Background Of The Invention Recent years have seen an explosion of interest in cytokines as a monitor of T lymphocyte function. Measurement, however, has remained a research tool, as assays have not been tailored for routine laboratory use and clinical value has remained unclear.

The concept of Thl/Th2 cells representing reciprocal polar patterns of cytokine secretion has been of value, and perhaps with surprise has increasingly reflected outcomes of host-parasite relationships in human disease. It is accepted that IFN-y and IL-4 are reliable markers of Thl and Th2 responses, respectively. However, low to non- detected cytokine levels in serum and plasma have discouraged clinical evaluation. Even formal antigen-stimulated T-cell cultures have used ratios rather than absolute supernatant levels to obtain useful information because of reliability issues. IFN-y is more stable and easier to measure but appears to be a less sensitive marker of cytokine shift than Th2 cytokines especially IL-4.

An improved method is required which has the ability to detect IL-4 in biological fluids and culture supernatants.

Summary Of The Invention ; It is an aim of the present invention to ameliorate one or more of the problems of the prior art methods or to at least provide a useful alternative.

The present invention is based at least in part on the surprising observation that significantly higher levels of IL-4 can be detected and measured if the IL-4 is captured at the time it is secreted or produced, by way of in situ capture of the produced/secreted IL-4.

Thus, according to the first aspect of the present invention there is provided a method for detecting and/or quantitating IL-4 comprising contacting a ligand capable of specifically binding IL-4 with a sample capable of producing or releasing IL-4, wherein the IL-4 is captured by the ligand in situ substantially at the time the IL-4 is produced or released.

Preferably, the ligand will be an IL-4 specific antibody and the preferred detection/quantitation format is an immunoassay such as for example ELISA.

However it will be understood that other formats and designs of the assay technique can be employed, as long as IL-4 is captured in situ (as it is being produced/secreted or shortly thereafter).

Preferably, the sample capable of producing or releasing IL-4 is a whole blood sample but it will be understood that any sample which contains cells capable of secreting IL-4 will be a suitable sample for the assay. For example such a sample may be a cellular component of whole blood, isolated cells or a tissue biopsy sample.

Biopsy tissues between 5-10 mg wet weight are preferred.

The preferred method ensures that substantially maximum secretion of IL-4 levels is achieved by CD40L ligation by platelets B cells and/or monocytes in whole

blood, although other mechanisms may also be applicable such as for example ligating CD40L by other natural or synthetic ligands. Prior to detection of IL-4 the whole blood, the cells or the tissue sample may be stimulated with a specific or non-specific antigen to enhance IL-4 production or release.

According, in another aspect of the present invention there is provided a method for detecting and/or quantitating IL-4 comprising contacting a ligand capable of specifically binding IL-4 with a sample capable of producing or releasing IL-4 which sample has been treated such that CD40L is ligated, wherein the IL-4 is captured by the ligand in situ substantially at the time it is produced or secreted.

Preferably, the CD40L is ligated by the addition of cells bearing CD40 such as platelets, fibroblasts, endothelial cells or dendritic cells, to the sample before, during or after contacting the sample with the ligand capable of specifically binding IL-4. Those skilled in the art will be aware of other ligands for CD40L and will be able to use them according to the present teaching. For example, the binding of an antibody specific for CD40L can activate secretion of IL-4. Further, enzymes such as neuraminidase can enhance the activation of IL-4 secretion/production driven by CD40L ligation.

Preferably, the method will comprise blocking the ligand capable of specifically binding IL-4 prior to contacting the sample to be tested with the ligand.

A method of the present invention may also comprise detecting and/or quantitating one or more other elements such as cytokines, antigens, enzymes and immunoglobulins (antibodies) in the same sample in conjunction with IL-4. This can be done most conveniently by collecting the sample after IL-4 has been captured and processing the sample in the usual way for other cytokine, antigen or antibody measurements. However, it will be understood that more than one capture antibody

may be coated onto wells of ELISA plates to capture the other molecule (s) in addition to IL-4 at the same time.

Hence, in another aspect of the invention there is provided a method for detecting and/or quantitating IL-4 in a sample in situ substantially at the time the IL-4 is produced or released as described above, and further comprising the detection and/or quantitation of another element such as cytokine, antigen and/or antibody in the sample.

In another aspect of the present invention there is provided a kit for use in a method of the invention. The kit may comprise one or more of for instance the ligand capable of specifically binding the IL-4, control reagents, wash reagents and instructions for use.

