WO1998037414A1

WO1998037414A1 - Immune response diagnostic test

Info

Publication number: WO1998037414A1
Application number: PCT/NZ1998/000024
Authority: WO
Inventors: Robert Bartlett Elliott; Hermann Elard Wasmuth; Jeremy Paul Hill
Original assignee: New Zealand Dairy Board; National Child Health Research Foundation
Priority date: 1997-02-21
Filing date: 1998-02-23
Publication date: 1998-08-27
Also published as: AU6313198A

Abstract

A diagnostic test and kit is disclosed to identify individuals who have susceptibility to an undesired immune response in the presence of β-casein. In particular, the test and kit are used to identify individuals susceptible to Type 1 diabetes induced by a diabetogenic variant of β-casein.

Description

IMMUNE RESPONSE DIAGNOSTIC TEST

TECHNICAL FIELD

This invention relates to a diagnostic test. More particularly it relates to a diagnostic test to identify individuals who have a susceptibility to an undesired immune response in the presence of a variant of β-casein. In a preferred embodiment it relates to a test to identify individuals susceptible to Type 1 diabetes induced by a diabetogenic variant of β-casein.

BACKGROUND ART

Type 1 diabetes, also known as insulin dependent diabetes melitis (IDDM), results from an environmentally triggered, genetically determined immune mediated destruction of the beta cells of the islets of Langerhans. A strong ecological correlation between national cow milk consumption and IDDM incidence suggests that milk might be such an environmental agent.

Indeed, in international application PCT/NZ95/00114 it is disclosed that Type 1 diabetes is induced by certain β-casein variants (most notably β-casein Al) and not by other β-casein variants (most notably β-casein A2) of milk. The invention claimed in the aforesaid application inter alia relates to the production of milk or milk products which do not contain the diabetogenic variant. It would be desirable to have a diagnostic test to identify individuals who are susceptible to the onset of Type 1 diabetes induced by the diabetogenic variant so that the individual involved will know that they should.avoid consuming the diabetogenic variant.

It has been observed that when each of the diabetogenic variants of β-casein (Al, B and C) is digested with the enzyme leucin-aminopeptidase the opioid peptide β- casomorphin-7 (BCM-7) is produced (Hartwig et al, 1995) which has immunological properties. The same study showed that BCM-7 was not produced by digesting each of the A2 and A3 variants. The authors stated that the presence of histidine at the 67 position in the A 1, B and C variants and its substitution by proline at position 67 in the A2 and A3 variants account for this. BCM-7 has opioid-like effects on intestinal motility in vitro (Yashikawa et al 1994) and in vivo (Schusdieziarra et al 1990) in animals and naloxone reversible inhibitory effects on human intestinal lymphocyte proliferative responses to the mitogen concanavalin-A (Elitsur and Luk 1991). It is therefore likely that the diabetes promoting effect of the cow casein could be related to undesired immunological effects of BCM-7. Indeed it has been shown by the inventors that naloxone (a μ-opioid receptor blocker) significantly reduces the diabetes promoting effect of Al casein, providing pharmacological evidence that the effect of Al β-casein is mediated by an opioid receptor mediated mechanism, which is likely

BCM-7 (published at the International Dairy Federation Meeting, Palmerston North, New Zealand, February 1997).

It would be advantageous if the BCM-7 peptide or other active peptide homologue of BCM-7 could be used in a diagnostic test.

It is an object of this invention to go some way towards achieving these desiderata or at least to offer the public a useful choice.

DISCLOSURE OF THE INVENTION

Accordingly, the invention may be said broadly to consist in a method of diagnosing the susceptibility of an individual of an undesired immune response which comprises:

reacting a cellular component of the human immune system of the individual being tested for such susceptibility with an antigen or stimulator in the presence of a μ-opioid receptor agonist, and

measuring the extent of the modulation of said immune response of said cellular component to said antigen or stimulator by said agonist.

Preferably, said, agonist is a peptide.

Preferably, said peptide is BCM-7 or any extension of BCM-7 from the N-terminal end thereof.

Most preferably, said peptide is BCM-7.

Preferably, BCM-7 is used at a concentration range of between 0.1-10 μg/ml.

Preferably, said cellular component is isolated from said individual.

Preferably, said cellular component comprises whole blood. Preferably, said cellular component comprises immune cells selected from the group comprising leucocytes, for example lymphocytes (β-lymphocytes and T-lymphocytes), granulocytes and monocytes.

Preferably, said immune cells are human blood adherent mononuclear cells.

Preferably, the number of cells required for the method of the invention is from 10⁴ to 10⁶.

