WO2007088349A2 - Use of anti-vimentin antibodies in diagnosis of hypertension or heart failure - Google Patents

Abstract

A method for predicting or diagnosing hypertension in a mammal, the method comprising detecting the presence and/or level of anti-vimentin antibodies in a suitable sample obtained from the mammal. A method for predicting or diagnosing heart disease in a mammal, the method 0 comprising detecting the presence and/or level of anti-vimentin antibodies in a suitable sample obtained from the mammal.

Description

ASSAY

This invention relates to a diagnostic assay, and in particular an assay for predicting and/or diagnosing hypertension and heart disease in a mammal.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge

As in humans, hypertension is an important medical condition in cats. Feline hypertension is commonly found as a complication of other underlying medical conditions (so-called 'secondary hypertension'), although primary hypertension (hypertension without any identifiable underlying disease) is also seen. The most common causes of secondary hypertension in cats are chronic kidney disease and hyperthyroidism, while more rare causes of hypertension include acromegaly and Cushing's disease. Hypertension associated with chronic renal failure (CRF) in cats is discussed by Syme et al (2002) and Mishina et al (199S).

Hypertension is damaging to the body, and may have serious effects in the eyes (choroidoretinopathy, bleeding into the anterior chamber of the eyes, and retinal changes such as oedema, haemorrhage and detachment can occur which may result in permanent blindness); brain and nervous system (neurological signs such as odd behaviour, ataxia, dullness, seizures, dementia and coma); heart (left ventricular thickening, congestive heart failure, breathlessness and lethargy); and kidneys (renal failure).

Clinically, since hypertension is often seen as an effect of other diseases, cats with hypertension may show signs attributable to their underlying problem. For example, in the case of hyperthyroid cats with high blood pressure, weight loss (in spite of a voracious appetite) and hyperactivity may be the major clinical signs. In many cats, no specific clinical signs of hypertension will be seen until the condition advances to the point where there is spontaneous bleeding into the eye or retinal detachment - these cats are often taken to a veterinary surgeon as they develop sudden onset blindness. Early recognition of hypertension is therefore important in order to minimise the severe and often permanently damaging effects of persistently high blood pressure on the eyes and other organs. Some cats with hypertension appear depressed, lethargic and withdrawn, and many owners notice an improvement in their cat's behaviour once hypertension has been successfully managed, even if signs of damage to other organs are not present.

A number of agents are available for treatment of hypertension although many are not specifically licensed for this use in cats. Examples of drugs commonly used are amlodipine (Istin^®) and, less commonly, benazepril (Fortekor^®). Feeding a low salt diet may also be of value although it is unlikely to be sufficient as a sole treatment of hypertension.

In primary hypertensive cases (where there is no underlying disease that has caused the high blood pressure) it is usually possible to manage the hypertension and prevent future complications such as damage to the eyes. In cases of secondary hypertension, the long-term outlook is very dependent on the nature and severity of the disease that has caused the high blood pressure. Cats with CRF that have developed hypertension have a worse prognosis than those where the cause of the high blood pressure is treatable, such as hyperthyroidism. It is important in all cases that the hypertension is monitored as accurately as possible on a regular basis in order to pre-empt problems such as blindness. In cats where blindness has occurred as an effect of hypertension, control of blood pressure can still be beneficial and affected cats may live for several years with a good quality oflife.

Since hypertension is now recognised as a common finding in geriatric cats, and because such cats may be asymptomatic, annual blood pressure screening is generally recommended for all geriatric cats (12 years or older) and at any age and/or more frequently in cats with known kidney or thyroid abnormalities.

A detailed eye examination by ophthalmoscopy is also usually performed since ocular disease is common in hypertensive cats. In mildly affected cats, subtle changes to the appearance of retinal blood vessels and to the retina itself may be seen. However, subtle changes in the retina may not be recognised by general veterinary practitioners and it can be difficult to get a good view of a cat's retina if the cat is uncooperative. In more severely affected cats, the changes can be dramatic and include retinal detachment and bleeding into the eye. Abnormalities are usually detected in both eyes although they may be more severe in one. In the absence of blood pressure measurement devices, a thorough ocular examination may enable a diagnosis of hypertension to be made and can be used to monitor progress once treatment has been started. However, proper blood pressure measurements are much preferred, both for diagnosis and for monitoring a cat's response to therapy.

Diagnosis of hypertension is ideally made following measurement of blood pressure. Various techniques and equipment are available and many veterinary clinics now have these facilities. The equipment used typically comprises an inflatable cuff placed around one of the front legs or the tail and a Doppler flow probe. Measuring blood pressure only takes a few minutes, and is completely pain-free. Nevertheless, taking a blood pressure reading from a cat is more difficult than in humans, since their arteries are very small and are covered with hair. Also, cats are easily stressed which can lead to a false reading. Since many cats display such white coat hypertension (Belew et al, 1999), adequate steps must be taken to ensure accurate measurement of systolic blood pressure by allowing acclimatisation and establishing repeatability of readings.

There is, therefore, a need in the art for alternative methods of diagnosing hypertension in cats which avoids the problems of white coat hypertension (false blood pressure readings due to stress in a clinical situation).

There is also a need in the art for methods of predicting hypertension in advance of a cat displaying clinical signs associated with hypertension.

In dogs as in humans, heart failure is a very serious, life threatening condition with adverse morbidity and mortality. Its dismal prognosis has led to efforts to identify the condition early in order to treat it more effectively. Biomarkers may have, utility in further improving the diagnostic and predictive capabilities that clinicians apply in routine practice. Heart disease and heart failure can occur in dogs of all breeds, weights and ages, and affects an estimated 15% of dogs in the UK. Heart disease in dogs typically includes chronic valvular disease (commonly seen in certain small breeds such as the Cavalier King Charles, Chihuahua, Miniature Schnauzer, Maltese, Pekinese and Whippet); myocardial .disease (commonly seen in certain large breeds such as Dobermans, Irish Wolfhounds, Great Danes, St Bernards and Newfoundlands); idiopathic pericardial effusion (commonly seen in certain large breeds such as St Bernards); and arrhythmias (particularly in Cavalier King Charles, Pugs, Miniature Schnauzers, Boxers, German Shepherds, Dobermans and Irish Wolfhounds). Heart disease is most common among older dogs, affecting 25% of all dogs over the age of 7. If left untreated, heart disease and subsequent heart failure can be fatal, making it the second most common cause of death after cancer. Dogs generally suffer from congestive heart failure (CHF), a relatively slow worsening of symptoms. 95% of all heart disease seen in dogs is acquired heart disease and this usually appears after the dog reaches middle age. By the time a dog shows obvious signs of acquired heart disease it is likely that irreversible damage has already been done. Clearly, the earlier that heart disease is diagnosed, the better a veterinarian will be able to manage the condition and to delay the onset of heart failure.

Heart disease can be present without signs of a failure to effectively pump blood around the body. For example, there may be a murmur detected or an enlarged heart seen by imaging techniques but no fluid retention, cough or exercise intolerance to suggest that the function of the heart is failing. Signs of heart failure include both congestive signs and low output signs e.g. collapse. Heart failure usually occurs secondary to heart disease.

There is, therefore, a need in the art for improved methods of diagnosing heart failure in dogs. There is also a need in the art for methods of predicting heart disease in advance of a dog displaying clinical symptoms associated with heart failure.

Vimentin is an intermediate filament polypeptide present in cells of mesenchymal origin. Along with the microfilaments (actins) and microtubules (tubulins), the intermediate filaments represent a third class of well-characterised cytoskeletal elements. The subunits of intermediate filaments display a tissue-specific pattern of expression, and vimentin is the sύbunit specific for mesenchymal tissue (OMIM Reference # 193060).

Although vimentin has been presumed to be important for stabilising the architecture of the cytoplasm, Mor-Vaknin et al (2003) found that monocyte- derived macrophages secrete vimentin into the extracellular space in vitro. Secretion of vimentin was enhanced by the proinflammatory cytokine TNF-α and inhibited by the anti-inflammatory cytokine ILlO, suggesting that vimentin is involved in the immune response.

Colucci-Guyon et al (1994) showed that mice lacking vimentin develop and reproduce without an obvious phenotype.

US5,716,787, GB2268935, Dunn et al (1992) and Wheeler et al (1995) describe the presence of certain anti-endothelial antibodies, including anti-vimentin antibodies, in patients following organ transplantation. The anti-endothelial antibodies were found in patients diagnosed clinically as showing rejection of a transplanted organ, but were rarely found in transplant patients not diagnosed as showing rejection. The presence of these anti-endothelial antibodies was stated to be a reliable indicator of chronic rejection of a transplanted organ or tissue, and of associated pathological conditions such as transplant-associated coronary artery disease. However, none of these four publications contemplate that these anti- endothelial antibodies (such as anti-vimentin antibodies) may be indicative of hypertension, or heart failure that is not associated with transplant rejection. Jurcevic et al (1998) describe an ELISA method for measuring anti-vimentin antibodies in serum from patients who have undergone heart transplantation.

Jurcevic et al (2001) disclose that the presence of anti-vimentin antibodies in serum from patients after cardiac transplantation is an independent predictor of transplant-associated coronary artery disease, and can be used to identify patients who are high risk of developing this disease.

