may be Toll-like receptor 2 (TLR 2) and/or Toll-like receptor 4 (TLR 4).
  • the invention also provides a method of identifying an agent for the prevention or treatment of metabolic or cardiovascular disease comprising:
  • the monitoring of the innate immune pathway may comprise monitoring of the level of (a) pro-inflammatory cytokines and/or (b) adiponectin, or the expression of (a) pro-inflammatory cytokines and/or (b) adiponectin, whereby decreased levels or expression of pro-inflammatory cytokines and/or increased levels or expression of adiponectin identifies a said agent.
  • the pro-inflammatory cytokines are preferably adipocytokines.
  • the pro-inflammatory cytokines or adipocytokines may be selected from one or more of lL-1. IL.-6, or TNF ⁇ .
  • the innate immune pathway may be monitored or additionally monitored by determining the level or expression of CD14, soluble CD14 and/or plasminogen activator inhibitor type 1 (PAI-1).
  • the immune pathway may be monitored or additionally monitored by determining activation of N F ⁇ Band/or translocation of NFKB to the nucleus.
  • said animal is a rodent, preferably a rat or a mouse.
  • the adipose tissue may be subcutaneous abdominal adipose tissue, preferably human subcutaneous abdominal adipose tissue.
  • the adipocyte is preferably a cultured adipocyte, more preferably a mature human adipocyte or a human subcutaneous abdominal adipocyte.
  • the various methods of the invention provides various technical advantages over the state of the art.
  • the invention permits a simple test for indicating the risk, onset or progression of a variety of metabolic and cardiovascular diseases, including insulin resistance, type 2 diabetes, atherosclerosis, coronary heart disease, obesity and non-alcoholic fatty liver disease. Hitherto it has been difficult to identify patients at risk before they develop clinical symptoms.
  • the methods of the invention also when combined with other routine examination and measurement of patients, e.g. height, weight, BMI, fat mass, gender, age and ethnicity, for example, permit earlier diagnoses of nascent medical conditions than has hitherto been possible.
  • the invention is predicated in part by a discovery and realisation of the inventors which is that the metabolic and cardiovascular diseases are linked by low-grade chronic inflammation related to circulating bacterial lipopolysaccharide (LPS, endotoxin) and that measuring the levels of LPS provides a quantitative biomarker for the risk of an individual developing such disease states, as well as the onset and progression (stage) of these diseases.
  • LPS circulating bacterial lipopolysaccharide
  • the invention is useful as a simple means of indicating individuals developing asymptomatic but progressive insulin resistance that often precedes frank type 2 diabetes, allowing therapeutic intervention at an early stage to reduce the risk of developing diabetes or cardiovascular disease in this high risk group.
  • the finding of raised serum LPS in an individual is closely linked to diseases related to the so-called 'metabolic syndrome'.
  • a finding of raised serum LPS is therefore useful in itself and, when taken together with the medical history, risk profile and other relevant biochemical and physical measures well-known in the art, offers an early diagnosis of hyperinsulinaemia, insulin resistance, type 2 diabetes, cardiovascular disease including coronary heart disease and atherosclerosis, and inflammatory liver disease such as non-alcoholic fatty liver disease (NAFLD), including its progressive stages of steatosis, steatohepatitis (non-alcoholic steatohepatitis - NASH), fibrosis and cirrhosis.
  • NAFLD non-alcoholic fatty liver disease
  • the inventors have shown a close relationship between circulating LPS and insulin levels; patients with increased insulin levels also show an increase in LPS levels. Furthermore they show that LPS works through the cell-surface receptor TLR-2 to initiate a pro-inflammatory response from adipose tissue.
  • a negative or negligible level of circulating LPS may be regarded as a 'well-being' marker in an otherwise high-risk individual.
  • Obese individuals are particularly at risk from this spectrum of diseases and so constitute a important target group for testing.
  • Obesity of the central, visceral or abdominal type characterised by a raised body mass index (BMI) but a disproportionately increased waist measurement, is a notable risk factor.
  • BMI raised body mass index
  • Assessment of obesity and significant waist measurement is dependent on an overall clinical judgement, but the American Heart Association guidelines suggest that a measurement of 40 inches (102 cm) for men, and 35 inches (88cm) for women as indicative of central obesity.
  • the invention provides a means of further defining the risk for such individuals. It is equally applicable to obese children and provides a particularly useful early indication of disease onset in this group.
  • LPS endotoxin
  • FIG 2 shows TLR 2 expression by quantitative Western blot in (A) obese and non-obese abdominal subcutaneous adipose tissue; and (B) abdominal subcutaneous adipose tissue from non-diabetic and type 2 diabetic individuals.