In one alternative, an antigen is used to stimulate an immune response, ie the production of antibodies.

Preferably, said antigen is ovalbumin (OVA) or tetanus toxoid.

In another alternative, a stimulator of cell oxidative burst is used to stimulate the effect of an increased rate of oxidative burst which is observed during an immune response.

Preferably, said stimulator is phorbol-12-myristate-13-acetate (PMA).

Preferably, said PMA is used in a concentration range of 0.1-1 Oμg/ml.

Preferably, the susceptibility being tested for is susceptibility to the induction of Type 1 diabetes.

Preferably, the extent of modulation of said immune response is measured by the extent of a redox reaction which effectively measures the rate of oxidation of the immune cells and gives a qualitive measure of immune cell activity and therefore immune reaction. This redox reaction may be measured by a colorimetric assay of the level of reduced nitro-blue tetrazolium (NBT) followed by the addition of potassium hydroxide and dimethy lsulphoxide .

Preferably, NBT is added at a concentration of lmg/ml, and incubated with the immune cells for 10-60 mins. The cells may then be fixed with methanol and the reduced NBT measured by change in optical density. In another embodiment the invention may be said broadly to consist in a method of diagnosing the susceptibility of an individual to an undesirable immune response to an antigen substantially as herein described with reference to any example thereof.

In another embodiment the invention may be said broadly to consist in a test kit for carrying out the method as defined above of diagnosing the susceptibility of an individual to an undesirable immune response which comprises an apparatus in which to conduct said reaction between said cellular component, said antigen or stimulator and said agonist, means for storing each of said antigen or stimulator and said agonist before use, optionally means to store any other reagent and optionally means for measuring the extent of modulation of said immune response.

Preferably, said apparatus comprises a microtiter plate having a plurality of reaction wells therein.

Preferably, said means for storing each of said antigen and said agonist comprises sealable containers.

Preferably, said optional means for storing any other reagent comprises a sealable container.

Preferably, said optional means for measuring the extent of modulation of said immune response is a colorimeter.

Preferably, said test kit is as herein described with reference to any example thereof.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 plots the percent inhibition/stimulation effect of BCM-7 on a PMA induced oxidative burst in mononuclear cells of pre-diabetic patients and controls. Figure 2 plots percentage inhibition/stimulation caused by BCM-7 on a PMA induced oxidative burst in mononuclear cells of non-obese diabetic (NOD) mice and Swiss mice.

Figure 3 plots change in optical density (OD) as a measure of the effect of BCM-7 on the primary immune response to ovalbumin (OVA) in NOD and Swiss mice.

MODES OF CARRYING OUT THE INVENTION

HUMAN STUDIES

EXAMPLE 1: Effects of BCM-7 on peripheral blood adherent mononuclear cells from prediabetic and normal children.

1. Cell Isolation:

Adherent mononuclear cells were isolated from children previously shown to have antibodies to pancreatic islet cells indicating islet auto immunity (prediabetic group) and from normal children. Samples were age matched.

2. Cell Adherence to Microtiter Plates:

Approximately one million mononuclear cells were placed in each well of a microtiter plate and incubated for two hours at 37°C in an atmosphere of 5.0% carbon dioxide.

The wells in the plate were washed three times so as to remove all cells which did not adhere to the plate.

3. Cell Stimulation to PMA: The cells in each well were then stimulated with PMA(0.1-10μg/ml) to initiate an oxidative burst reaction. Said oxidative burst is a normal reaction of these cells to some foreign materials.

4. Cell Stimulation in the presence of BCM-7: In some experiments BCM-7 at a concentration of between 0.1-10 μg/ml was added simultaneously with the PMA to the cells. 5. Assay for Cell Oxidative Burst Following Stimulation:

Following an incubation period of 0.5 hours with PMA or PMA + BCM-7. NTB (lmg/ml) was added to the wells and the cells further incubated for one hour. The cells in each well were then fixed by methanol treatment at 56-58°C.

The degree of cell stimulation was determined by colorimetric assay at 630 nm of the level of reduced NBT following the addition of potassium hydroxide and dimethylsulphoxide.

In Figure 1 the results of this assay are plotted. It will be seen that in the prediabetic patient group there is a pronounced inhibition of the PMA induced oxidative burst for each of the sets of cells which are treated. In contrast, for the controls there is one minor inhibition and otherwise stimulation of each of the sets of cells.

EXAMPLE 2: Effect of BCM-7 on PMA induced oxidative burst of peripheral blood adherent mononuclear cells.