Yang et al (2002) detected anti-vimentin antibodies in the sera of patients with idiopathic pulmonary fibrosis and non-specific interstitial pneumonia, and suggested that anti-vimentin antibodies may be involved in the process of lung injury in these conditions.

We have now detected significantly elevated levels of anti-vimentin antibodies in hypertensive cats compared to normal, non-hypertensive cats. We have also shown significantly elevated levels of anti-vimentin antibodies in cats up to 500 days prior to diagnosis of secondary hypertension. We have therefore concluded that the presence of anti-vimentin antibodies in cats is a diagnostic and predictive marker for hypertension. Without wishing to be bound by theory, we consider that feline hypertension causes release of endothelial cell antigens such as vimentin, and the subsequent formation of anti-vimentin antibodies. The development of anti-vimentin antibodies precedes development of measurable systolic hypertension and retinal lesion formation in feline CRF, and detecting anti-vimentin antibodies is expected to be a useful clinical tool for the early prediction and diagnosis of hypertension in cats.

Furthermore, many features are shared between hypertension in humans and cats, including the frequent occurrence of retinopathy in untreated cases, increased risk of sudden collapse due to cardiovascular events (Elliott et al, 2001), neurological disease possibly caused by cerebrovascular bleeds (Maggio et al, 2000), heart murmur, gallop rhythm (Elliott et al, 2001), left ventricular hypertrophy (Snyder et al, 2001; Chetboul et al, 2003), heart failure and epistaxis (Liftman 1994). Feline and human hypertension both possibly occur as a result of consuming well in excess of the natural requirements of sodium chloride (most prepared cat foods are very high in salt to make them more palatable), and both feline and human hypertension occur most frequently in older individuals and are associated with kidney disease and low plasma potassium, particularly in ageing patients.

Thus, since feline systolic hypertension is also a useful clinical model of human hypertension, detecting anti-vimentin antibodies is also expected to be a useful clinical tool for the early prediction and diagnosis of hypertension in other mammals including humans.

Accordingly, a first aspect of the invention provides method for predicting or diagnosing hypertension in a mammal, the method comprising detecting the presence of anti-vimentin antibodies in a suitable sample obtained from the mammal.

This aspect of the invention includes a method for predicting or diagnosing hypertension in a mammal, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative of hypertension in the mammal. In other words, this invention includes a method of aiding in the prediction or diagnosis of hypertension.

It is appreciated that the invention includes a method of assessing whether a mammal is suffering from hypertension, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative of hypertension in the mammal. It is appreciated that this assessment may aid diagnosis, and may be used in association with other tests, or observations by the physician, in reaching a diagnosis.

Typically, the level of anti-vimentin antibodies in the sample that is indicative of hypertension is a level equal to or greater than the mean +2 standard deviations (SD) of the level of anti-vimentin antibodies found in a population of control mammals that do not have hypertension.

Alternatively, if the data from the control population is normally distributed (i.e. approximates a normal distribution), the level of anti-vimentin antibodies in the sample that is indicative of hypertension may be considered to be a level equal to or greater than the 97.5^th percentile of the level of anti-vimentin antibodies found in the population of control mammals that do not have hypertension.

When deteπnining the value of a diagnostic test, decision levels (i.e. cut-off values) can be defined by a Receiver Operating Characteristic (ROC) curve, which is a plot of the true positive rate against the false positive rate for the different possible cut-off points for a diagnostic test. Thus using methods well known in the art, such as MMtab Release 12 and S-plus software, suitable efficient cut-off values can be determined with the desired levels of sensitivity and specificity.

In an embodiment, antibody titres can be measured rather than the concentration of anti-vimentin antibodies (as described in Jurcevic et al, 2001). In humans, normal sera (n=20) gave a titre of 53±32.1 (mean ± SE), and a cut-off value of > 120 was chosen as indicative of transplant-associated coronary artery disease with a sensitivity of 63% and a specificity of 76% (Jurcevic et al, 2001).

The minimum size of control population used to define the reference range can readily be determined by a statistician. The composition of the control population is also important since a better match of the control population to the at-risk population (which includes the individual from whom the sample is taken) will reduce the size of the control population required for statistical significance. Thus, it is preferred if the population of control mammals is matched as closely as possible for age, gender and breed (where applicable) to the individual from whom the sample is taken. From a power calculation that we carried out using data from our pilot studies on cats, we calculated that a comparison of at least 40 normal non-hypertensive individuals and at least 40 hypertensive individuals would be required for a cut-off level to have true statistical significance. Preferably, at least 50 and more preferably at least 100 individuals are used in one or both groups when determining significant cut-off levels that indicate hypertension.

It is also appreciated that the invention includes a method of assessing whether a mammal may develop hypertension, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative that the mammal may develop hypertension. It is appreciated that this assessment may aid a predictive diagnosis, and may be used in association with other tests, or observations by the physician, in reaching a predictive diagnosis.

Typically, the level of anti-vimentin antibodies in the sample that is indicative that the mammal may develop hypertension is a level equal to or greater than the mean +2 SD of the level of anti-vimentin antibodies found in a population of control mammals that do not have hypertension and have been shown thereafter not to develop hypertension for a period of at least 3 months, preferably at least 6 months or one year, and more preferably for a period of two years. However, it is appreciated that other cut-off values may be used depending upon the desired levels of sensitivity and specificity for the assay, as discussed above.

In a first embodiment, the mammal is a cat.

The sample may be from a cat that has previously been diagnosed with a medical condition associated with hypertension, such as chronic renal failure, hyperthyroidism, acromegaly or Cushing's disease. The sample may be taken from a cat that has a clinical sign that may result from hypertension, such as retinopathy, ocular bleeding, retinal swelling, retinal detachment, blindness, odd behaviour, hyperactivity, ataxia, dullness, seizures, dementia, coma, left ventricular thickening, congestive heart failure, breathlessness, weight loss, and lethargy. It is appreciated that a sample can be from a cat that has no symptoms of hypertension, for example as a part of a routine health check up on a geriatric cat (12 years or older, and preferably from 8 years of age and upwards), in order to allow an early prediction or diagnosis of hypertension. Indeed older cats represent the major at-risk population, and about 17% of cats diagnosed as hypertensive do not have an identifiable underlying disease process.

The sample can be from a cat which has been identified as having high blood pressure but which has no lesions, or can be from a cat in which a blood pressure reading is borderline. In these cases, the test may predict those individuals at risk of developing lesions if their blood pressure is left untreated, distinguishing them from cats that are transiently hypertensive due to stress.

Conversely, the sample can be from a cat that has been shown not to have hypertension, at or around the time the sample was taken, in order to allow an early prediction of the condition.

As used herein, a cat that has a systolic blood pressure of less than 170mm Hg₅ and preferably on two consecutive occasions 7-14 days apart, does not have hypertension. Conversely, a cat that has a systolic blood pressure greater than 170mm Hg, and preferably on two consecutive occasions 7-14 days apart, has hypertension. According to the ACVIM Consensus Statement on Hypertension (2003), cats with a systolic blood pressure <150 mm Hg are at minimal risk of end-organ damage; those with blood pressure >150 but <160 mm Hg are at low risk; those between 160 and 180 mm Hg are at moderate risk; and those with a systolic blood pressure >180 mm Hg are at high risk of end-organ damage.

In an embodiment, at least in cats, the mammal does not have a thyroid disorder.

In Example 1, the positive cut-off for an ELISA for the presence of anti-vimentin antibodies in cat plasma that is indicative of hypertension was determined to be 125.8μg/ml from pilot experiments using a population of control cats (n=20), although this figure may be adjusted once samples from a larger cat population have been tested. Thus, in an embodiment, the cut-off value for a positive finding of anti-vimentiα antibodies in a cat plasma sample that is indicative of hypertension is at least 75μg/ml, more preferably at least lOOμg/ml, and still more preferably at least 125μg/ml.

In cats, the level of anti-vimentin antibodies in the sample that is indicative that the cat may develop hypertension is > 125μg/ml., based on the current reference range.

In other embodiments, the mammal may be a human patient, a dog (in which hypertension has been recognised as a clinical problem), or a horse or a pony (especially laminitic horses and ponies which have higher blood pressure than normal ponies).

The cut-off value for a positive finding of anti-vimentin antibodies in a human sample which is indicative of transplant rejection is a titre of at least 100, and more preferably a titre of at least 120 (measured according to Jurcevic et al, 2001), and similar levels are expected to be indicative of heart failure.

In an embodiment, the sample may be from a human patient who has previously been diagnosed with a medical condition associated with hypertension, such as obesity, metabolic syndrome, diabetes mellitus (type I or type II) or renal failure, or who has one or more clinical symptoms that may result from hypertension, such as stroke, myocardial infarction, heart failure, renal failure and retinopathy.

The sample may be from a human patient who has white coat hypertension, prehypertension (systolic blood pressure between 120-139 mm Hg), or stage 1 hypertension (systolic blood pressure between 140-159 mm Hg), according to the JNC VII guidelines (Chobanian et al, 2003).

It is appreciated that a sample can be from a human patient who has no symptoms of hypertension, for example as a part of a routine health check up, in order to allow an early prediction or diagnosis of hypertension. Similarly, the sample can be from a human patient who has been shown not to have hypertension, at or around the time the sample was taken, in order to allow an early prediction of the condition.