  • Figure 3 shows the increase in TLR 2 expression in response to LPS in abdominal subcutaneous adipose tissue (relative fold increase, quantitative Western blotting).
  • Figure 4 compares IL-6 and TNF- ⁇ production in response to LPS in cultured adipocytes.
  • CC Case Controls
  • FLD Fatty Liver Disease
  • Figure 8 shows the elevation in sCD14 levels in NAFLD; highest in cirrhosis.
  • Figure 9 shows the correlation between sCD14 levels and severity of liver cirrhosis.
  • Figure 10 shows that sCD14 is higher in diabetes than non-diabetics with NAFLD.
  • Figure 11 shows sCD14 correlates with fasting blood glucose
  • Figure 12 shows distribution of LPS levels before (A) and after (B) by transformation.
  • Figure 14A shows relationships between circulating plasma endotoxin levels and metabolic variables in men (White; African origin; South Asian).
  • Figure 14B shows relationships between circulating plasma endotoxin levels and metabolic variables in women (White; African origin; South Asian).
  • Figure 15 shows the correlation between TNF- ⁇ and log LPS levels in type 2 diabetes patients.
  • Figure 16 shows the correlation between circulating LPS levels and fasting insulin levels in a non-diabetic cohort (see Example 2).
  • Example 1 LPS activates the innate immune response in human adipose tissue in obesity and type 2 diabetes
  • the aforementioned serum samples were analyzed for the determination of insulin, (Linco Research Inc. Missouri, USA) leptin, (Unco Research Inc. Missouri, USA), IL-6, (Bender MedSystems, Vienna, Austria) TNF- ⁇ , (Bender MedSystems, Vienna, Austria) and sCD14 (R&D Systems, Abingdon, UK) protein concentrations. Insulin, leptin, sCD14, TNF- ⁇ and IL-6 levels were analyzed by a solid phase enzyme linked immunosorbent assay.
  • anthropometric data was collected-detailed in tables: 1 and 2.
  • Fasting glucose was analyzed using a glucose oxidase method (YSL 200 STAT plus).
  • Insulin resistance (HOMA IR) was derived using the HOMA equation (Matthews etal, 1985, Diabetologia 28: 412). Serum endotoxin was assayed using a QCL-1000 LAL Endpoint Assay (Cambrex, New Jersey, USA).
  • the cells were maintained in this medium for 14h either treated with 100ng/mL LPS (Sigma Aldrich, Gillingham, UK) or untreated to act as matched paired controls. Following treatment, the conditioned media and adipocytes were separated by centrifugation. Conditioned medium was removed and stored at -8O 0 C. Protein was extracted from the adipocytes using RIPA buffer (McTernan et al, 2000, lnt J Obes Relat Metab Disord 24:875) containing; 15OmM NaCI, 1 -0% IGEPAL® CA-630, 0-5% sodium deoxycholate, 0-1% SDS, and 5OmM Tris.
  • Conditioned media 100 ⁇ l) from untreated (control) and LPS treated abdominal subcutaneous adipocytes, were assayed for IL-6 and TNF- ⁇ (QuantiGlo ELISA, R&D Systems, Abingdon, UK) using a solid phase enzyme linked immunosorbant assay: IL-6 intra-assay CV 3-1%, inter-assay CV 2-7%; TNF- ⁇ intra-assay CV 6-7%, inter-assay CV 11 -0%.
  • Total protein was extracted from homogenized abdominal subcutaneous tissue from the obese, lean and diabetic cohorts, as previously described, using the RIPA buffer technique. Homogenized whole human adipose tissue (cohort as discussed) was extracted using a 10% RIPA buffer method. Western blot analysis was performed and relative expression was standardized using densitometry quantification software (GeneTools, Geneflow, Fradley, UK). In brief, 10-40 ⁇ g of protein was loaded onto a 10% polyacrylamide gel (Geneflow, Fradley, UK).
  • TLR-2 human monoclonal toll-like receptor 2 primary antibody
  • TLR-4 human polyclonal anti-TLR 4
  • HRP horseradish-peroxidase
  • Equal protein loading for these samples was confirmed by examining ⁇ - tubulin expression using Western blotting, as previously described (McTeman et al, 2000, lnt J Obes Relat Metab Disord 24:875). Protein concentrations were determined using the Bio-Rad DC (Detergent Compatible) protein assay kit.
  • TLR protein expression is higher in obese and Type 2 diabetic subjects
  • TLR-2 protein expression is upregulated by LPS in human isolated abdominal subcutaneous human adipocytes.
  • TLR-2 and 4 protein expression was shown in isolated subcutaneous adipocytes.