The features and advantages of the invention will become further apparent from the following detailed description of preferred embodiments of the present invention.

Brief Description Of The Accompanying Figures Figure 1 is a graph illustrating the effect of blocking on IL-4 detection in whole blood culture using capture ELISA; Figure 2 is a graph illustrating the effect of incubation time on IL-4 secretion in whole blood culture; Figure 3 is a graph showing a comparison of IL-4 secretion between whole blood and peripheral blood lymphocyte cultures; Figure 4 is a graph illustrating a correlation between IL-4 secretion in mucosal tissue and whole blood cultures; Figure 5 is a graph illustrating the effect of defibrination of whole blood on IL-4 secretion;

Figure 6 is a graph illustrating IL-4 and IFN-y secretion in whole blood from subjects with coronary artery disease; Figure 7 is a graph illustrating increase in secreted IL-4 levels in whole blood cultures from subjects with H. pylori infection following eradication therapy ; Figure 8 is a graph illustrating IL-4 secretion in whole blood cultures from H. pylori infected subjects with failed eradication (resistance) following antibiotic treatment; Figures 9A and 9B are graphs illustrating IL-4 and IFN-y secretion in gastric biopsy cultures, respectively; Figure 10 shows graphs illustrating higher levels of secreted IL-4 in a subject with rye grass allergy compared to a non-allergic subject, irrespective of an allergen or Con A stimulation; Figure 11 is a graph illustrating inhibition of IL-4 production by treatment of whole blood cultures with anti-CD40 monoclonal antibody; and Figure 12 is a graph illustrating C. pneumoniae status and IL-4 secretion in whole blood from patients with coronary artery disease.

Detailed Description Of Preferred Embodiments Of The Present Invention Recognising the value of monitoring Thl/Th2 status at a clinical level, the applicants have developed a new'capture'IL-4 assay using innovative ELISA-based technology for monitoring Thl/Th2 status at a clinical level.

The preferred format of the capture assay is based on short term whole blood culture within standard ELISA plate wells. Without wishing to be bound by any particular mechanism of action, the use of whole blood in preferred embodiments of the

invention can avoid the requirement of an activation step, probably as a result of ligation of CD40L on Thl and Th2 CD4+ cells by platelets.

Preferred embodiments of the assay are simple, inexpensive, can provide same-day results and may be readily employed in diagnostic laboratories. Evidence suggests the absolute IL-4 level in this system reflects both mucosal and systemic events, and provides direct evidence on Thl/Th2 balance in particular clinical circumstances.

Accordingly, the assay finds application in a variety of clinical situations.

The present invention covers a range of modifications of the assay for specific circumstances where either stimulation or collateral diagnostic information is of interest.

The assay can be performed on small quantities of blood (e. g. 0.1 ml) but can also utilise tissue (e. g. gastric biopsy) which maybe of benefit in particular circumstances (e. g. in gastric cancer or pre-cancer changes in H. pylori infection).

A number of specific applications of the assay of the present invention will now be described in more detail below, although it will be clear to those skilled in the art that many other applications of the assay can be easily derived from the teachings provided herein.

1. H. pylori infection. The assay may be used to identify subjects at risk of not responding to routine eradication therapy. It is clinically recognised that about 30% of subjects fail to eradicate infection in clinical practise, and this'failure'cannot be understood simply in terms of antibiotic resistance. In particular, it has been found that about 30% of all subjects fail to secrete IL-4'spontaneously'from blood cultures and that a significant difference exists between levels of IL-4 in those who eradicate H. pylori with the use of antibiotics and those that do not. A good correlation exists between IL-4 production from blood and gastric mucosa (Figure 4), emphasising flexibility of the assay. The ability to predict who will, and who will not, respond to short term simplified

and low side effect eradication therapy, would make a major difference in the strategic approach to eradication of H. pylori infection. The data obtained indicates that those with a low spontaneous (or stimulated) Th2 response are prone to eradication failure and require more intense and more prolonged eradication therapy.

A further use of the IL-4 capture assay relates to evolution of gastric cancer in some patients infected with H. pylori. More specifically, normal Thl dominance within the gastric mucosa shifts to one of profound Th2 dominance in gastric cancer, with high IL-4 values from mucosal biopsies found in those with pre-cancerous lesions. In addition, a combination'IL-4/urease'modification (discussed further below) can be used as a routine assay at endoscopy to diagnose H. pylori, assess likely response to eradication therapy, and to detect early shifts that may predict cancer risks.