In a further embodiment of the first aspect of the invention, the level of one or more further clinical markers of hypertension is measured in a sample from the mammal, or is assessed in the mammal, and a determination is made whether the level of the one or more further markers is indicative of hypertension. Suitable further markers that can be tested include elevated serum lipids and microalbuminurea, which are both known risk factors for hypertension. Other suitable further tests for confirming a diagnosis of hypertension include ambulatory blood pressure monitoring.

Conveniently, the level of anti-vimentin antibodies and of the further hypertension marker are measured in the same sample taken from the mammal. Alternatively, the levels may be measured in separate samples taken from the mammal. Conveniently, the samples are taken from the mammal at substantially the same time, or within several hours of each other.

In a preferred embodiment, the presence and/or level of anti-vimentin antibodies and of the one or more further markers of hypertension are all taken into account when assessing whether the mammal has hypertension or may develop hypertension. It is believed that using a combination of markers may improve the accuracy of the assessment (e.g. diagnosis or prediction), but nevertheless deterrnining the level of anti-vimentin antibodies alone in a sample from the mammal is useful.

We have also detected significantly elevated levels of anti-vimentin antibodies in plasma samples from dogs in heart failure compared to normal dogs, and we expect to find significantly elevated levels of anti-vimentin antibodies in dogs prior to clinical symptoms of heart failure. We have therefore concluded that the presence of anti-vimentin antibodies in dogs is a diagnostic and predictive marker of heart failure. Without wishing to be bound by theory, we consider that heart failure in dogs causes the release of endothelial cell antigens such as vimentin, and the subsequent formation of anti-vimentin antibodies. The development of anti- vimentin antibodies is expected to precede clinical symptoms of heart failure in dogs, and detecting anti-vimentin antibodies is expected to be a useful clinical tool for the early prediction and diagnosis of heart in dogs.

Furthermore, congestive heart failure syndrome in both dogs and humans has an element related to endothelial cell dysfunction and a lack of vasodilator substance, nitric oxide, tonically released by the endothelium. The anti-vimentin antibody level might act as a marker of endothelial ill-health, and consequently is likely to be present in heart failure regardless of species.

Since canine heart failure is a useful clinical model of human heart failure, detecting anti-vimentin antibodies is also expected to be a useful clinical tool for the early prediction and diagnosis of heart failure in other mammals including humans.

Thus a second aspect of the invention provides a method for predicting or diagnosing heart failure in a mammal, the method comprising detecting the presence of anti-vimentin antibodies in a suitable sample obtained from the mammal.

This aspect of the invention includes a method for predicting or diagnosing heart failure in a mammal, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative of heart failure in the mammal. In other words, this invention includes a method of aiding in the prediction or diagnosis of heart failure.

It is appreciated that the invention includes a method of assessing whether a mammal is suffering from heart failure, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative of heart failure in the used in association with other tests, or observations by the physician, in reaching a diagnosis.

Typically, the level of anti-vimentin antibodies in the sample that is indicative of heart failure is a level equal to or greater than the mean +2 SD of the level of anti- vimentin antibodies found in a population of control mammals that do not have heart failure. However, it is appreciated that other cut-off levels may be used depending upon the desired sensitivity and specificity of the assay, such as those discussed above with reference to assays for diagnosing hypertension.

It is also appreciated that the invention includes a method of assessing whether a mammal may develop heart failure, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample and assessing whether the level is indicative that the mammal may develop heart failure. It is appreciated that this assessment may aid a predictive diagnosis, and may be used in association with other tests, or observations by the physician, in reaching a predictive diagnosis of heart failure.

Typically, the level of anti-vimentin antibodies in the sample that is indicative that the mammal may develop heart failure is a level equal to or greater than the mean

+2 SD of the level of anti-vimentin antibodies found in a population of control mammals that do not have heart failure and have been shown thereafter not to develop heart failure for a period of at least 3 months, preferably at least 6 months or one year, and more preferably for a period of two years. However, it is appreciated that other cut-off levels may be used depending upon the desired sensitivity and specificity of the assay, such as those discussed above with reference to assays for diagnosing hypertension.

In an embodiment, the mammal is a dog.

In an embodiment, the sample may be from a dog that has previously been diagnosed with a medical condition associated with heart failure, such as chronic valvular disease, myocardial disease, pericardial disease or arrhythmia, or that has a clinical symptom that may result from heart failure.

It is appreciated that a sample can be from a dog that has no symptoms of heart failure, for example as a part of a routine health check up on an older dog (7 years or older) in order to allow an early prediction or diagnosis of heart failure. Alternatively, the sample can be from a breed of dog that commonly suffers from heart disease, as listed above, in order to allow an early prediction of heart failure.

In Example 2, the positive cut-off for an ELISA for the presence of anti-vimentin antibodies in canine plasma that is indicative of heart failure was determined to be 896ng/ml from pilot experiments using a control dog population (n=l 1), although this figure may be adjusted once a larger normal dog population has been tested. Indeed, since only a single dog in the control population had any measurable level of anti-vimentin antibodies, the cut-off value is likely to be reduced once a larger number of normal animals are tested. Thus, in an embodiment, the cut-off value for a positive finding of anti-vimentin antibodies in a canine plasma sample that is indicative of heart failure is at least 500ng/ml, more preferably at least 700ng/ml, and still more preferably at least 900ng/ml.

In dogs, the level of anti-vimentin antibodies in the sample that is indicative that the dog may develop heart failure is at least 900ng/ml.

In another embodiment, the mammal may be a human patient or a cat, both of which suffer from heart failure.

Typically, the cut-off value for a positive finding of anti-vimentin antibodies in a human sample which is indicative of heart failure is a titre of at least 100, and more preferably a titre of at least 120 (measured according to Jurcevic et al, 2001).

In an embodiment, the sample may be from a human patient who has previously been diagnosed with a medical condition associated with heart failure, or who has one or more clinical symptoms that may result from heart failure. It is appreciated that the sample may be from a human patient with no symptoms or mild symptoms of heart failure, i.e. level I or II according to the New York Heart Association Guidelines (Hunt et al, 2005).

It is further appreciated that a sample can be from a human patient who has no symptoms of heart failure, for example as a part of a routine health check up, in order to allow an early prediction or diagnosis of heart failure.

In an embodiment, at least in human patients, the heart failure is not associated with transplant rejection.

In a further embodiment of the second aspect of the invention, the level of one or more further clinical markers of heart failure is measured in a sample from the mammal, or is assessed in the mammal, and a determination is made whether the level of the one or more further markers is indicative of heart failure. Suitable further markers that can be tested include complete blood count, urinalysis, serum electrolytes (including calcium and magnesium), blood urea nitrogen, serum creatinine, fasting blood glucose (glycohemoglobin), lipid profile, liver function tests, and thyroid-stimulating hormone. Other suitable further tests for confirming a diagnosis of heart disease include a twelve-lead electrocardiogram and chest radiograph (PA and lateral) (Hunt et al, 2005).

Conveniently, the level of anti-vimentin antibodies and of the further heart failure marker are measured in the same sample taken from the mammal. Alternatively, the levels may be measured in separate samples taken from the mammal. Conveniently, the samples are taken from the mammal at substantially the same time, or within several hours of each other.

In a preferred embodiment, the presence and/or level of anti-vimentin antibodies and of the one or more further markers of heart failure are all taken into account when assessing whether the mammal has heart failure or may develop heart failure. It is believed that using a combination of markers may improve the accuracy of the assessment (e.g. diagnosis or prediction), but nevertheless determining the level of anti-vimentin antibodies alone in a sample from the mammal is useful.

It is appreciated that a physician can readily differentiate between a patient that has hypertension and one that has heart failure based on the clinical presentation of the patient, as discussed above. However, as discussed above, it may be preferable to measure one or more additional clinical markers of hypertension and/or heart failure, or perform one or more suitable further tests, in order to strengthen the diagnosis or prediction of these conditions.

In addition, an assay for anti-vimentin antibodies has previously been suggested for use in diagnosing idiopathic pulmonary fibrosis and interstitial pneumonia (Yang et al, 2002). However, it is appreciated that a physician can readily differentiate between both hypertension and heart failure, and idiopathic pulmonary fibrosis and interstitial pneumonia, based on the clinical presentation of the patient, including crackling upon chest auscultation, and abnormal chest radiograph at the time of presentation (American Thoracic Society, 2000).

In particular embodiments of the invention, the method of both the first and second aspects may further comprise the step of obtaining a suitable sample from the mammal, and this sample is provided for analysis of the presence and/or levels of anti-vimentin antibodies.

Conveniently, the sample is a fluid sample and suitable samples include whole blood, serum and plasma. It is particularly convenient if the sample is a plasma sample which may be prepared from a blood sample in a standard way (for example by collection in EDTA tubes followed by centrifugatiori). It is also possible that tissue biopsies may contain anti-vimentin antibodies.

The presence and/or level of anti-vimentin antibodies may be determined in the sample by any suitable means. One convenient way of deteπrώώig the presence and/or level of anti-vimentin antibodies in the sample is to make use of a reagent which can identify the anti-vimentin antibodies. Conveniently, the reagent is one which is selectively bound by the anti-vimentin antibodies, but it may be any other type of suitable reagent.

Reagents which are selectively bound by anti-vimentin antibodies include vimentin itself, and antigenic fragments thereof. If fragments of vimentin are used, it is preferred if multiple overlapping fragments that span most, if not all, of the full length vimentin polypeptide are used.