  • Treatment of abdominal subcutaneous adipocytes with LPS increased the expression of TLR-2 two-fold compared with control (p ⁇ 0 05; Figure 3).
  • There was no change observed in TLR-4 expression with LPS treatment (Data not shown).
  • IL-6 and TNF- ⁇ were increased in the in vitro cultured adipocytes treated with LPS (IL-6: Control p ⁇ 0-01; TNF- ⁇ : p ⁇ 001; Figure 4). This corresponded with the increase in TLR-2 expression as previously discussed (Fig. 3).
  • Serum endotoxin is significantly reduced by the insulin sensitizer, rosiglitazone, and change in serum endotoxin correlates with the fall in insulin observed in the diabetic patients.
  • RSG rosiglitazone
  • RSG-treated type 2 diabetes patients showed an absolute change in insulin levels that also correlated with the change in circulating LPS levels.
  • RSG is known to have direct immunomodulatory properties on adipose cells and appear to reduce inflammation in both in vitro and in vivo model systems (Cuoco et al, 2002, Hepatogastroenterology 49:1582).
  • the reduction in inflammation seen during RSG treatment may, at least in part, be due to the change in circulating LPS levels.
  • Type 2 diabetes patients have significantly higher levels of serum endotoxin than non-diabetic subjects and the innate immune system is upregulated within obese and type 2 diabetes adipose tissue. Serum insulin and endotoxin and are closely related.
  • Example 2 chronic endotoxaemia in non-alcoholic fatty liver disease
  • the sub- categories of FLD were determined by liver biopsies and liver function tests by ballooning and/or fibrosis (Brunt etal, 1999). Diabetic status was also ascertained by glucose and insulin levels.
  • Serum endotoxin was assayed using a commercially available QCL-1000 LAL Endpoint Assay (Cambrex, New Jersey, USA). Intra-assay CV 3.9 ⁇ 0.46, inter- assay CV 9.6 ⁇ 0.75. Assessment of inflammatory markers and adiponectin.
  • Serum was analysed by enzyme-linked immunosorbent assay (ELISA) for quantitative detection of the inflammatory markers: soluble CD14, soluble TNF- ⁇ Receptor Il (TNFRII), Resistin, TNF- ⁇ (R&D Systems, UK) and Leptin (Biogenesis Ltd, UK) and of the anti-inflammatory cytokine: Adiponectin (Biogenesis Ltd, UK); according to manufacturer's instructions.
  • ELISA enzyme-linked immunosorbent assay
  • Adiponectin was determined by an enzyme immunoassay (Linco Research, Inc. Missouri, USA) with a sensitivity of 0.78ng/mL and an intra- and interassay CV of 3.3% and 5.5% respectively.
  • Endotoxin levels were significantly higher in patients with FLD compared with CC
  • FLD 11.06 ⁇ 0.96EU/mL (mean ⁇ SEM); CC: 5.27 ⁇ 0.45EU/mL, p ⁇ 0.01; Figure 5A).
  • FLD alone produced comparable endotoxin levels to T2DM (FLD:T2DM:10.82 ⁇ 1.15EU/mL; Non-Diabetic: 11.16 ⁇ 0.90EU/mL; Figure 5B).
  • Analysis of the different stages of NAFLD demonstrated that endotoxin levels were raised at all stages of NAFLD when compared to CC (FL: 12.95 ⁇ 1.37 EU/mL, NASH: 10.19 ⁇ 0.77 EU/mL and CRR: 8.24 ⁇ 2.10 EU/mL, Figure 6).
  • Circulating endotoxin levels and relationship with fasting insulin are associated with Circulating endotoxin levels and relationship with fasting insulin.
  • sCD14 circulating levels were raised in FLD compared to CC (FLD: 1645.87 ⁇ 46.12 ng/ml (mean ⁇ SEM) and CC: 1450.57 ⁇ 69.17ng/mL, p ⁇ 0.05, Figure 7A).
  • the levels of sCD14 in FLD were higher in FLD with diabetes (FLD:T2DM:1903 ⁇ 0.12ng/ml; Non-Diabetic: 1558 ⁇ 0.05ng/ml; Figure 10) and correlated with fasting blood glucose levels (Figure 11).
  • LPS levels were significantly higher in patients with FLD compared with case control subjects.
  • FLD alone produced similar LPS levels to type 2 diabetes subjects with FLD.
  • Type 2 diabetes patients are known to have gut dysmotility (Zietz et al, 2000) which may affect circulating LPS levels, however increased LPS was also identified in the non-diabetic FLD patients.