2. Detection of atheroma load. Atherosclerosis remains the major cause of death in the western world. It is now recognised as an inflammatory response to injury. The applicants have shown that atheroma is a'Th2 disease'with levels of blood T-cell secreted IL-4 correlating with the extent of coronary artery disease. This impressive correlation fits well with the emerging data relating to T-cell driven inflammation mediated by ligation of CD40L on CD4+ T cells by CD40 on a range of structural and circulating cells including platelets. The present invention provides a unique non- invasive approach to measuring/detecting'high atheroma load', enabling routine assessment of asymptomatic subjects over 50 years to identify a population at risk of developing clinical complications, which requires energetic attention to remediation of risk factors.

3. Atopic disease. Numerous studies now identify a reduction in Th2 cytokines from allergen stimulated T-cells as the most reliable monitor of response to desensitisation.

The capture assay of the present invention (stimulated and non-stimulated) for IL-4 has

been assessed in this context as a marker of need to continue or restart therapy. Fine tuning of desensitisation therapy enables better prediction of clinical outcome, usage of less inappropriate amounts and duration of antigen therapy, and less side effects. This capacity to monitor therapy can lead to an increase in use of antigen therapy. It also increases validity and use of mucosal therapy (e. g. sublingual drops) since it allows for the first time a reliable surrogate marker to be measured/detected. A variant of the present invention, linking both the capture antibody and allergen (eg. rye grass antigen) coated to wells, is also contemplated herein.

4. Additional values. Assays of the invention may also be used for diagnostic and monitoring value of IL-4 to assess Thl/Th2 balance in a range of chronic infections and neoplastic conditions. The present application covers a range of modified tests for maximising diagnostic and monitoring information.

The preferred assay design for detection and/or measurement of IL-4 is ELISA.

This involves coating microtitre plates with a capture antibody specific for IL-4 and applying a sample of whole blood. Following a period of incubation, during which IL- 4 released by the cellular component of the blood sample is captured by the antibody, the captured IL-4 is detected by another IL-4 specific antibody conjugated to a detectable marker.

Using this assay system with samples from normal individuals, from patients with coronary heart disease and from patients with Helicobacter pylori infection, it was possible to demonstrate detectable IL-4 levels, even in normal subjects, whereas IL-4 could not be detected in these subjects if serum or plasma were used.

The measurement/detection of IL-4 using an assay of the present invention may be conducted in conjunction with measurement of other cytokines, for example

cytokines which are markers of Thl response, such as IFN-y, to enable calculation of Thl/Th2"balance"as absolute values or ratios.

IL-4 measurements may also be used in conjunction with measurement of specific diagnostic markers, such as H. pylori antibody, to enable both diagnosis of H. pylori infection and chance of failure of standard eradication therapy. Other antibodies may also be measured in conjunction with IL-4, for example those which are markers and indicators of difficult-to-treat infections (e. g. Chlamydia, tuberculosis, etc.).

In addition, other diagnostic marker may be measured in conjunction with IL-4 measurement, such as an antigen or an enzyme, particularly urease from gastric biopsy for H. pylori. In the later case a single biopsy sample may be used to measure both urease and IL-4.

The IL-4 assay of the present invention. can therefore be applied in conjunction with determination of other specific and characteristic markers, to provide useful diagnostic and predictive tools. For example, to detect poor IL-4 response in circumstances of persistent infection, e. g. H. pylori, tuberculosis, Chlamydia etc., to detect high IL-4 response in diagnosis of pre-cancer/cancer of the stomach, high atheroma load, and the like. The assay of the present invention can also be used to monitor therapy for allergy (e. g. the Thl/Th2 ratio falls with successful desensitisation therapy. Further, completion, re-institution of desensitisation (injection/mucosal) can be meaningfully determined with measurement of IL-4/IFN-y combination.

The measurement of combinations of IL-4 with other markers mentioned above can be performed on the same sample. The sample is most conveniently whole blood but the method of the present invention may also be applied to biopsy samples and to selected cell populations. In this regard, whole blood may be fractionated and/or depleted of certain cell populations and components, as long as it still contains cellular

populations capable of producing/secreting IL-4 which can be detected in situ as described herein.

The invention will now be described more particularly with reference to a number of non-limiting examples.