Human vimentin is well known and was first described by Ferrari et al (l 986). By human vimentin we mean a polypeptide having the amino acid sequence listed in Genbank Accession No NP_003371. The sequence of a polynucleotide encoding human vimentin is listed in Genbank Accession No NM_003380. The amino acid sequence of mouse vimentin is listed in Genbank Accession No NP_O35831, the amino acid sequence of rat vimentin is listed in Genbank Accession No NP_112402, the amino acid sequence of dog vimentin is listed in Genbank Accession No XP_849561, and the amino acid sequence of cow vimentin is listed in Genbank Accession No NP_776394.

Anti-vimentin antibodies in the sample may be detected using any of the well- known immunoassay techniques, such as an enzyme linked immunosorbent assay (ELISA). Alternatively, Luminex or other bead-based assays for analysis of multiple biomarkers simultaneously in one sample may be used to detect anti- vimentin antibodies in the sample. Western blotting may also be used but this is less-preferred.

In other words, the first aspect of the invention includes the use of an assay, e.g. an immunoassay, for the presence of anti-vimentin antibodies to predict or diagnose hypertension in a mammal.

Correspondingly, the second aspect of the invention includes the use of an assay, e.g. an immunoassay, for the presence of anti-vimentin antibodies to predict or diagnose heart failure. An immunosorbent assay for detecting anti-vimentin antibodies typically comprises a solid phase coated with vimentin, and a detectable-label conjugate which will bind to the antibodies bound to the solid phase. Typically, the solid phase is a microtitre well. It is preferred if the vimentin is from the same species as the mammal from whom the sample was taken, although this is not essential since there is a high degree of sequence identity between vimentin from various mammalian species (over 97% amino acid sequence identity between human and mouse vimentin, and between human and rat vimentin). Indeed, anti-vimentin antibodies from mice, cats and dogs have been shown to bind to human vimentin.

The detectable-label conjugate usually contains a secondary antibody specific for the mammalian species being tested, for example an anti-dog antibody, an anti-cat antibody, an anti-human antibody and so on, as is well known in the art. Many such antibodies are commercially available. The detectable-label in the conjugate is typically an enzyme, for example horseradish peroxidase, and possibly also a substrate for the enzyme.

Typically, the vimentin protein is coated on microtitre plates overnight at 4°C. Unbound protein is washed off with a wash buffer such as phosphate buffered saline or Tris buffered saline. Serum or other samples are incubated on the plate, typically at 2O⁰C for between 1 and several hours. Unbound material is washed off, the plates are incubated with species specific enzyme-labelled (e.g. horseradish peroxidase) antibody, typically anti-IgG or IgM for serum samples, for 1 to several hours at 20⁰C. Unbound antibody is washed off and plates are incubated with a substrate such as TMB for about 5 minutes, and the optical density measured in a photometer.

Preferably, the vimentin protein is made by expression of a suitable DNA construct encoding the protein using recombinant DNA technology. Suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and purification of expressed proteins, are well known in the art and are described for example in Sambrook et al (2001), incorporated herein by reference. Alternatively, suitable vimentin be obtained and purified from, e.g. cats and dogs, using affinity purification using a column bound with a monoclonal antibody directed against vimentin (e.g. vim 13.2 clone from Sigma)(Harlow & Lane "Antibodies a Laboratory Manual" 1988 Cold Spring Harbor Laboratory Press).

Suitable methods, immunoassays and test kits for detecting anti-vimentin antibodies from a human sample as described in US 5,716,787 and GB 2 268 935. Once the anti-human secondary antibody has been replaced with an appropriate secondary antibody for the species being tested, the methods, immunoassays and test kits described in US 5,716,787 and in GB 2268 935 are suitable for detecting anti-vimentin antibodies in a sample from a mammal other than humans.

Yang et al (2002) describe an ELISA for detecting anti-vimentin antibodies in human sera. The secondary antibody used was a peroxidase-conjugated mouse anti-human IgG antibody. Again, once the anti-human secondary antibody has been replaced with an appropriate secondary antibody for the species being tested, this ELISA is suitable for detecting anti-vimentin antibodies in a sample from other mammalian species.

Jurcevic et al (1998) and Jurcevic et al (2001) describe an ELISA for detecting anti-vimentin antibodies in human sera. The secondary antibody used was a horseradish peroxidase-conjugated rabbit anti-human IgM antibody. Again, once the anti-human secondary antibody has been replaced with an appropriate secondary antibody for the species being tested, this ELISA is suitable for detecting anti-vimentin antibodies in a sample from other mammalian species.

A large number of suitable conjugated and unconjugated secondary antibodies are readily available. For example, a variety of bovine, cat, dog, goat, guinea pig, hamster, horse, human, monkey, mouse, pig, rabbit, rat, and sheep secondary antibodies and conjugates are commercially available from Sigma-Aldrich and from Jackson Immunoresearch Laboratories, Inc. In addition to horseradish peroxidase, detectable labels suitable for use on the secondary antibody include FITC, biotin and alkaline phosphatase, and RPE for luminex applications.

A third aspect of the invention provides the use of a reagent which selectively identifies anti-vimentin antibodies in the diagnosis or prediction of hypertension in a mammal. The invention includes the use of a reagent which selectively identifies anti-vimentin antibodies in the assessment of whether a mammal is suffering from hypertension or may develop hypertension.

A fourth aspect of the invention provides the use of a reagent which selectively identifies anti-vimentin antibodies in the diagnosis or prediction of heart failure in a mammal. The invention includes the use of a reagent which selectively identifies anti-vimentin antibodies in the assessment of whether a mammal is suffering from heart failure or may develop heart failure.

Typically the use of both the third and fourth aspects of the invention is an in vitro use which is performed on a suitable sample obtained from the mammal. Suitable reagents for use in the third and fourth aspects of the invention are disclosed above, and it is particularly preferred to use a reagent that is selectively bound by an anti-vimentin antibody in the diagnosis or prediction of hypertension or heart failure, respectively. Preferably the reagent is vimentin, or less preferably one or more fragments of vimentin. More preferably, the reagent is human vimentin, or one or more fragment thereof. As discussed above, it is preferred if the vimentin is from the same species as the mammal which is being assessed, although this is not essential due to the high degree of sequence identity between vimentin from various mammalian species, and the fact that anti-vimentin antibodies from other mammals has been shown to bind to human vimentin. Typically, the reagent is used in the methods described above.

A fifth aspect of the invention provides a kit of parts comprising vimentin attached to a solid substrate, and detectably labelled anti-cat secondary antibodies. A sixth aspect of the invention provides a kit of parts comprising vimentin attached to a solid substrate, and detectably labelled anti-dog secondary antibodies.

Typically, the solid substrate is a microtitre plate. The vimentin attached to the solid substrate is preferably human vimentin, but may be from another mammalian species. The detectable label may be an enzyme, and the Mt may further comprise a substrate for the enzyme. Optionally, the kit may also comprise suitable wash buffers, instructions for carrying out the assay, and/or positive and negative serum controls appropriate for the species.

A seventh aspect of the invention provides a solid substrate with cat vimentin attached thereto. In an embodiment, the solid substrate with cat vimentin attached may be present in a kit of parts further comprising detectably labelled anti-cat secondary antibodies.

An eighth aspect of the invention provides a solid substrate with dog vimentin attached thereto. In an embodiment, the solid substrate with dog vimentin attached may be present in a kit of parts further comprising detectably labelled anti-dog secondary antibodies.

In the seventh and eighth aspects of the invention, typically, the solid substrate is a microtitre plate. In the kit of parts embodiments of the seventh and eighth aspects of the invention, the detectable label may be an enzyme, and the kit may further comprise a substrate for the enzyme. Optionally, the kit may also comprise suitable wash buffers, instructions for carrying out the assay, and/or positive and negative serum controls appropriate for the species.

A ninth aspect of the invention provides a method of combating hypertension in a mammal the method comprising diagnosing or predicting hypertension in the mammal according to the method of the first aspect of the invention, and treating or preventing the hypertension. By "combating" a particular disease or condition we include the meaning of treating, preventing or ameliorating the symptoms of that particular disease or condition.

In an embodiment, the mammal is a cat and treating or preventing the hypertension comprises administering a therapeutic agent selected from amlopidine or benazepril to the cat.

The invention thus includes the use of amlopidine or benazepril in the preparation of a medicament for treating or preventing hypertension in a cat which has been diagnosed as having hypertension or which has been predicted to develop hypertension by the method of the first aspect of the invention.

In another embodiment, the mammal is a cat and preventing the hypertension comprises placing the cat on a lower salt diet and/or administering a therapeutic agent selected from amlopidine or benazepril to the cat.

In an embodiment, the mammal is a human patient and treating the hypertension comprises administering a therapeutic agent such as a thiazide-type diuretic, an ACE-inhibitor (ACEI), an angiotensin receptor blocker (ARB), a β-blocker or a calcium channel blocker (CCB). More typically, treating the hypertension comprises administering two of these therapeutic agents usually a thiazide-type diuretic and an ACEI, ARB₅ β-blocker, or CCB. Also, lifestyle modifications, such as weight reduction, diet modification including a reduction of dietary sodium, increased physical activity, limiting alcohol consumption and stopping smoking, are recommended treatments for hypertension (JNC VII recommendations; Chobanian et al, 2003).