PBMC were isolated by Ficoll-Hypaque density gradient centrifugation. Adherent cells were isolated by 2 h incubation at 37 C, 5% C0₂. Cells were stimulated with PMA in the presence or absence of BCM-7 in different concentrations (0.01-lμg/ml). After 20 min NBT was added and cells were incubated for another 60 min NBT reduction was determined as described above.

Children (age 11-15 years; n-10) who had been previously found to have islet cell antibodies <20 units together with one or other of significant GAD, IA2 or insulin autoantibodies were deemed "diabetes prone". Normal control children (n=9) were age and sex matched. The results are shown below in Table 1.

Table 1

BCM-7 addition (μg/ml)

0.01 0.1

Normal controls (n=9)

% increase/decrease of NBT reduction mean +2.8 +2 +7.5

95% CI -7.6.+13.2 -11.6,+15.6 -3.3.+18.3

'Diabetes prone' children (n=10)

% increase/decrease of NBT reduction mean -14* -16.7* -22.4***

95% CI -28.2,+0.2 -35.9,+2.5 -37.6,-7.2 statistical note (Mann-Whitney U test) *p=0.0001 vs Controls

**p=0.001 vs Controls ***p=0.0001 vs Controls

Inhibition of the monocyte/macrophage stimulation by BCM-7 was only seen in the samples from 'diabetes prone' individuals. This difference from controls was apparent at all BCM-7 concentrations used.

EXAMPLE 3: Effect of BCM-7 on proliferative response of human Peripheral blood adherent mononuclear cells to tetanus toxoid -

Peripheral blood adherent mononuclear cells (PBMC) were isolated as described in Example 2 from 'diabetes prone' (age 10-17 years, n=15) and control (age 11-19 years, eventually n=7) children. PBMC (10⁵ cells/well) were stimulated with tetanus toxoid (lOμg/ml) in the absence or presence of BCM-7 (0.01-1 μg/ml). After 3 days ³H- Thymidine was added to the cultures. Cells were harvested after an additional 16 h of incubation and ³H-Thymidine incorporation was measured by liquid scintillation counting. Results are expressed as % increase/decrease of stimulation index to tetanus toxoid in the presence of BCM-7 compared to its absence. The results from samples which gave a proliferative index of less than three (without BCM-7) to tetanus toxoid were discarded. The results are shown below in Table 2. Table 2

BCM-7 addition (μg/ml)

0.01 0.1 normal controls (n=7) mean stimulation index to tetanus toxoid without BCM 7(95% CI:0, 16.8)

%increase/decrease of stimulation Index mean +119.5 +64.1 +92.6

95% CI -100,+361.9 -35.5.+163.7 -88.4,+272.5

'Diabetes prone' children (n=15) mean stimulation Index to tetanus toxoid without BCM 8.1(95%CI:0,22.7)

%increase/decrease of stimulation Index mean -15* -12.7** -12.3***

95%CI -45.6,+15.6 -64.7,+41.3 -64.7.+41.3 statistical note (Mann- Whitney U test) *p=0.001 vs Controls **p=0.0001 vs Controls ***p=0.007 vs Controls

BCM-7 inhibited the proliferative response to tetanus toxoid in the samples obtained from 'diabetes prone' children, but not in controls. Although the variance of the results was higher compared to the other experiments, this difference was statistically significant at all concentrations of BCM-7 used.

This is strong evidence that BCM-7 inhibits immune cell function in prediabetic humans but not in normal humans.

MOUSE STUDIES

EXAMPLE 4: Effects of BCM-7 on normal and NOD mice cells

The procedure of Example 1 was repeated using splenic adherent cells isolated from normal mice (Swiss mice) and Non Obese diabetic (NOD) mice, a strain of mice derived from the Swiss mouse. The results of the assay of this example are presented in Figure 2 and in Table 3 below. Results are expressed as % increase/decrease of NBT reduction in the presence or absence of BCM-7. As with the cells of prediabetic humans, cells of NOD mice exhibit a consistent and strong inhibition of PMA induced oxidation burst indicating an inhibition of immune cell function in the presence of BCM-7. The normal or Swiss mice show a minor inhibition in one case and stimulation of PMA induced oxidative burst in all other cases indicating that BCM-7 has no effect on the PMA stimulation of immune cell function in normal mice.

This confirms the BCM-7 inhibition of immune cell function in NOD mice which is not present in Swiss mice.