  • Other factors appear to alter LPS levels prior to the onset of type 2 diabetes. The data here appears to confirm that whilst diabetic status may raise LPS levels compared with case control subjects, the impact of liver disease in a pre-diabetic state also represents a significant contributor to raised LPS levels.
  • Example 3 Circulating LPS levels as a marker for cardiovascular and metabolic risk in ethnic minority groups
  • 62 were white (30 women)
  • 68 were of African origin (33 women)
  • 63 were of South Asian origin (33 women).
  • the Local Ethics Committee approved the study. All participants gave their informed consent to participate.
  • LAL Test Chromogenic Limulus Amoebocyte Lysate (Cambrex, New Jersey, USA), on heparinised plasma, which had been stored at -40 0 C and defrosted at room temperature prior to analysis.
  • LAL Test is a quantitative test for gram-negative bacterial endotoxin. A sample is mixed with the LAL supplied in the test kit and incubated at 37°C ( ⁇ 1°C) for 10 minutes. The reaction is stopped after a further minute incubation (37°C [ ⁇ 1 0 C]) with the substrate solution.
  • the amount of endotoxin present in the sample is proportional to the yellow colour that develops, which is determined spectrophotometrically at 405-410 nm and can be calculated by comparison with a standard curve. All the samples were diluted 1 in 7 with LAL water using a standard curve in the range of 0 to 5 EU/mL. All samples were run in duplicate within the same 96-well plate. To prevent contamination all items used in the assay were sterilised (by autoclave or treatment with microsol). Intra-assay coefficient of variation was 3-9 ⁇ 0-46 and inter-assay CV 9-6 ⁇ 0-75.
  • Plasma levels of endotoxin were not normally distributed. Therefore analyses were performed on log-transformed data (Figure 12). Differences between groups (with described adjustments) were tested using analysis of co-variance. Results are presented as geometric means and 95% confidence intervals (Cl). General Linear Models were used to estimate the relationship between endotoxin levels and cardiovascular risk factors adjusting for confounders. Interaction test was used to compare slopes between groups. Given the degree of multiple testing, only a p ⁇ 0.005 (Bonferroni's correction) was considered as statistically significant.
  • endotoxin levels are associated with the expected differences in atherosclerotic vascular disease risk. That is endotoxin levels are higher in men than women and highest in South Asians and lowest in people of African origin. In addition, endotoxin levels are associated with cardiovascular risk factors such as waist-hip ratio and serum lipid levels.
  • Example 4 LPS as a mediator of sub-clinical inflammation in obese children
  • HOMA-IR homeostasis model assessment
  • ISI Stumvoll index
  • the vascular injury markers for these obese children were measured using a multiplex assay system that analysed interleukin - (IL-) 1 ⁇ , 6, 8 and 10, interferon- Y (IFN- Y), TNF- ⁇ , monocyte chemotactic protein-1 (MCP-1), n-terminal brain natriuretic peptide (NT-proBNP), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor 1 (PAI-1), C-reactive protein (CRP), fibrinogen, serum amyloid A (SAA), serum amyloid P component (SAP), E-selectin, soluble intercellular adhesion molecule type 1 (slCAM-1), soluble vascular cell adhesion molecule type 1 (sVCAM-1), matrix metalloproteinase -9 (MMP9), myeloperoxidase, lipid peroxide modified protein (MPO) and adiponectin.
  • IL- interleukin -
  • IFN- Y
  • BMI vascular endothelial growth factor
  • Waisfchip ratio Men 0.91 (0.89 to 0.93) 0.91 (0.89 to 0.93) 0.94 (0.92 to 0.96)
  • Serum triglycerides Men 0.84 (0.73 to 0.97) 1.14 (0.98 to 1.33) 1.30 (1.11 to 1.53)
  • HDL cholesterol (mmol/L) Men 1.3 (1.2 to 1.4) 1.3 (1.2 to 1.4) 1.2 (1.1 to 1.3)
  • Serum Insulin (mU/L)* Men 7.1 (6.0 to 8.5) 6.6 (5.6 to 7.9) 10.4 (8.6 to 12.5)
  • Results are means f*Geometric means) or percentages (95% CD .
  • P values are for test of heterogeneity between ethnic groups by analysis of co-variance. There were no more than two values missing from any cell.
  • Example 5 Elevated Endotoxin levels as a mediator of continued Chronic sub-clinical inflammatory risk in coronary artery bypass graft (CABG) patients.
  • CABG coronary artery bypass graft
  • endotoxin levels are significantly raised in CABG.
  • the associated endotoxinaemia appears to mediate chronic inflammation observed by raised HsCRP levels.
  • the unaltered sCD14 and TNFRII levels in disease indicate these factors correlate with the early developmental phases of this disease.