The invention thus includes the use of a thiazide type diuretic and a further therapeutic agent selected from an ACEI, an ARB, a β-blocker and a CCB in the preparation of a medicament for treating or preventing hypertension in a human patient who has been diagnosed as having hypertension or who has been predicted to develop hypertension by the method of the first aspect of the invention. In another embodiment, the mammal is a human patient and preventing the hypertension comprises lifestyle modifications as above, and possibly administering a thiazide-type diuretic, and less preferably an ACEI, ARB, β- blocker or CCB, or a combination thereof (JNC VII recommendations, Chobanian et α/, 2003).

A tenth aspect of the invention provides a method of combating heart failure in a mammal the method comprising diagnosing or predicting heart failure in the mammal according to the method of the second aspect of the invention, and treating or preventing the heart failure.

In an embodiment, the mammal is a dog and treating the heart failure comprises administering one or more therapeutic agents selected from an ACEI, a diuretic, an antidysrhythmic drug, and a positive inotopic drug, depending on the underlying cause of the heart failure.

The invention thus includes the use of a therapeutic agent selected from an ACEI, a diuretic, an antidysrhythmic drug and a positive inotopic drug in the preparation of a medicament for treating heart failure in a dog which has been diagnosed as having heart failure by the method according to the second aspect of the invention.

In another embodiment, the mammal is a dog and preventing the heart failure comprises administering a therapeutic agent such as an ACE inhibitor, especially for subclinical acquired mitral valve disease (Abbott, 2001).

The invention thus includes the use of an ACEI in the preparation of a medicament for preventing heart failure in a dog which has been predicted to develop heart failure by the method according to the second aspect of the invention.

In an embodiment, the mammal is a human patient and treating the heart failure comprises lifestyle modifications, and/or administering one or more therapeutic agents such as a diuretic, an ACEI₃ a β-blocker, an aldosterone antagonist, an AEB, digitalis, and hydralazine/nitrates; and/or an invasive measure including biventricular pacing, implantable defibrillators, and a heart transplant (Hunt et al, 2005).

The invention thus includes the use of at least one therapeutic agent selected from a diuretic, an ACEI, a β-blocker, an aldosterone antagonist, an ARB, digitalis and hydralazine/nitrates in the preparation of a medicament for treating heart failure in a human patient who has been predicted to develop heart failure by the method according to the second aspect of the invention.

In another embodiment, the mammal is a human patient and preventing the heart failure comprises lifestyle modifications, and/or administering one or more therapeutic agents such as an ACEI and an ARB (Hunt et al, 2005).

The invention thus includes the use of at least one therapeutic agent selected from an ACEI and an ARB in the preparation of a medicament for preventing heart failure in a human patient who has been predicted to develop heart failure by the method according to the second aspect of the invention.

All of the documents referred to herein are incorporated herein, in their entirety, by reference.

The invention will now be described in more detail by reference to the following Examples and Figures.

Figure 1: Detection of anti-vimentin antibodies in plasma from hypertensive cats with chronic renal failure (CRF)

Cats were retrospectively classified into four groups (hypertensive with mild renal failure, n=40; normotensive with moderate renal failure, n=40; normotensive with mild renal failure, n =40; and normal, n=39) according to systolic blood pressure and plasma creatinine. Presence of anti-vimentin antibodies was analysed by ELISA after dilution of plasma 1:2000 as described in the methods. The positive cut-off for the ELISA was determined to be 126μg/ml from pilot experiments using normal cat plasma (n=20).

Figure 2: Immunoreactivity to vimentin in plasma from hypertensive cats with CRF

Recombinant human (hu) .vimentin, murine (mu) vimentin or lysates from human umbilical vein endothelial cells were resolved by SDS-PAGE and blotted onto PVDF. The blots were immuno-probed with an anti-vimentin monoclonal antibody (viml3.2), plasma from a hypertensive cat diluted 1:2000, or plasma from a normal cat diluted 1 :2000.

Figure 3: Development of anti-vimentin antibodies prior to measurable hypertension

Cats from which plasma samples had been stored from before and after clinical diagnosis of hypertension were selected. Plasma samples were analysed by ELISA as above. The positive cut-off for the ELISA was determined to be 126μg/ml from pilot experiments using normal cat plasma (n=20).

Figure 4: Detection of anti-vimentin antibodies in plasma from dogs in heart failure

Dogs were classified into two groups (heart failure, n=34; normal, n=ll). Presence of anti-vimentin antibodies was analysed by ELISA after dilution of plasma 1:2000. The positive cut-off for the ELISA was determined to be 896.0 ng/ml.

Example 1: Detection of anti-vimentin antibodies in a feline clinical population with hypertension secondary to chronic renal failure

Summary Plasma samples from 159 cats were analysed retrospectively for antibodies to vimentin by ELISA, using human recombinant vimentin as the capture antigen. Patients were segregated into four groups according to systolic blood pressure and plasma creatinine: 1. Hypertensive mild CRF (SBP>170mmHg; creatinine 177-250μmol/L; n=40)

2. Normotensive moderate CRF (SBP<170mmHg; creatinine>250μmol/L; n=40)

3. Normotensive mild CRF (SBP<170mmHg; creatinine 177-250μmol/L; n=40)

4. Age matched normals (SBP<170mmHg; creatinine <177μmol/L; n=39).

Significantly elevated levels of anti-vimentin antibodies (p=0.007) were detected in hypertensive cats (12/40; 30%) compared to normal cats (3/39; 7.7%). The development of anti-vimentin antibodies prior to diagnosis of secondary hypertension was analysed in sequential samples from 9 cats, and significantly elevated levels of antibody were detected in 3 (33%) up to 500 days before clinical diagnosis of hypertension.

We concluded that feline hypertension causes release of endothelial cell antigens such as vimentin, and anti-vimentin antibody formation. The development of antibodies precedes development of measurable systolic hypertension and retinal lesion formation in feline CRF and may be a useful clinical tool. Feline systolic hypertension is also a useful clinical model of human hypertension.

Background Hypertension is a multifactorial disease, and high blood pressure is recognised as a complication of chronic renal disease both in humans and in animal models of chronic renal insufficiency (Adamczak et al, 2002). Feline chronic renal failure is reported to occur in approximately 30% of cats between the age of 10-15 years (equivalent to middle age in humans) (Lulich et al, 1992) with the prevalence increasing with age. In feline medicine renal disease is diagnosed on the development of azotaemia (plasma creatinine concentration >177umol/L) supported by inadequate urinary concentrating ability (US G< 1.035). Systolic hypertension in the cat can be either essential or secondary to a variety of clinical conditions including chronic renal failure, hyperthyroidism (Kobayashi et al, 1990; Stiles et al, 1994), chronic anaemia (Morgan 1985), erythropoietin treatment (Cowgill et al, 1998) and primary hyperaldosteronism (Flood et al, 1999; Maggio et al, 2000). Systolic hypertension is reported to occur in approximately 20% of cats with chronic renal failure (Syme et al, 2002). However, white coat hypertension has been reported in cats (Belew et al, 1999) and adequate steps must therefore be taken to ensure accurate measurement of systolic blood pressure through allowing acclimatisation and establishing repeatability of readings. Guidelines for the diagnosis of systolic hypertension in cats have evolved from clinical experience. In line with other studies, we currently diagnose systolic hypertension in cats presenting with systolic blood pressure >170mmHg on two consecutive visit or on one occasion if in association with clinical manifestations of hypertension. In cats hypertensive retinopathy has been reported to occur in up to 60-80% of cats varying in severity from focal bullous retinal detachments and small intra-retinal haemorrhage to complete retinal detachment, gross hyphaema and resulting blindness (Stiles et al, 1994; Elliott et al, 2001).

Many features are shared between hypertension in humans and cats, including the frequent occurrence of retinopathy in untreated cases, increased risk of sudden collapse due to cardiovascular events (Elliott et al, 2001), neurological disease possibly caused by cerebrovascular bleeds (Maggio et al, 2000), heart murmur, gallop rhythm (Elliott et al, 2001), left ventricular hypertrophy (Snyder et al,

2001; Chetboul et al, 2003), heart failure and epistaxis (Littman 1994). For many cats being treated for CRF, systolic hypertension is first diagnosed by the appearance of hypertension related retinopathy resulting in sudden onset blindness and this severely diminishes their quality of life. Therefore it is a major goal of veterinary clinical hypertension research to identify suitable non-invasive markers of hypertension before clinical signs develop. A suitable predictive marker would also be of relevance for diagnosis of human hypertension.

There is increasing evidence that there is an immunological component contributing to the pathogenesis of human hypertension, including increased serum concentrations of immunoglobulins and increased T cell activation (Fu 1995). Several autoantibodies have recently been identified in the serum of hypertensive patients, including antibodies directed against phospholipids (Wu et al, 2005), and nuclear membrane proteins (Fu 1995), cell surface receptors including anti-angiotensin II type 1 receptor antibodies (Dechend et al, 2000; Liao et al, 2002; Dechend et al, 2004; Dragun et al, 2005).

Hypertension and endothelial dysfunction are intimately linked, and there is irreversible damage or necrosis of a proportion of endothelial cells (Gustafsson 1997). We hypothesised that there will be release of some endothelial cytoplasmic proteins into the microenvironment of the blood vessel wall, and that these proteins could act as autoantigens for self-reactive B-cells.