Table 3

BCM-7 addition (μg/ml)

0.1 10

Swiss mice (n=5)

% increase/decrease of NBT reduction mean +13.9 +5.7 +7

95% CI -11.9.+39.7 -9.7.+21.1 -5.2.+19.2

non-diabetic NOD mice (n=5)

% increase/decrease of NBT reduction mean -20.3* -27.1** -19.5***

95% CI -51.1.+10.5 -57.7.+10.5 -39.3,+0.3 statistical note (Mann- Whitney U test) *p=0.047 vs Swiss mice

**p=0.009 vs Swiss mice ***p=0.009 vs Swiss mice

EXAMPLE 5: Effect of BCM-7 in vivo on the immune systems of normal and NOD mice

To test the effect of BCM- 7 on the immune system in vivo, NOD and Swiss mice were injected with the antigen ovalbumm (OVA) in saline and incomplete Freund's

Adjuvant: On day one four groups of approximately 10 mice were given the following treatments:

Group 1. NOD mice were injected with OVA. Group 2. NOD mice were injected with OVA + BCM-7. Group 3. Swiss mice were injected with OVA.

Group 4. Swiss mice were injected with OVA + BCM-7.

On day two the same groups were given these additional treatments:

Group 1. NOD mice were injected with a placebo.

Group 2. NOD mice were injected with BCM-7 (no OVA).

Group 3. Swiss mice were injected with a placebo.

Group 4. Swiss mice were injected with BCM-7 (no OVA).

Blood samples were taken from the retroorbital sinus of all mice prior to immunisation and on day eight following the treatments to determine the primary immune response. The blood samples were centrifuged to separate blood cells from the blood serum and the serum then stored at -20°C prior to anti-OVA-antibody determination.

The presence of Anti-OVA-antibodies was determined using an ELISA assay.

The results of this example are presented in Table 4, below and in Figure 3. It will be seen that OVA-antibodies are produced in NOD mice in the absence of BCM-7 but are not produced when BCM-7 is present indicating that the BCM-7 has inhibited the immune response. In the control using Swiss mice it is seen that the immune response to OVA is stimulated by the addition of BCM-7.

Therefore, the marked inhibitory effect of BCM-7 on prediabetic human and NOD mouse immune cell function illustrated in Examples 1, 2, 3 and 4 is supported by the in vivo experiment in NOD and Swiss mice in Example 5. Table 4

without BCM-7 with BCM-7

Swiss mice (n=9) anti-ovalbumin-antibodies (Δ OD) mean 0.173 0.229 95% CI 0,0.549 0,0.697

NOD mice (n=10) anti-ovalbumin-antibodies (Δ OD) mean 0.08* 0** 95% CI 0,0.286 0

Statistical note (Mann- Whitney U test) *p=0.013 vs NOD mice with BCM-7 **p=0.0001 vs Swiss mice with BCM-7

Discussion

The treatment of whole blood or of isolated mononuclear cells with an agonist of this invention in the presence of the cell stimulator PMA and the measurement of modulation of the cellular oxidative burst is a diagnostic test for the determination of individuals likely to develop Type 1-diabetes. In practice, cells isolated from samples of human blood at birth would preferably be subjected to the test so that the diet of individuals diagnosed as being susceptible could be adjusted to avoid the consumption of anti-diabetic variants of β-casein.

Those skilled in the art will know that a test kit can be manufactured containing apparatus in which the immune reaction can be conducted and containing the reagents including the active peptide for conducting the reaction.

For example, the test kit could comprise separate compartments for the sample to be tested, the agonist and any other reagent used. The compartments would be sealed from one another with a frangible seal that could be easily broken to allow the immune response reaction. The extent of modulation of the reaction can be measured by any technique which measures the extent of an oxidation reduction reaction. When colorimetry is used at least a qualitative test can be effected with a colour change strip within the compartments which changes colour with different oxidation numbers. It is known that the Al variant of β -casein induces a Type 1 diabetes immune response. However, it is believed on the basis of what is known in general about immune responses that the Al variant may induce other immune responses of importance to the health of individuals. The present invention is not limited to determining the susceptibility of individuals to Type 1 diabetes but includes diagnosis of any other immune condition which might be caused by the presence of the active peptide.

INDUSTRIAL APPLICABILITY

The present invention is directed to a diagnostic test and kit for carrying out the diagnostic test to identify individuals who have susceptibility to type I diabetes induced by a diabetiogenic variant of β-casein. This test is particularly useful to test susceptible neonates so that the onset type I diabetes in some neonates may be avoided by dietary planning to avoid the consumption of diabetiogenic variant.

REFERENCES:

A. Hartwig, W. Lehmann, H.-J. Teschemacher, G. Erhardt (Germany, 1995). Institute of Veterinary Genetics, Justus-Liebig-University Giessen, Germany; German Cancer

Research Institute, Heidelberg, Germany; Rudolf-Buchheim-Institute of Pharmacology, University of Giessen, Germany. Relationship between genetic variants of cow's milk proteins and release of biologically active peptides in the cow's digestive system.