Examples Example 1: Determination of IL-4 in a blood sample of human subjects Wells of 96-well microtitre plates are coated with 2 , g/mL of monoclonal anti- IL-4 capture antibody (IL-4 MoAb) in sodium bicarbonate buffer pH 8.5. After removal of antibody and washing with PBS/Tween 20,100 pL of heparinised whole blood is added to each well containing an equal volume of AIM-V medium. After incubation at 37°C, in a 5% CO2 atmosphere for 24-48 hours the plasma supernatant is removed for measurement of other cytokines or immunoglobulins (e. g. IFN-y or various antibody levels). The amount of IL-4 captured by IL-4 MAb in each well is detected by reaction with biotinylated murine anti-IL-4 antibody at 0. 5u. g/ml (Endogen) and strepavidin-peroxidase conjugate. The amount of IL-4 can be measured by colour development with tetramethyl benzidine substrate at 1: 400 dilution, (TMB, Sigma-Aldrich, Castle Hill, NSW, Australia), using the appropriate IL-4 standards (recombinant IL-4 from Endogen). Absorbance is read at 450nm in an ELISA plate reader (Biorad 450, Japan).

A blocking step using BSA, casein or other agents commonly used for this purpose may be introduced after coating the wells with the capture antibody. However better results, at least with whole blood samples, can be achieved if this step is omitted (see Fig. 1). This may be due to the effect of plasma proteins which act as blocking proteins.

In particular, IL-4 capture ELISA using microtitre wells coated with monoclonal anti-IL-4 without blocking with 3% bovine serum albumin or casein to prevent non- specific binding to plastic surface gave the best results with low background values. In contrast, wells blocked with BSA gave higher background values.

The limit of sensitivity for IL-4 in this experiment was 9.4pg/ml. The amount of IL-4 in the samples was determined using a Softmax program (Version 2.3 FPU, USA).

In this system a polyclonal capture antibody typically raised in goat, sheep or rabbit may also be effectively used.

Further, not entire microtitre plate need be coated with the capture antibody.

Strips of microwells can be coated (75ul/well at 5ug/ml in 0. 1M sodium bicarbonate buffer pH 9; 2 hours at 37°C, remove solution) and after incubating with BSA (3% solution, 200ul/well at 37°C for 45 minutes, remove solution) and drying at 37°C for 20 minutes, stored at 4°C until required.

Experiments performed to optimise the assay indicate that incubation of plates with the whole blood samples for 24 to 48 hours is not necessary. Shorter periods of time can provide good results as shown in Fig. 2. In circumstances where a short incubation period is desirable, a 2 hour incubation may be sufficient to obtain valuable data. Specifically, heparinised whole blood from subjects with atheroma were cultured in microtitre wells at 37°C for 2,4,6 and 24 hrs, after which time the amount of IL-4 secretion was measured by capture ELISA. Maximal levels of IL-4 were detected by 2 hrs and the levels remained stable of a 24 hr period.

IL-4 was not detectable in supernatants of whole blood or of isolated PBL (peripheral blood leukocytes) but was easily measurable in whole blood, whether or not it was stimulated by H. pylori antigen (see Fig. 3). Levels of IL-4 could also be

detected in isolated PBL cultures after stimulation with the H. pylori antigen. In particular, whole blood and peripheral blood lymphocytes (PBL) from the same subject were cultured in microtitre wells at 37°C for 24 hrs after which time IL-4 secretion was measured by the capture ELISA assay and inculture supernatants. Whole blood cultures produced the highest level of IL-4 by capture assay whereas low to undetectable levels were obtained in PBL culture or culture supernatants.

The flexibility of the assay is also shown by a good correlation between IL-4 production from blood and mucosal (biopsy) (Fig. 4). For this study, gastric biopsy tissue (5-10 mg) and whole blood were cultured at 37°C for 24 hrs in microtitre wells coated with monoclonal anti-IL-4. The amounts of IL-4 secreted were measured by capture ELISA assay. The correlation between mucosal IL-4 and blood IL-4 indicates IL-4 secretion in whole blood reflects that secreted in local tissue.

Example 2: Effect of CD40L ligation on IL-4 production/secretion Without wishing to be bound by any particular mode or mechanism of action, platelets appear to be an important factor for the production of IL-4 on the basis that T-cell inflammation is driven by ligation of CD40L expressed on activated T-cells by CD40 which may be expressed on platelets.

The present study compared whole blood collected in heparin-coated tubes with defibrinated blood. In the latter, 5 mL of freshly collected whole blood from subjects with atheroma was defibrinated by gently mixing the blood in a tube containing glass beads for 10 mins. After defibrination was completed, whole blood were separated by aspiration and then cultured with equal volume of AIM V serum-free medium (150 uL) in wells coated with or without IL-4, as described in Example 1 above.