Antibodies against the intermediate filament protein vimentin have previously been described in the serum of patients during chronic rejection of cardiac transplants (transplant associated vasculopathy) (Jurcevic et al, 1998; Jurcevic et al, 2001). This disease is primarily a vascular complication and involves endothelial damage at the time of transplant followed by immunological attack (Rose 2005).

The aim of this study was to determine whether antibodies against vimentin are a feature of feline hypertension, and whether their presence in plasma can be predictive of the onset of hypertension in cats with CRF.

Methods

Patient demographics

Cats included in this study were recruited from geriatric clinics (> 9 years old) established at two first opinion practices in Central London (PDSA, Bow and Beaumont Animal's Hospital, Camden). At presentation all cats received a full physical examination, plasma biochemistry and urinalysis. Where clinical signs (palpable goitre, tachycardia, polyphagia and weight loss, vomiting, diarrhoea) and biochemical results (elevated alkaline phosphatase activity and increased alanine transferase concentrations) were suggestive of hyperthyroidism a total T4 (thyroxine assay) was also evaluated. Systolic blood pressure (SBP) was measured routinely using the Doppler technique. All cats with systolic blood pressure measurements >170mmHg received an indirect fundic examination. Systolic hypertension was diagnosed with a SBP >170mm Hg on two consecutive visits 7-14 days apart or on one occasion if in association with hypertensive retinopathy. Cats identified with hypertension were initially treated with 0.625mg amlodipine besylate once daily. Cats were re-examined 7-14 days after initiating treatment and if the systolic blood pressure was inadequately controlled (SBP>160mm Hg) the dose was increased to 1.25mg once daily. Cats were diagnosed with mild renal disease if their starved plasma creatinine concentration was between 177-250 μmol/L, and with moderate renal disease if their starved plasma creatinine concentration was > 250 μmol/L.

Cats were selected retrospectively and were classified into four groups on the basis of their systolic blood pressure and renal function.

Group 1

Mild renal failure, hypertensive (n=40). Plasma creatinine levels of 197+73 μmol/L; SBP pre-treatment 211±19mmHg, SBP on medication (amlodipine) 146±15mmHg, mean age 15.1+4.4 years.

Group 2

Moderate renal failure, normotensive (n=40). Plasma creatinine levels of

382.3+134.6 μmol/L; SBP 143+13mmHg, mean age 12.1+4.6 years.

Group 3 Mild renal failure, normotensive (n=40). Plasma creatinine levels 204.3±17.8 μmol/L; SBP 143.0±17.8mmHg; mean age 13.3+4.9 years;

Group 4

Normal (n=39). Plasma creatinine levels 125.1+27.8μmol/L; SBP

123.8+14.8mmHg; mean age 11.4+4.9 years.

Cats were excluded from the study if they presented with severe inflammatory disease or severe cardiovascular disease thought not to be associated with hypertension both of which may have falsely elevated levels of plasma anti- vimentin antibody. Anti-Vimentin ELISA

Recombinant human vimentin was purified from BL21 bacteria harbouring the full length human vimentin gene cloned into pET15b (Novagen), using a nickel column according to the manufacturers instructions (Novagen). Recombinant murine vimentin was similarly purified using a nickel column. Recombinant vimentin was stored at lmg/ml in 6M urea to maximise solubility. Recombinant human or mouse vimentin was diluted to lOμg/ml in PBS and used to coat untreated Nunc flat bottomed polystyrene 96 well plates (code 269620) overnight at 4°C (lOOμl/well). Control wells were coated with PBS alone. The following day vimentin (or PBS) was removed, and wells were washed three times with PBS/0.2% Tween-20. Wells were blocked with 200μl 5% non-fat dried milk powder diluted in PBS-0.1% Tween (blocking buffer), which was removed after incubation for two hours at room temperature.

Feline plasma samples were diluted 1 :2000 in blocking buffer and lOOμl added in triplicate to vimentin-coated and control wells. A mouse monoclonal antibody clone vim 13.2 (Sigma Catalogue No V5255; Adams & Watt, 1988) with a starting IgM concentration of 1.5mg/ml was used as a standard, and serial dilutions in blocking buffer from 1:10,000 to 1:32,000 were prepared. lOOμl of each dilution was added in triplicate to vimentin-coated or control wells and used to generate a standard curve. Samples were incubated for two hours at room temperature followed by five washes in PBS-0.1% Tween-20 (wash buffer). HRP-conjugated anti-feline-Ig (Jackson Laboratories, Catalogue No 102-035-003) was diluted 1:250 in blocking buffer. lOOμl was added to each test well (feline samples). HRP-conjugated anti-mouse-Ig diluted 1:1000 in blocking buffer was added to standard wells. Secondary antibodies were incubated for one hour at room temperature then wells were washed five times in wash buffer. 1 OOμl pre- prepared TMB solution (Sigma) was added to each well and after a five minute incubation the reaction was stopped by the addition of 50μl IM sulphuric acid. Plates were read at 450nm on a microtitre plate reader (Wallac). Vimentin western blot

IOng murine or human recombinant vimentin or lOμg cell Iy sates from human umbilical vein endothelial cells (prepared as described previously (Lawson et al, 1999) were resolved by 10% SDS-PAGE and blotted onto PVDF membrane in triplicate, using a No vex minigel system (Invitrogen). The membrane was blocked for 18h at 4°C in 5% non-fat dried milk ρowder/PBS-0.1% Tween-20 (blocking buffer). Blots were probed with a vim 13.2 monoclonal antibody diluted 1:1000, negative feline plasma diluted 1:2000 or positive feline plasma diluted 1:2000 and incubated for 2h at room temperature with agitation. Blots were washed three times in PBS-0.1% Tween-20 (wash buffer), incubated for Ih with either HRP-conjugated anti-feline-Ig (diluted 1:250 in blocking buffer) or HRP-conjugated anti-mouse-Ig (diluted 1:1000 in blocking buffer), washed three times and then incubated with ECL (Pierce), followed by exposure to autoradiograph film for 5 minutes.

Statistical analysis

SPSS (SPSS, Inc) and Prism (Graphpad Software, Inc) were used to carry out one way ANOVA, independent T tests, and Normality test. Altaian's nomogram (Altaian, 1982) was used to determine the power of the assay. All data are presented ± SD using "antibody equivalent units" (equivalent to monoclonal anti- vimentin IgM) in μg/ml. ^"

Results

Optimisation of ELISA for detection of anti-vimentin antibodies in feline plasma In pilot studies, the presence of antibodies that bound to either human or murine vimentin-coated microtitre plates was determined in plasma from 6 cats with previously diagnosed hypertension (SBP >170mm Hg) or 6 normal cats without evidence of CRF or hypertension, after serial dilutions from 1 :50 and 1 :5000. Human vimentin was chosen as a suitable antigen and 1:2000 was chosen as a suitable dilution to eliminate non-specific binding of feline Ig. Dilutions of mAb clone vim 13.2 (IgM) were diluted between 1:10,000 to 1:320,000 and from which the "equivalent concentration of antibody" was determined on each plate, to standardise all assays performed. Plasma from 20 age-matched normal cats was then analysed for the presence of anti-vimentin antibodies using the above conditions. The mean "equivalent concentration of antibody" was determined to be 41.18+42.33 μg/ml.

Using these values, the cut off value for plasma with vimentin-binding activity was determined to be 125.84 μg/ml (mean+2sd). One cat was excluded from the normal group after detection of anti-vimentin antibodies in excess 350μg/ml and subsequent diagnosis of systolic hypertension by Doppler flow detection.

Anti-vimentin binding activity in plasma from hypertensive cats with renal insufficiency compared to normotensive cats

Anti-vimentin antibodies were detected in plasma from cats with CRF with or without hypertension. Significantly elevated levels of anti-vimentin antibodies were detected in the mild CRF/hypertensive cats (Group 1) when compared with other groups (mean (n=40) 108.9+100.6 μg/ml vs normal cats (Group 4) (n=39) 56.3+46.6μg/ml p=0.004; vs moderate CRF/normotensive (Group 2) (n=40) 60.6±63.0 μg/ml p=0.012). Mild CRF/normotensive cats (Group 3) also had elevated levels of anti-vimentin (73.0+80μg/ml) although this was not statistically significant. As shown in Figure 1, 12/40 (30%) mild CRF/hypertensive (Group 1) cats had elevated levels of anti-vimentin, compared to 5/40 (12.5%) mild CRF/normotensive cats (Group 3); 5/40 (12.5%) moderate CRF/normotensive cats (Group 2); and 3/39 (7.7%) normal cats (Group 4). Using Altaian's nomogram to perform power calculations, analysis of 40 cats per patient group was found to be statistically adequate.

Immunodetection of recombinant vimentin after Western blotting using positive plasma.

To confirm that the effects described above were due to specific binding of anti- vimentin antibodies in the plasma to vimentin epitopes, recombinant vimentin was resolved by SDS-PAGE, blotted onto PVDF membrane and subjected to immunodetection by plasma that was previously found to have vimentin-binding activity by ELISA. Negative plasma was used as a negative control. Vim 13.2 mAb was used as a positive control. As shown in Figure 2, plasma with activity by ELISA was able to specifically bind to both human and murine recombinant vimentin, whilst negative plasma did not bind.