  • the continued reduction in adiponectin levels indicates that the inflammatory cytokine profile arises through elevated endotoxin levels which may mediate the continued inflammatory state.
  • Example 6 South Asians with impaired glucose tolerance and subclinical inflammation have reduced endoxtin levels and adiponectin levels with increased weight loss using orlistat.
  • Sub-clinical inflammation is an important factor in the pathogenesis of type 2 diabetes and may be influenced by ethnicity and the degree of glucose intolerance.
  • the study compared inflammatory markers and adipocytokines in South Asian individuals with impaired glucose tolerance (IGT-Group) to age-, sex- and BMI-matched individuals with normal glucose tolerance (NGT-Group).
  • ITT-Group impaired glucose tolerance
  • NTT-Group normal glucose tolerance

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Abstract

Pour surveiller la progression des maladies métaboliques et cardiovasculaires chez les individus prédisposés, on mesure les niveaux de lipopolysaccharides (LPS) dans des échantillons de tissus. Une recherche des lipopolysaccharides du sang permet de surveiller la progression ou de diagnostiquer le déclenchement de la résistance à l'insuline, de l'obésité, des stéatopathies non alcooliques, de l'athérosclérose, de la coronaropathie et du diabète de type 2, particulièrement chez des individus obèse. Pour la recherche systématique d'agents actifs contre les maladies métaboliques et cardiovasculaires, on procède par dosage impliquant l'expression de récepteurs de type Toll, et la surveillance réponse immunitaire innée dans les cellules et tissus adipeux.
PCT/GB2006/002714 2006-07-20 2006-07-20 Prédiction de maladie par dosage des lipopolysaccharides Ceased WO2008009869A1 (fr)

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WO2010052334A1 (fr) * 2008-11-10 2010-05-14 F. Hoffmann-La Roche Ag Pertinence de la fonction intestinale des adipocytes dans le diabète de type 1
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WO2015007464A1 (fr) * 2013-07-18 2015-01-22 Nestec S.A. Escherichia coli en tant que marqueur de l'hypertriglycéridémie
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JPWO2014104305A1 (ja) * 2012-12-28 2017-01-19 持田製薬株式会社 プレセプシン測定による炎症性腸疾患の診断
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JP2022074806A (ja) * 2020-11-05 2022-05-18 カゴメ株式会社 健康状態評価システム、健康状態評価プログラム及び健康状態評価方法、並びに、食生活評価システム、食生活評価プログラム及び食生活評価方法

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010052334A1 (fr) * 2008-11-10 2010-05-14 F. Hoffmann-La Roche Ag Pertinence de la fonction intestinale des adipocytes dans le diabète de type 1
EP2624844A4 (fr) * 2010-10-04 2014-03-26 Immuron Ltd Méthodes et compositions utilisant des anticorps anti-ligand lps destinées au traitement et à la prévention de troubles inflammatoires
JPWO2014104305A1 (ja) * 2012-12-28 2017-01-19 持田製薬株式会社 プレセプシン測定による炎症性腸疾患の診断
WO2015007464A1 (fr) * 2013-07-18 2015-01-22 Nestec S.A. Escherichia coli en tant que marqueur de l'hypertriglycéridémie
US20150024422A1 (en) * 2013-07-18 2015-01-22 Health Diagnostics Laboratory, Inc. METHOD OF DETERMINATION OF RISK OF 2 HOUR BLOOD GLUCOSE EQUAL TO OR GREATER THAN 140 mg/Dl
US10475536B2 (en) * 2013-07-18 2019-11-12 True Health Ip Llc Method of determination of risk of 2 hour blood glucose equal to or greater than 140 mg/dL
US11143659B2 (en) 2015-01-27 2021-10-12 Arterez, Inc. Biomarkers of vascular disease
US11821905B2 (en) 2015-01-27 2023-11-21 Arterez, Inc. Biomarkers of vascular disease
JP2022074806A (ja) * 2020-11-05 2022-05-18 カゴメ株式会社 健康状態評価システム、健康状態評価プログラム及び健康状態評価方法、並びに、食生活評価システム、食生活評価プログラム及び食生活評価方法
JP7604181B2 (ja) 2020-11-05 2024-12-23 カゴメ株式会社 健康状態評価システム、健康状態評価プログラム及び健康状態評価方法、並びに、食生活評価システム、食生活評価プログラム及び食生活評価方法
JP2025038040A (ja) * 2020-11-05 2025-03-18 カゴメ株式会社 健康状態評価システム、健康状態評価プログラム及び健康状態評価方法、並びに、食生活評価システム、食生活評価プログラム及び食生活評価方法

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