Yoshikawa M; Suganuma H; Takahashi M; Fukedome SL; Chiba H (1994): Enzymatic release of pro-β-casomorhin-9 and pro-β-casomorhin-9 from bovine β-casein

In: Branti V; Teschemacher H (eds) β-casomorhins and related peptides. Recent developments VHC, Weinheim, Germany, pp 38-42

Schusdiziarra V; Schmid R; Schulte-Frohlinde E; Reiser S; Branti V (1990): Effect of β-casomorhin on gastrointestinal molity and pancreatic endocrine function

In: Nyberg F; Branti V (eds) β-casomorhins and related peptides. Uppsala, Schweden Fyris-Tryck AB; pp 109-110

Elitsur Y and Luk GD (1991): Beta-casomorphin (BCM) and human lamina propris lymphocyic proliferation

Clin. Exp.Immunol. 85: 493-497

Claims

CLAIMS:

1. A method of diagnosing the susceptibility of an individual of an undesired immune response which comprises:

reacting a cellular component of the human immune system of the individual being tested for such susceptibility with an antigen or stimulator in the presence of a ╬╝-opioid receptor agonist, and

measuring the extent of the modulation of said immune response of said cellular component to said antigen of stimulator by said agonist.

2. A method according to claim 1 wherein said agonist is a peptide.

3. A method according to claim 2 wherein said peptide is BCM-7 or any extension of BCM-7 from the N-terminal end thereof.

4. A method according to claim 3 wherein said peptide is BCM-7.

5. A method according to claim 3 or 4 wherein BCM-7 is used at a concentration range of between 0.1-10 ╬╝g/ml.

6. A method according to any preceding claim wherein said cellular component is isolated from said individual.

7. A method according to claim 6 wherein said cellular component comprises whole blood.

8. A method according to claim 6 wherein said cellular component comprises immune cells selected from the group comprising leucocytes.

9. A method according to claim 8 wherein the leucocytes are lymphocytes (╬▓-lymphocytes and T-lymphocytes), granulocytes or monocytes.

10. A method according to any one of claims 6-9 wherein said immune cells are human blood adherent mononuclear cells.

11. A method according to any preceding claim wherein the number of cells required of the cellular component to measure the susceptibility to an undesired immune response is from 10⁴ to 10⁶

12. A method according to claim 1 wherein an antigen is used to stimulate an immune response.

13. A method according to claim 12 wherein said antigen is ovalbumin (OVA) or tetanus toxoid.

14. A method according to claim 1 wherein a stimulator of cell glycolysis is used to stimulate the effect of an increased rate of oxidative burst which is observed during an immune response.

15. A method according to claim 14 wherein said stimulator is phorbol-12- myristate-13-acetate (PMA). ^"

16. A method according to claim 15 wherein said PMA is used in a concentration range of 01.- 1 O╬╝g/ml.

17. A method according to any preceding claim wherein the susceptibility being tested for is susceptibility to the induction of Type 1 diabetes.

18. A method according to any preceding claim, wherein the extent of modulation of said immune response is measured by the extent of a redox reaction.

19. A method according to claim 18 wherein said redox reaction is measured by a colorimetric assay of the level of reduced nitro-blue tetrazolium (NBT) followed by the addition of potassium hydroxide and dimethylsulphoxide.

20. A method according to claim 19 wherein said NBT is added at a concentration of lmg/ml, and incubated with the immune cells for 10-60 mins before the cells are fixed with methanol and the reduced NBT measured by change in optical density.

21. A method of diagnosing the susceptibility of an individual to an undesirable immune response to an antigen substantially as herein described with reference to any example thereof.

22. A test kit for carrying out the method of diagnosing the susceptibility of an individual to an undesirable immune response according to claim 1 which comprises an apparatus in which to conduct said reaction between said cellular component, said antigen or stimulator and said agonist, means for storing each of said antigen or stimulator and said agonist before use, optionally means to store any other reagent and optionally means for measuring the extent of modulation of said immune response.

23. A kit according to claim 21 wherein said apparatus comprises a microtiter plate having a plurality of reaction wells therein.

24. A kit according to claim 21 or 22 wherein said means for storing each of said antigen and said agonist comprises sealable containers.

25. A kit according to any one of claims 21-23 wherein said optional means for storing any other reagent comprises a sealable container.

26. A kit according to any one of claims 21-24 wherein said optional means for measuring the extent of modulation of said immune response is a colorimeter.

27. A kit according to claim 21 as herein described with reference to any example thereof.