Presence ofanti-vimentin antibodies prior to onset of hypertension

Consecutive plasma samples from nine cats taken prior to onset and after diagnosis and ongoing treatment of hypertension were analysed for the presence of anti-vimentin antibodies. As seen in Figure 3, anti-vimentin antibodies were elevated above 126 μg/ml in 3/9 cats at least 30 days before hypertension was diagnosed, and in one cat by more than 500 days. In a fourth cat (PS 814) anti- vimentin levels were elevated with each subsequent visit to the clinic for 76 days prior to measurable elevated SBP, although the levels did not reach the predetermined threshold of 126μg/ml.

Discussion

This study has identified development of anti-vimentin antibodies as a novel marker of hypertension in a feline clinical population. Antibodies were detectable in 30% cats with mild renal failure and hypertension, compared to 7.7% of normal cats. The detection of plasma anti-vimentin antibodies has been shown to precede measurable elevation of SBP outside of the normal range, from between 30 days to more than one year.

In this study cats were segregated according to both SBP, and plasma creatinine levels which are indicative of renal function. It has previously been shown that cats with moderate renal failure (>250mg/L plasma creatinine) are less likely to develop hypertension than those whose plasma creatinine remains between 177- 250mg/l (Syme et al, 2002). This may explain why we found that normotensive cats with mild renal failure had higher level of anti-vimentin antibodies than normotensive cats with moderate renal failure.

It has been documented that cats are susceptible to white coat or office hypertension (Belew et al, 1999) and we (at The Royal Veterinary College, UK) have established clinical protocols to treat systolic hypertension in cats presenting with systolic blood pressure >170mmHg on two consecutive visits when no clinical manifestations of hypertension are apparent. However, one drawback of this approach is that some cats only begin treatment when retinal damage has occurred. Since blood is routinely sampled from cats with CRF₅ the presence of anti-vimentin antibodies could be used as part of a routine diagnostic test, to start anti-hypertensive treatment prior to end-organ damage. From the results presented here, this could be useful for up to 30% cats which develop hypertension and CRF.

The presence of autoantibodies directed against endothelial antigens, other than vimentin, have recently been described in patients with pre-eclampsia and with malignant hypertension (Fu, 1995; Wu et al, 2005; Dechend et al, 2000; Liao et al, 2002; Dechend et al, 2004; and Dragun et al, 2005). The response to injury hypothesis proposed by Ross et al (1997) first highlighted the importance of the endothelium for initiation of atherosclerotic lesion formation. The presence of measurable levels of autoantibodies in hypertension suggests that the immune system is also activated to some degree and therefore it is attractive to propose that the endothelium has a dual role in the pathogenesis of hypertensive disease, firstly due to endothelial dysfunction with regard to impaired release of vasodilators (Puddu et al, 2000; Vanhoutte et al, 2005) but also by contributing to the immune response. Autoantibodies against ATI receptors have been shown to cause signal transduction and vasoconstriction of intact vessels, suggesting that they contribute to the disease process (Wallukat et al, 1999; Dechend et al, 2000; Dechend et al, 2003; Dechend et al, 2004; Dragun et al, 2005). The role of anti-vimentin antibodies in the pathology of hypertension remains to be established.

There is a wealth of literature looking at the prevalence of white coat hypertension in humans and the negative impact this may have on early diagnosis and treatment of hypertension (Celis & Fagard 2004). We consider that the prevalence of anti- vimentin antibodies in serum from human patients with hypertension could also be a useful diagnostic marker of human hypertension. Example 2: Detection of anti-vimentin antibodies in a canine clinical population with heart failure

Summary Plasma samples from 45 dogs were analysed for antibodies to vimentin by ELISA using human recombinant vimentin as the capture antigen. Patients were segregated into two groups: dogs with heart failure (n=34) and normal controls (n=ll). Significantly elevated levels of anti-vimentin antibodies (p=0.028) were detected in dogs with heart failure (8/34; 23.5%) compared to normal dogs (1/11; 9.1%). We concluded that canine heart failure causes release of endothelial cell antigens such as vimentin, and anti-vimentin antibody formation. Canine heart disease is also a useful clinical model of human heart disease.

Background Heart failure (HF) is a major and growing public health issue in the Western world. It is estimated that approximately 5 million patients in the USA have HF and more than 550,000 patients are newly diagnosed each year. The incidence of HF is approximately 1% (10 per 1000) in the population over the age of 65, and it is widely recognised that the ageing population contributes to the increasing incidence of HF (American Heart Association, Heart Disease and Stroke Statistics: 2005 Update, Dallas, Texas: American Heart Association; 2005). The American Heart Association defines HF as a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood. The cardinal manifestations of HF are dyspnea and fatigue, which may limit exercise tolerance, and fluid retention, which may lead to pulmonary congestion and peripheral edema (Hunt et al, 2005). A number of cardiac conditions lead to HF in human and other mammals. Whilst a proportion of HF cases in humans are due to poor lifestyle and western diet, there is likely to be a familial component, however subtle, as well as the established association between ageing and HF. The similarities between the common causes of canine and human HF suggest that canine HF may be a useful clinical model of human HF. Mitral valve endocardiosis (MVE) is the most common cardiac disease in the dog, being present in approximately 30% of dogs 13 years and over. Tricuspid and aortic valves are less often affected. Mitral valve incompetence may result in progressive cardiac enlargement and HF. Post-mortem evidence of advanced degenerative valvular disease was found in 58% dogs over 9 years and more than 90% dogs over 13 years (Abbott, 2001).

Primary myocardial diseases (diseases of the heart muscle of unknown aetiology) including dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are also recognised clinically in the dog. In both diseases there appears to be a hereditary component since most dogs with cardiomyopathy are pure bred. In particular, there are common familial trends among typical "giant breed dogs" such as Doberman pinschers for DCM (Calvert, 2001). The disease has also been recognised in medium sized dogs including spaniels and Dalmatians, rarely in small breed dogs. Between 0.35% and 1.1% of dogs referred for secondary diagnosis have been diagnosed with DCM, and sudden death may also be the first (and last) sign of DCM (O'Grady, 2004).

The aim of this study was to determine whether antibodies against vimentin are a feature of canine heart failure, and whether their presence in plasma can be predictive of the onset of heart failure in dogs.

Methods

Dogs were selected and were classified into two groups: severe congestive heart failure (Group 1) (n=34), and normal (Group 2) (n=l 1).

ELISAs to detect the presence of anti-vimentin antibodies were performed as described above in Example 1, using conjugated anti-canine Ig (Jackson Laboratories Catalogue No 304-035-003) as the secondary antibody. All data are presented ± SD using "antibody equivalent units" (equivalent to monoclonal anti- vimentin IgM) in μg/ml. Results

The cut-off value for plasma with vimentm-binding activity was determined to be 896.0 ng/ml (mean+2sd). Significantly elevated levels of anti-vimentin antibodies were detected in plasma from dogs with heart failure when compared with normal dogs (heart failure (n=34) 1517±599.2 ng/ml vs normal (n=ll) 117.38+117.38; p=0.028). As shown in Figure 4, 8/34 (23.5%) of heart failure dogs had elevated levels of anti-vimentin, compared to 1/11 (9.1%) normal dogs.

Discussion This study has identified development of anti-vimentin antibodies as a novel marker of heart failure in a canine clinical population. Antibodies were detectable in 23.5% (8/34) of dogs with heart failure compared to 9.1% (1/11) of normal dogs. Furthermore, the detection of plasma anti-vimentin antibodies is expected to precede clinical diagnosis of heart failure, from between 30 days to more than one year.

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Claims

1. A method for predicting or diagnosing hypertension in a mammal, the method comprising detecting the presence of anti-vimentin antibodies in a suitable sample obtained from the mammal.

2. A method for diagnosing hypertension in a mammal, the method comprising providing a sample from the mammal, determining the level of anti- vimentin antibodies in the sample, and assessing whether the level is indicative of hypertension in the mammal.

3. A method according to Claim 2 wherein the level of anti-vimentin antibodies in the sample that is indicative of hypertension is a level equal to or greater than the mean +2 standard deviations (SD) of the level of anti-vimentin antibodies found in a population of control mammals that do not have hypertension.

4. A method for predicting whether a mammal may develop hypertension, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample, and assessing whether the level is indicative that the mammal may develop hypertension.

5. A method according to Claim 4 wherein the level of anti-vimentin antibodies in the sample that is indicative that the mammal may develop hypertension is a level equal to or greater than the mean +2 SD of the level of anti- vimentin antibodies found in a population of control mammals that do not have hypertension and have been shown thereafter not to develop hypertension for a period of at least 6 months.

6. A method according to any of Claims 1 -5 wherein the mammal is a cat.

7. A method according to Claim 6 wherein the cat has been diagnosed with a medical condition associated with hypertension, such as chronic renal failure, hyperthyroidism, acromegaly or Cushing's disease.

8. A method according to Claim 6 wherein the cat has one or more clinical symptoms that may result from hypertension, such as retinopathy, ocular bleeding, retinal swelling, retinal detachment, blindness, odd behaviour, hyperactivity, ataxia, dullness, seizures, dementia, coma, left ventricular thickening, congestive heart failure, breathlessness, weight loss, and lethargy.

9. A method according to Claim 6 wherein the cat does not have any clinical symptoms of hypertension.

10. A method according to Claim 6 wherein the cat has been shown not to have hypertension at the time the sample was obtained.

11. A method according to any of Claims 6-9 wherein the cat is 8 or more years old.

12. A method according to any of Claims 1-5 wherein the mammal is a human patient.

13. A method according to Claim 12 wherein the human patient has been diagnosed with a medical condition associated with hypertension.