As shown in Fig. 5, IL-4 secretion in defibrinated blood was significantly reduced compared with that in heparinised whole blood, indicating that cells bearing

CD40, such as platelets, or at least ligation of CD40L, may be required for optimal production of IL-4 in whole blood. It is believed that this ensures a constant near maximal secretion rate enabling a more realistic application of stable state, i. e. reduces variation otherwise seen. Thus, a system based on the CD40L/CD40 interaction (i. e.

CD40L ligated by CD40) can enable stable, maximum secretion of IL-4 and mimics the possible in vivo interaction.

Other cells which may also be able to ligate CD40L because they possess CD40, are fibroblasts, B cells, macrophages, smooth muscle cells, endothelial cells and dendritic cells.

Example 3: A cytokine-based whole blood assay for detection of activated T-cells Whole blood was obtained from five groups of subjects undergoing planned diagnostic coronary angiogram.

Group 1. Normal coronary arteries (10 subjects-four male (45-60 yrs) and six female (32-64 yrs)).

Group 2. Mild coronary artery atherosclerosis (28 subjects-16 male (46-76 yrs) and 12 female (44-75 yrs)).

Group 3.19 subjects with one vessel disease (9 male (51-76 yrs) and 10 female (48-77 yrs)).

Group 4.15 subjects with two vessel disease (10 male (54-75 yrs) and five female (45-76 yrs)).

Group 5.23 subjects with three vessel disease (16 male (52-81 yrs) and 6 female (60-77 yrs)).

3.1 Cytokine assay: Heparinised blood from the subjects was diluted 1 : 1 (v/v) in AIM-V medium in wells of a 96-well round-bottomed microtitre plate. For measuring the production of IL-4, some wells were pre-coated with a capture monoclonal anti-IL-4

antibody as described above. The cultures were incubated at 37°C in a 5% CO2 atmosphere for 24-48 hours after which time the plasma supernatants were collected for IL-5, IL-10 and IFN-y assays (Endogen kits, CSL). Captured IL-4 together with appropriate standards were directly determined in the wells as described above. The whole blood assay for measuring antigen-specific cytokine production profiles has been validated for studies in human subjects with H. pylori infection (Ren et al, Helicobacter, 2000,5 (3): 135-141, incorporated in its entirety herein by reference).

As shown in Fig. 6, an increase in IL-4 production was detected in the disease groups with significantly higher levels of IL-4 detected in subjects with two vessel disease (p < 0.05) and three vessel disease (p < 0.01). IL-4 secretion was undetectable at zero time or in culture incubated at room temperature. These results demonstrated that IL-4 secreted from circulating cells correlated with the extent of coronary heart disease. CD4+ T cells are the source of IL-4 in whole blood culture. No such correlation was noted IFN-y with lower levels of secretion in culture from subjects with three vessel disease. However, an inverse relationship between IFN-y and IL-4 was found in all subjects with p<0.001.

Various relevant antibody levels can also be measured in the same sample. This can be readily accomplished by ELISA or other immunoassay technique for any antibody in the supernatants.

Example 4: Diagnostic applications of IL-4 assay 4.1 Increase in secreted IL-4 as an indicator of successful eradication therapy in subjects with Hpylori infection Secreted IL-4 capture ELISA, as described above, was used to monitor the effect of H. pylori eradication therapy on IL-4 secretion in unstimulated whole blood or whole blood stimulated with H. pylori antigens which were prepared by glycine-acid

extract of whole bacteria. The extract consists of surface antigens as well as cytoplasmic antigens by SDS gel electorphoresis.

Heparinised whole blood was cultured with AIM-V medium (Life Science Technology), with or without Hpylori antigens for 24 hrs at 37°C after which time secreted IL-4 was measured by capture IL-4 ELISA assay, as above. Fig. 7 shows that the levels of secreted IL-4 before and after antibiotic treatment from 13 subjects.

There was an increase in IL-4 production in whole blood cultures irrespective of antigen stimulation following eradication therapy, compared with levels before eradication (p < 0.05). IFN-y production was unchanged after eradication therapy, suggesting that IL-4 is a more sensitive marker of cytokine shift.

4.2 Low secreted IL-4 in subjects with H. pylori infection following failed eradication therapy (resistance).

Secreted IL-4 was measured in antigen-stimulated or unstimulated whole blood cultures from subjects whom had failed to eradicate the organisms after antibiotic treatment. Fig. 8 shows that subjects who successfully eradicated the organisms had higher levels of secreted IL-4 compared with those before eradication therapy. In contrast, resistant subjects whom failed to eradicate had low levels of secreted IL-4 irrespective of antigen-stimulation in culture. There was no change in IFN-y.