14. A method according to Claim 12 wherein the human patient has one or more clinical symptoms that may result from hypertension.

15. A method according to Claim 12 wherein the human patient does not have any clinical symptoms of hypertension.

16. A method according to Claim 12 wherein the human patient has been shown not to have hypertension at the time the sample was obtained.

17. A method for predicting or diagnosing heart failure in a mammal, the method comprising detecting the presence of anti-vimentin antibodies in a suitable sample obtained from the mammal.

18. A method for diagnosing heart failure in a mammal, the method comprising providing a sample from the mammal, determining the level of anti- vimentin antibodies in the sample, and assessing whether the level is indicative of heart failure in the mammal.

19. A method according to Claim 18 wherein the level of anti-vimentin antibodies in the sample that is indicative of heart failure is a level equal to or greater than the mean +2 SD of the level of anti-vimentin antibodies found in a population of control mammals that do not have heart failure.

20. A method for predicting whether a mammal ^"may ^"develop heart failure, the method comprising providing a sample from the mammal, determining the level of anti-vimentin antibodies in the sample, and assessing whether the level is indicative that the mammal may develop heart failure.

21. A method according to Claim 20 wherein the level of anti-vimentin antibodies in the sample that is indicative that the mammal may develop heart failure is a level equal to or greater than the mean +2 SD of the level of anti- vimentin antibodies found hi a population of control mammals that do not have heart failure and have been shown thereafter not to develop heart failure for a period, of at least 6 months.

22. A method according to any of Claims 17-21 wherein the mammal is a dog.

23. A method according to Claim 22 wherein the dog has been diagnosed with a medical condition associated with heart failure, such as chronic valvular disease, myocardial disease, pericardial disease or arrhythmia.

24. A method according to Claim 22 wherein the dog has one or more clinical symptoms that may result from heart failure.

25. A method according to Claim 22 wherein the dog does not have any clinical symptoms of heart failure.

26. A method according to Claim 22 wherein the dog does not have heart failure at the time the sample was obtained.

27. A method according to any of Claims 22-26 wherein the dog is 7 or more years old.

28. A method according to any of Claims 22-27 wherein the dog is selected from a Cavalier King Charles, Chihuahua, Miniature Schnauzer, Maltese, Pekinese, Whippet, Doberman, Irish Wolfhound, Great Dane, St Bernard, Newfoundland, ^"Golden Hefriever, Pug, Boxer or a German ^"Shepherd.

29. A method according to any of Claims 17-21 wherein the mammal is a human patient.

30. A method according to Claim 29 wherein the human patient has been diagnosed with a medical condition associated with heart failure.

31. A method according to Claim 29 wherein the human patient has one or more clinical symptoms that may result from heart failure.

32. A method according to Claim 29 wherein the human patient does not have any clinical symptoms of heart failure.

33. A method according to Claim 29 wherein the human patient does not have heart failure at the time the sample was obtained.

34. A method according to any of Claims 29-33 wherein the heart failure is not associated with transplant rejection.

35. A method according to any of Claims 1-34 further comprising the step of obtaining a suitable sample from the mammal.

36. A method according to any of Claims 1-35 wherein the suitable sample is whole blood, serum or plasma.

37. A method according to any of Claims 1-36 wherein detecting the presence and/or level of anti-vimentin antibodies in the sample comprises the steps of: contacting the sample with vimentin, or one or more fragments thereof, under conditions that allow binding of anti-vimentin antibodies the vimentin, and detecting the presence and/or level of anti-vimentin antibodies bound to the vimentin, or the one or more fragments thereof.

38. A method according to Claim 37 wherein the vimentin is from the same species as the mammal from whom the sample was obtained.

39. A method according to Claim 37 wherein the vimentin is human vimentin.

40. A method according to any of Claims 37-39 wherein the vimentin is attached to a solid substrate, such as a microtitre plate.

41. A method according to any of Claims 37-40 wherein detecting the presence of anti-vimentin antibodies bound to the vimentin comprises washing the sample from the vimentin, and using species-specific secondary antibodies to detect the presence of anti-vimentin antibodies bound to the vimentin.

42. A method according to Claim 41 wherein the secondary antibodies are detectably labelled.

43. A method according to Claim 42 wherein the detectable label is horseradish peroxidase.

44. Use of an immunoassay for anti-vimentin antibodies to predict or diagnose hypertension in a mammal.

45. Use of an immunoassay for anti-vimentin antibodies to predict or diagnose heart failure in a mammal.

46. The use of a reagent which selectively identifies anti-vimentin antibodies in the diagnosis or prediction of hypertension in a mammal.

47. The use of a reagent which selectively identifies anti-vimentin antibodies in the diagnosis or prediction of heart failure in a mammal.

48^". ^~ Use according to Claim^' 46 or 47 wherein the reagent is one which is selectively bound by anti-vimentin antibodies.

49. Use according to Claim 48 wherein the reagent comprises vimentin, or one or more fragments thereof.

50. Use according to Claim 49 wherein the vimentin is human vimentin.

51. Use according to any of Claims 44-50 wherein the use is an in vitro use.

52. A kit of parts comprising vimentin attached to a solid substrate, and detectably labelled anti-cat secondary antibodies.

53. A kit of parts comprising vimentin attached to a solid substrate, and detectably labelled anti-dog secondary antibodies.

54. A kit of parts according to Claim 52 or 53 wherein the vimentin is human vimentin.

55. A kit of parts according to any of Claims 52-54 wherein the detectable label is an enzyme, and the kit further comprises a substrate for the enzyme.

56. A solid substrate with cat vimentin attached thereto.

57. A kit of parts comprising the solid substrate according to Claim 56 and detectably labelled anti-cat secondary antibodies.

58. A solid substrate with dog vimentin attached thereto.

59. A kit of parts comprising the solid substrate according to Claim 58 and detectably labelled anti-dog secondary antibodies.

60. A solid substrate or kit of parts according to any of claims 56-59 wherein the solid substrate is a microtitre plate. - -

61. A method of combating hypertension in a mammal the method comprising diagnosing or predicting hypertension in the mammal according to any of Claims 1-16 and 35-43 (when dependent on Claims 1-16), and treating or preventing the hypertension.

62. A method according to Claim 61 wherein the mammal is a cat and treating or preventing the hypertension comprises administering a therapeutic agent selected from amlopidine or benazepril to the cat.

63. Use of amlopidine or benazepril in the preparation of a medicament for treating or preventing hypertension in a cat which has been diagnosed as having hypertension or which has been predicted to develop hypertension by the method according to any of Claims 6-11 and 35-43 (when dependent on Claims 6-11).

64. A method according to Claim 61 wherein the mammal is a human patient and treating or preventing the hypertension comprises administering to the patient a thiazide type diuretic and a further therapeutic agent selected from an ACE- inhibitor (ACEI), an angiotensin receptor blocker (ARB), a β-blocker and a calcium channel blocker (CCB).

65. Use of a thiazide type diuretic and a further therapeutic agent selected from an ACEI, an ARB, a β-blocker and a CCB in the preparation of a medicament for treating or preventing hypertension in a human patient who has been diagnosed as having hypertension or who has been predicted to develop hypertension by the method according to any of Claims 12-16 and 35-43 (when dependent on Claims 12-16).

66. A method of combating heart failure in a mammal the method comprising diagnosing or predicting heart failure in the mammal according to any of Claims 17-34 and 35-43 (when dependent on Claims 17-34), and treating or preventing the heart failure.

67. A method according to Claim 66 wherein the mammal is a dog and treating the heart failure comprises administering a therapeutic agent selected from an ACEI, a diuretic, an antidysrhythmic drug and a positive inotopic drug to the dog.

68. Use of a therapeutic agent selected from an ACEI, a diuretic, an antidysrhythmic drug and a positive inotopic drug in the preparation of a medicament for treating heart failure in a dog which has been diagnosed as having heart failure by the method according to any of Claims 22-28 and 35-43 (when dependent on Claims 22-28).

69. A method according to Claim 66 wherein the mammal is a dog and preventing the heart failure comprises administering an ACEI to the dog.

70. Use of an ACEI in the preparation of a medicament for preventing heart failure in a dog which has been predicted to develop heart failure by the method according to any of Claims 22-28 and 35-43 (when dependent on Claims 22-28).

71. A method according to Claim 66 wherein the mammal is a human patient and treating the heart failure comprises administering to the patient at least one therapeutic agent selected from a diuretic, an ACEI₅ a β-blocker, an aldosterone antagonist, an ARB, digitalis, and hydralazine/nitrates.

72. Use of at least one therapeutic agent selected from a diuretic, an ACEI, a β-blocker, an aldosterone antagonist, an ARB, digitalis, and hydralazine/nitrates in the preparation of a medicament for treating heart failure in a human patient.

73 A method according to Claim 66 wherein the mammal is a human patient and p ^r-eevveennttiinngg tthhee heart failure comprises administering to the patient a therapeutic agent selected from an ACEI or an ARB

74. Use of an ACEI or an ARB in the preparation of a medicament for preventing heart failure in a human patient who ^"has ^"been predictecl^'to^" develop heart failure by the method according to any of Claims 29-34 and 35-43 (when dependent on Claims 29-34).

75. Any novel kit of parts as herein disclosed.