4.3 IL-4 measurement as predictor ofprogression to gastric carcinoma in subjects with or without H. pylori infection.

The potential value of IL-4 assay of the present invention in the diagnosis of pre- cancer lesion (dysplasia) and gastric carcinoma in subjects with or without H. pylori infection is illustrated by the data shown in Fig. 9. Gastric biopsies taken from subjects with H. pylori infection with histological evidence of dysplasia (pre-cancer) were cultured overnight after which the level of secreted IL-4 was measured by the capture

ELISA described above. Subjects with gastric carcinoma had low to undetectable levels of IFN-y compared to the high levels of secreted IL-4 in mucosal cultures, whereas the reverse was true in H. pylori-infected subjects with chronic gastritis. In marked contrast, subjects with pre-cancer lesions had higher levels of IFN-y with increased levels of secreted IL-4, representing a shift to a type 2 CD4+ T helper cell response seen in gastric carcinoma. In this H. pylori-gastritis-dysplasia-gastric carcinoma model, IL-4 measurement can be used as a diagnostic/monitoring test for long-term prospective and horizontal studies.

4.4 Detection of secreted IL-4 in whole blood culture stimulated with ryegrass allergen.

An IL-4 assay in accordance with the instant invention was used to detect secreted IL-4 in whole blood cultures from subjects with ryegrass allergy. Whole blood cultures were either unstimulated or stimulated with either Con A (polyclonal T cell activator, Pharmacia-Upjohn) or ryegrass allergen extract (Greers Laboratory, Lenoir, NC, USA) at various concentrations at 37°C for 1 or 2 days, after which secreted IL-4 levels were measured by the capture assay as described above. As shown in Fig. 10, higher levels of secreted IL-4 were detected in subject 1 with ryegrass allergy compared to non-allergic subject 2, irrespective of allergen or Con A stimulation.

4.5 Other applications.

Many studies have shown that desensitisation by specific immunotherapy (SIT) with allergen in atopic disease leads to a profound downregulation of Th2 response, characterised by a decrease in the production of IL-4. The IL-4 assay of the present invention can be used to monitor SIT with better prediction of clinical outcome and compliance. In addition, the IL-4 assay of the present invention can be adopted for use in allergen stimulation by co-coating specific allergen as stimulator of secreted IL-4

and capture anti-IL-4 antibody. Other uses include infection (e. g. Chlamydia), neoplasia and autoimmune diseases where monitoring IL-4 is of value to assess Thl/Th2 balance.

Example 5: IL-4 production in whole blood cultures f om patients with coronary artery disease is inhibited by anti-CD40 monoclonal antibody.

Heparinised blood was collected from subjects with coronary artery disease and cultured in equal volume of serum-free AIM-V medium (300 pL total volume) containing graded concentrations of anti-CD40L or anti-CD40 antibody in a 96-well flat- bottomed coated with anti-IL-4 antibody. Control cultures contained medium alone or a mouse IgGl isotype control. After incubation for 24hrs, the amount of IL-4 secreted was measured by the capture ELISA assay as described in Example 1. As shown in Fig. 11, IL-4 production was inhibited by anti-CD40 in a dose-dependent manner compared with control (p<0.05 for 9 subjects) while the addition of mouse IgGl isotype control or anti- CD40L (data not shown) had no effect. The result showed that the engagement of CD40 is critical for the production of IL-4 whole blood culture.

Example 6: Relationship between G'pneumoniae antibody status and IL-4 secretion in whole blood, from patients with coronary artery disease.

IL-4 secretion in whole blood was compared with Cpneumoniae serum antibody status in groups of subjects with normal, mild disease or 1,2 or 3 vessel disease. As shown in Fig. 12, level of secreted IL-4 were higher in subjects with positive serum antibody to Cpneumoniae than in subjects with negative antibody in all subject groups.

In subjects with 2-3 vessel disease, higher level of secreted IL-4 were detected. While higher levelsof IFN-y were detected in normal and subjects with mild or 1-2 vessel disease positive for C pneumoniae antibody, there was no relationship between antibody status and IFN-y levels in subjects with 3 vessel disease.

Although the present invention has been described with reference to a number of preferred embodiments, the skilled addressee will understand that variations and- modifications are possible without departing from the scope of the invention.